Bioprobes at a Glance

Hiroyuki Koshino; Takeo Usui

doi:10.1007/978-4-431-56529-1_6

Bioprobes pp 149-371 | Cite as

Bioprobes at a Glance

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Hiroyuki Koshino
Takeo Usui

Chapter

First Online: 17 May 2017

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Abstract

This chapter provides the fact database of bioprobes that have been reported.

1.

The “Bioprobes at a Glance” section is ordered alphabetically by entry name.
2.

A compound with an asterisk (*) at the entry name is the synthetic compound.
3.

Molecular weight is rounded off to two decimal places.
4.

Solubility is represented by symbols: +++, easily soluble; ++, soluble; +, sparingly soluble; ±, barely soluble; −, insoluble.

Keywords

Discovery Isolation Structure Chemical study Target identification Biological activity

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16F16*

Keywords: [Protein disulfide isomerase (PDI)] [Misfolded proteins]

Structure

	16F16	16F16A
Molecular formula	C₁₆H₁₇ClN₂O₃	C₁₈H₁₇ClN₂O₃
Molecular weight	320.77	344.80
Solubility	DMSO, ++; H₂O, ±; MeOH, +	DMSO, ++; H₂O, ±; MeOH, +

Discovery/Isolation/Chemical Studies

16F16 was identified as a small-molecule suppressor of polyglutamine-induced apoptosis in a PC12 cell model of Huntington’s disease from 68,887 compounds (small-molecule libraries containing natural products, natural product analogs, synthetic drug–like compounds, and annotated biologically active compounds) [1]. An analog of 16F16 lacking the chloro substituent was inactive, and the alkyne analog of 16F16 (16F16A) suppressed Q103-induced apoptosis, suggesting that 16F16 covalently binds its target proteins. By coupling azidofluorescein to PC12-Q103 lysates treated with 16F16A, two isoforms of protein disulfide isomerase (PDIA1 and PDIA3) were identified as specific targets of 16F16A.

Biological Studies

16F16 restored “normal” cell morphology to induced Q103 (the first exon of the human huntingtin gene (HTT) containing mutant polyglutamine repeats fused to EGFP)–expressing cells without affecting the accumulation of the HTT-Q103 inclusion body. 16F16 inhibited the catalytic activity of PDI, and suppressed the PDI-induced MOMP (mitochondrial outermembrane permeabilization ) and mitochondrial release of cytochrome c. In a rat corticostriatal brain-slice model, PDI inhibitors including 16F16 suppressed neurotoxicity induced by mutant huntingtin exon 1 and amyloid β. These results indicate the linking of protein misfolding and apoptotic cell death, and the proapoptotic function of PDI [1].

Biological Activity of 16F16

IC₅₀ values for 16F16 and 16F16A in insulin aggregation assay: 74 μM and 108 μM, respectively [2]

GI₅₀ for 16F16 and 16F16A against several cancer cell lines: ~ 4 μM [2]

References

1.

Hoffstrom BG, Kaplan A, Letso R, Schmid RS, Turmel GJ, Lo DC, Stockwell BR (2010) Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat Chem Biol 6: 900–906
2.

Ge J, Zhang CJ, Li L, Chong LM, Wu X, Hao P, Sze SK, Yao SQ (2013) Small molecule probe suitable for in situ profiling and inhibition of protein disulfide isomerase. ACS Chem Biol 8:2577–2585

Aplyronine A and C

Keywords: [Antitumor] [Actin polymerization inhibitor] [Actin-tubulin complex inducer]

Structure

	Aplyronine A	Aplyronine C
Molecular formula	C₅₉H₁₀₁N₃O₁₄	C₅₃H₉₀N₂O₁₂
Molecular weight	1076.46	947.31
Solubility	DMSO, +++; H₂O, −; MeOH, +++	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation

Aplyronine A was isolated as a potent antitumor compound from the sea hare Aplysia kurodai from the Pacific coast of Mie Prefecture, Japan [1].

Biological Studies

Aplyronine A inhibited actin polymerization and quickly depolymerized F-actin by forming a 1:1 complex with G-actin [2]. X-ray crystallography revealed that aplyronine A binds to a hydrophobic cleft composed of subdomains 1 and 3 of actin by intercalating its aliphatic tail part into the actin molecule [3]. Interestingly, the trimethylserine moiety of aplyronine A extends to the outside of the actin protein. Because aplyronine A shows potent cytotoxicity compared with aplyronine C which lacks trimethylserine moiety, this moiety is thought to be important for cytotoxicity. Kita et al. noticed that both aplyronine A and C disrupt actin network in cells at the same concentration, but only aplyronine A induced apoptosis at low concentrations [4]. Further chemical biological analyses revealed that aplyronine A forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with actin synergistically binding to tubulin, and inhibits tubulin polymerization. This complex formation results in the inhibition of the spindle formation.

Biological Activity of Aplyronine A and C

IC₅₀ values for aplyronine A and C on cell growth of HeLa-S3 cells: 0.039 and 159 ng/ml, respectively [1]

Antitumor activity in vivo of aplyronine A against P388 murine leukemia: 0.08 mg/kg, T/C = 545% [1]

K_d for aplyronine A to G-actin: 0.10 μM [2]

Inhibition of actin network in HeLa cells by aplyronines A and C: 100 nM [4]

Induction of abnormal spindle formation by aplyronine A: 0.1 nM [4]

References

1.

Yamada K, Ojika M, Ishigaki T, Yoshida Y, Ekimoto H, Arakawa M (1993) Aplyronine-A, a Potent Antitumor Substance, and the Congeners Aplyronine-B and Aplyronine-C Isolated from the Sea Hare Aplysia kurodai. J Am Chem Soc 115:11020–11021
2.

Saito S, Watabe S, Ozaki H, Kigoshi H, Yamada K, Fusetani N, Karaki H (1996) Novel actin depolymerizing macrolide aplyronine A. J Biochem 120:552–555
3.

Hirata K, Muraoka S, Suenaga K, Kuroda T, Kato K, Tanaka H, Yamamoto M, Takata M, Yamada K, Kigoshi H (2006) Structure basis for antitumor effect of aplyronine A. J Mol Biol 356:945–954
4.

Kita M, Hirayama Y, Yoneda K, Yamagishi K, Chinen T, Usui T, Sumiya E, Uesugi M, Kigoshi H (2013) Inhibition of microtubule assembly by a complex of actin and antitumor macrolide aplyronine A. J Am Chem Soc 135:18089–18095

Apoptolidin

Keywords: [Apoptosis inducer] [F₀/F₁ ATPase inhibitor]

Structure

	Apoptolidin
Molecular formula	C₅₈H₉₆O₂₁
Molecular weight	1129.39
Solubility	DMSO, +++; H₂O, ±; MeOH, +++

Discovery/Isolation/Chemical Studies

Apoptolidin was isolated as an apoptosis inducer in adenovirus E1A–transformed rat glia cells from Nocardiopsis sp. [1]. The structure of apoptolidin was determined by detailed NMR studies including its stereochemistry [2]. The first total synthesis was done by the Nicolau group [3–5].

Biological Studies

Apoptosis induced by 1 μM apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9 in a mouse lymphoma cell line. From a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action, the target molecule of apoptolidin was revealed as mitochondrial F₀F₁-ATP synthase [6]. Correlation analysis of F₀F₁-ATP synthase inhibitors and gene expression suggested that pyruvate kinase and aspartate aminotransferase are drug sensitivity determinants, suggesting that the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Indeed, inhibitors of both lactate formation and carbon flux through the Embden–Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to apoptolidin [7].

Biological Activity of Apoptolidin

IC₅₀ for glia cells: >100 μg/ml [1]

IC₅₀ for RG-E1A-7, RG-E1A19K-2, RG-E1A54K-9, or RG-E1-4 cells: 10–13 ng/ml [1]

K_i for yeast mitochondria F₀F₁-ATPase: 4–5 μM [6]

References

1.

Kim JW, Adachi H, Shin-ya K, Hayakawa Y, Seto H (1997) Apoptolidin, a new apoptosis inducer in transformed cells from Nocardiopsis sp. J Antibiot (Tokyo) 50:628–630
2.

Hayakawa Y, Kim JW, Adachi H, Shin-ya K, Fujita K, Seto H (1998) Structure of apoptolidin, a specific apoptosis inducer in transformed cells. J Am Chem Soc 120:3524–3525
3.

Nicolaou KC, Li Y, Fylaktakidou KC, Mitchell HJ, Wei HX, Weyershausen B (2001) Total Synthesis of Apoptolidin: part 1. Retrosynthetic Analysis and Construction of Building Blocks. Angew Chem Int Ed Eng. 40:3849–3854
4.

Nicolaou KC, Li Y, Fylaktakidou KC, Mitchell HJ, Sugita K (2001) Total Synthesis of Apoptolidin: part 2. Coupling of Key Building Blocks and Completion of the Synthesis. Angew Chem Int Ed Eng. 40:3854–3857
5.

Nicolaou KC, Li Y, Sugita K, Monenschein H, Guntupalli P, Mitchell HJ, Fylaktakidou KC, Vourloumis D, Giannakakou P, O″Brate A (2003) Total synthesis of apoptolidin: completion of the synthesis and analogue synthesis and evaluation. J Am Chem Soc 125:15443–15454
6.

Salomon AR, Voehringer DW, Herzenberg LA, Khosla C (2001) Apoptolidin, a selective cytotoxic agent, is an inhibitor of F₀F₁-ATPase. Chem Biol 8:71–80
7.

Salomon AR, Voehringer DW, Herzenberg LA, Khosla C (2000) Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F₀F₁-ATPase. Proc Natl Acad Sci U S A 97:14766–14771

Apratoxin A

Keywords: [Hsc70/Hsp70] [JAK/STAT signaling] [N-glycosylation ] [Secretory pathway]

Structure

	Apratoxin A	Oz-apratoxin A
Molecular formula	C₄₅H₆₉N₅O₈S	C₄₅H₆₉N₅O₉
Molecular weight	840.13	824.07
Solubility	DMSO, ? ; H₂O, ? ; MeOH, ++	DMSO, ? ; H₂O, ? ; MeOH, ++

Discovery/Isolation/Chemical Studies

Apratoxin A was isolated as a potent cytotoxin from the marine cyanobacterium Lyngbya majuscule Harvey ex Gomont [1].

Using an oxazoline analog of apratoxin A (oz-apraA) which has similar potency with respect to tumor toxicity as aprotoxin A, Hsc70, Hsp70, RARS (arginyl-tRNA synthease protein), and SRP68 were identified as the binding proteins of oz-apraA [2].

Biological Studies

Apratoxin A mediates its antiproliferative activity through the induction of G1-phase cell cycle arrest and apoptosis, which is at least partially initiated through antagonism of FGF signaling via STAT3 [3]. Down-regulation of signal transduction was caused by the degradation of several receptor kinases, which lack N-glycosylation. Using in vitro cell-free systems revealed that apratoxin A prevents cotranslational translocation of proteins destined for the secretory pathway [4].

Oz-apraA induces the accumulation of polyubiquitin-positive proteins, and promotes the degradation of Hsp90 clients through chaperone-mediated autophagy. Because apratoxin A binds to Hsc70/Hsp70, it is speculated that apratoxin A inhibits Hsp90 function by stabilizing the interaction of Hsp90 client proteins with Hsc70/Hsp70 and thus prevents their interactions with Hsp90 [2].

Biological Activity of Apratoxin A

IC₅₀ for cytotoxicity in vitro against human tumor cell lines: from 0.36 to 0.52 nM [1]

G1 cell cycle arrest for 24- and 48-hour treatment in U2OS cells: 5 nM [3]

Caspase-3 activation and inhibition of STAT3 phosphorylation in U2OS cells: 50 nM [3]

Inhibition of in vitro angiogenesis of HUVECs: 10 nM [3]

Inhibition of N-linked glycosylation of several proteins in U2OS cells: 50 nM [4]

Inhibition of cotranslational translocation in vitro: 10 nM [4]

Accumulation of ubiquitinated proteins and formation of aggresome in HEK293 cells by oz-apra A: 100 nM [2]

References

1.

Luesch H, Yoshida WY, Moore RE, Paul VJ, Corbett TH (2001) Total structure determination of apratoxin A, a potent novel cytotoxin from the marine cyanobacterium Lyngbya majuscula. J Am Chem Soc 123:5418–5423
2.

Shen S, Zhang P, Lovchik MA, Li Y, Tang L, Chen Z, Zeng R, Ma D, Yuan J, Yu Q (2009) Cyclodepsipeptide toxin promotes the degradation of Hsp90 client proteins through chaperone-mediated autophagy. J Cell Biol 185:629–639
3.

Luesch H, Chanda SK, Raya RM, DeJesus PD, Orth AP, Walker JR, Izpisua Belmonte JC, Schultz PG (2006) A functional genomics approach to the mode of action of apratoxin A. Nat Chem Biol 2:158–167
4.

Liu Y, Law BK, Luesch H (2009) Apratoxin a reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol Pharmacol 76:91–104

Artemisinin

Keywords: [Antimalarial] [PfATP6 inhibitor]

Structure

	Artemisinin
Molecular formula	C₁₅H₂₂O₅
Molecular weight	282.34
Solubility	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation

Artemisinin was extracted by Youyou Tu, a principal investigator at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, as the most potent antimalarial compound from sweet wormwood (Artemisia annua), which has been used by Chinese herbalists for more than 2000 years [1].

Biological Studies

Artemisinin rapidly kills all asexual stages of Plasmodium falciparum. Activated artemisinin form adducts with a variety of biological macromolecules, including haem [2], translationally controlled tumor protein (TCTP) [3], and other higher molecular weight proteins. The crystal structure of the TCTP–arteminisin complex revealed that there are two binding sites on TCTP which are close to amino acid residues important for TCTP function, suggesting that TCTP may be one of the target proteins in P. falciparum [4].

Eckstein-Ludwig et al. reported that artemisinin, but not quinine or chloroquine, inhibits the SERCA ortholog (PfATP6) of P. falciparum in Xenopus oocytes with similar potency to thapsigargin [5]. Together with several results, they concluded that PfATP6 is a target protein of arteminisin; however, there are also controversial reports. Therefore, the actual mechanism of action of arteminisin remains ambiguous.

Biological Activity of Arteminisin

K_i for endogenous Ca²⁺-ATPase activity in PfATP6-expressing oocytes: 162 ± 31 nM [5]

K_d for PfTCTP measured by SPR: 77 ± 58 μM [4]

References

1.

Miller LH, Su X (2011) Artemisinin: discovery from the Chinese herbal garden. Cell 146:855–858
2.

Hong YL, Yang YZ, Meshnick SR (1994) The interaction of artemisinin with malarial hemozoin. Mol Biochem Parasitol 63:121–128
3.

Bhisutthibhan J, Pan XQ, Hossler PA, Walker DJ, Yowell CA, Carlton J, Dame JB, Meshnick SR (1998) The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. J Biol Chem 273:16192–16198
4.

Eichhorn T, Winter D, Buchele B, Dirdjaja N, Frank M, Lehmann WD, Mertens R, Krauth-Siegel RL, Simmet T, Granzin J, Efferth T (2013) Molecular interaction of artemisinin with translationally controlled tumor protein (TCTP) of Plasmodium falciparum. Biochem Pharmacol 85:38–45
5.

Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O″Neill PM, Bray PG, Ward SA, Krishna S (2003) Artemisinins target the SERCA of Plasmodium falciparum. Nature 424:957–961

AGK2*

Keywords: [SIRT2 inhibitor]

Structure

	AGK2
Molecular formula	C₂₃H₁₃Cl₂N₃O₂
Molecular weight	434.28
Solubility	DMSO, +; H₂O, ? ; MeOH, ?

Discovery/Isolation

AGK2 is a selective synthetic SIRT2 inhibitor, which was identified among compounds that increase intracellular α-synuclein inclusions from numerous small aggregates to large inclusions [1].

Biological Studies

AGK2 induces hyperacetylation of α-tubulin, a substrate of SIRT2, and rescues α-synuclein toxicity both in vitro and in a Drosophila model of Parkinson’s disease [1]. Pharmacological inhibition of SIRT2 by AGK2 reduces viability of merlin-mutant mouse Schwann cells [2]. AGK2 induces cell death and decreases the intracellular ATP levels in microglial cells. Both the AGK2-induced cell death and the AGK2-induced ATP decline are mediated by activation of poly(ADP-ribose) polymerase (PARP) [3]. Protective effects of AGK2 on LPD-induced neuroinflammation and brain injury have also been reported [4].

Biological Activity of AGK2

In vitro IC₅₀ for SIRT2: 3.5 μM [1]

In vitro IC₅₀ for SIRT1 and SIRT3: >50 μM [1]

Increase of α-tubulin acetylation: 10–50 μM [1]

References

1.

Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet JC, McLean PJ, Young AB, Abagyan R, Feany MB, Hyman BT, Kazantsev AG (2007) Sirtuin 2 inhibitors rescue α-synuclein-mediated toxicity in models of Parkinson’s disease. Science 317:516–519
2.

Petrilli A, Bott M, Fernández-Valle C (2013) Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis. Oncotarget 4:2354–2365
3.

Li Y, Nie H, Wu D, Zhang J, Wei X, Ying W (2013) Poly(ADP-ribose) polymerase mediates both cell death and ATP decreases in SIRT2 inhibitor AGK2-treated microglial BV2 cells. Neurosci Lett 544:36–40
4.

Wang B, Zhang Y, Cao W, Wei X, Chen J, Ying W (2016) SIRT2 Plays Significant Roles in Lipopolysaccharides-Induced Neuroinflammation and Brain Injury in Mice. Neurochem Res 41:2490–2500

Aurilide

Keywords: [Cytotoxic] [OPA1-mediated apoptosis] [Prohibitin]

Structure

	Aurilide
Molecular formula	C₄₄H₇₅N₅O₁₀
Molecular weight	834.11
Solubility	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

Aurilide is a 26-member cyclodepsipeptide isolated from the Japanese sea hare Dolabella auricularia [1, 2]. Enantioselective synthesis and SAR studies showed that modifications at the C35 hydroxyl group had a limited impact on the biological activity [2, 3].

Biological Studies

Aurilide induces apoptosis in cultured human cells at the picomolar to nanomolar range. Using an aurilide probe whose C35 hydroxyl group was conjugated with biotin molecule through a protease-cleavable polyproline linker, prohibitin 1 (PHB1) was identified as one of the binding proteins [4]. PHB1 is a mitochondria protein negatively regulating cell proliferation, and aurilide–fluorescein conjugate accumulated in mitochondria in HeLa cells. Aurilide accumulation in mitochondria induces the cleavage of OPA1 (one of the proteins essential for mitochondria fusion) and mitochondria-induced apoptosis.

Biological Activity of Aurilide

IC₅₀ for cytotoxicity against HeLa S₃: 0.011 μg/ml [2]

Mitochondria fragmentation and apoptosis induction in HeLa cells: 100 nM [4]

References

1.

Suenaga K, Mutou T, Shibata T, Itoh T, Kigoshi H, Yamada K (1996) Isolation and stereostructure of aurilide, a novel cyclodepsipeptide from the Japanese sea hare Dolabella auricularia. Tetrahedron Lett 37:6771–6774
2.

Suenaga K, Mutou T, Shibata T, Itoh T, Fujita T, Takada N, Hayamizu K, Takagi M, Irifune T, Kigoshi H, Yamada K (2004) Aurilide, a cytotoxic depsipeptide from the sea hare Dolabella auricularia: isolation, structure determination, synthesis, and biological activity. Tetrahedron 60:8509–8527
3.

Suenaga K, Kajiwara S, Kuribayashi S, Handa T, Kigoshi H (2008) Synthesis and cytotoxicity of aurilide analogs. Bioorg Med Chem Lett 18:3902–3905
4.

Sato S, Murata A, Orihara T, Shirakawa T, Suenaga K, Kigoshi H, Uesugi M (2011) Marine natural product aurilide activates the opa1-mediated apoptosis by binding to prohibitin. Chem Biol 18:131–139

Avermectin, Ivermectin

Keywords: [Biocidal] [Glutamate-gated chloride ion channel opener]

Structure

	Avermectin B_1a (R = Et)	Avermectin B_1b (R = Me)
Molecular formula	C₄₈H₇₂O₁₄	C₄₈H₇₀O₁₄
Molecular weight	873.09	859.06
Solubility	DMSO, +++; H₂O, −; MeOH, +++	DMSO, +++; H₂O, −; MeOH, +++

	Ivermectin (8:2 mixture of 22,23-dihydro avermectin B_1a and B_1b)
	22,23-dihydro avermectin B_1a (R = Et)	22,23-dihydro avermectin B_1b (R = Me)
Molecular formula	C₄₈H₇₄O₁₄	C₄₈H₇₂O₁₄
Molecular weight	875.11	861.08
Solubility	DMSO, +++; H₂O, −; MeOH, +++	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

By using a helminth-infected mouse model, the avermectins were isolated from Streptomyces avermitilis as a complex of chemically related compounds (A_1a, A_1b, A_2a, A_2b, B_1a, B_1b, B_2a, and B_2b ) which exhibit extraordinarily potent anthelmintic activity [1]. Different from most macrolide or polyene antibiotics, avermectins lack significant antibacterial or antifungal activity [2].

Ivermectin, which is a reduction product of the C22–C23 double bond of avermectin B_1a and B_1b, improved both the spectrum of activity and safety [3]. Methyl group at terminal next to R group is up stereochemistry for avermectins and ivermectin.

Biological Studies

Because avermectin quickly immobilized parasitic nematodes at low doses, it was thought that its anthelmintic activity may be due to effects on the nervous system. Following this suggestion, Fritz et al. investigated the effects on the lobster stretcher neuromuscular junction, and speculated that avermectins induced the opening of membrane Cl⁻ channels including those regulated by γ-aminobutyric acid at the inhibitory synapse [4]. In addition to the effects on the GABA receptor–Cl⁻ channel, Scott and Duce found that ivermectin also affected the glutamate H receptor–Cl⁻ channel complex [5]. Further investigations identified the avermectin-sensitive glutamate-gated chloride channels by expression cloning using Xenopus oocytes [6]. These results strongly suggest that avermectin/ivermectin interacts with glutamate-gated chloride channels and prevents their closure, leading to hyperpolarization of the neuronal membrane.

Biological Activity of Avermectin B_1a

>95% effective in reducing the numbers of Haemonchus placei, Ostertagia ostertagi, Trichostrongylus axei, T. colubriformis, Cooperia oncophora, C. punctata, Oesophagostomum radiatum, and Dictyocaulus viviparous in infected cattle with a single oral dose: 0.1 mg/kg [2]

References

1.

Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, Kong YL, Monaghan RL, Olson G, Putter I, Tunac JB, Wallick H, Stapley EO, Oiwa R, Omura S. (1979) Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother 15:361–367
2.

Egerton JR, Ostlind DA, Blair LS, Eary CH, Suhayda D, Cifelli S, Riek RF, Campbell WC. (1979) Avermectins, new family of potent anthelmintic agents: efficacy of the B1a component. Antimicrob Agents Chemother 15:372–378
3.

Chabala JC, Mrozik H, Tolman RL, Eskola P, Lusi A, Peterson LH, Woods MF, Fisher MH, Campbell WC, Egerton JR, Ostlind DA. (1980) Ivermectin, a new broad-spectrum antiparasitic agent. J Med Chem 23:1134–1136
4.

Fritz LC, Wang CC, Gorio A. (1979) Avermectin B1a irreversibly blocks postsynaptic potentials at the lobster neuromuscular junction by reducing muscle membrane resistance. Proc Natl Acad Sci U S A 76:2062–2066
5.

Scott RH, Duce IR. (1985) Effects of 22,23-dihydroavermectin B1a on locust (Schistocerca gregaria) muscles may involve several sites of action. Pestic Sci 16:599–604
6.

Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LH, Schaeffer JM, Arena JP. (1994) Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371:707–711

Bafilomycin (Setamycin)

Keywords: [Antifungal] [V-ATPase inhibitor]

Structure

	Bafilomycin A₁	Bafilomycin B₁ (Setamycin)
Molecular formula	C₃₅H₅₈O₉	C₄₄H₆₅NO₁₃
Molecular weight	622.84	816.00
Solubility	DMSO, +++; H₂O, +; MeOH, +++	DMSO, +++; H₂O, +; MeOH, +++

Methanol inactivates bafilomyins by methylation.

Discovery/Isolation

The first bafilomycin to be isolated was setamycin [1], which was shown to have antifungal and antinematodal activities in 1981 [2]. In 1983 and 1984, L-681,110, another bafilomycin, was isolated as an inhibitor of Na⁺/K⁺-ATPases from Streptomyces sp. MA-5038 [3, 4]. Bafilomycin A₁, A₂, B₁, B₂, C₁, and C₂ were isolated as antibacterial and antifungal compounds from Streptomyces griseus strains TÜ1922, TÜ2473, and TÜ2599 [5]. Setamycin and L-681,110 are the same compounds with bafilomycin B₁ and C₁, respectively [2].

Biological Studies

Because bafilomycin C₁ is a moderate Na⁺/K⁺-ATPase inhibitor, the effects of bafilomycin A₁ on various membrane ATPases were investigated and it was found that this compound is a specific potent inhibitor of vacuolar ATPases (V-ATPases) [6]. Bafilomycin A₁ blocks vacuole acidification and causes protein sorting/secretion deficiencies [7]. V-ATPase inhibition by bafilomycins resulted in several biological phenotypes including inhibitions of bone resorption by osteoclasts [8], of protein degradation in lysosomes [9], and of the accumulation of lipid droplets in macrophages [10].

A biochemical approach using a purified V₀ domain of V-ATPase revealed that the binding site of bafilomycins is located in the V₀ domain [11], and further genetic analysis suggests that bafilomycins locate on the pocket formed by helices 1, 2, and 4 of subunit c [12].

Biological Activity of Bafilomycins

MIC for Sacchromyces sake: 0.78 μg/ml [1]

I₅₀ values for membrane-bound ATPases from Neurospora vacuoles, chromaffin granules, and corn vacuoles: 0.4, 0.05, and 0.004 nmol/mg, respectively [6]

IC₅₀ for osteoclast bone resorption: 1 nM [8]

Inhibition of oxidized low-density lipoprotein (LDL)–induced accumulation of lipid droplets in macrophage J774: 3 nM [10]

References

1.

Omura S, Otoguro K, Nishikiori T, Oiwa R, Iwai Y (1981) Setamycin, a new antibiotic. J Antibiot (Tokyo) 34: 1253–1256
2.

Otoguro K, Nakagawa A, Omura S (1988) Setamycin, a 16-membered macrolide antibiotic. Identification and nematocidal activity. J Antibiot (Tokyo) 41: 250–252
3.

Hensens OD, Monaghan RL, Huang LY, Albersschonberg G (1983) Structure of the sodium and potassium-ion activated adenosine-triphosphatase inhibitor-L-681,110. J Am Chem Soc 105: 3672–3679
4.

Huang L, Albers-Schonberg G, Monaghan RL, Jakubas K, Pong SS, Hensens OD, Burg RW, Ostlind DA, Conroy J, Stapley EO (1984) Discovery, production and purification of the Na⁺, K⁺ activated ATPase inhibitor, L-681,110 from the fermentation broth of Streptomyces sp. MA-5038. J Antibiot (Tokyo) 37: 970–975
5.

Werner G, Hagenmaier H, Drautz H, Baumgartner A, Zahner H (1984) Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J Antibiot (Tokyo) 37: 110–117
6.

Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A 85: 7972–7976
7.

Banta LM, Robinson JS, Klionsky DJ, Emr SD (1988) Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol 107: 1369–1383
8.

Sundquist K, Lakkakorpi P, Wallmark B, Vaananen K (1990) Inhibition of osteoclast proton transport by bafilomycin A₁ abolishes bone resorption. Biochem Biophys Res Commun 168: 309–313
9.

Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991) Bafilomycin A₁, a specific inhibitor of vacuolar-type H⁺-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266: 17707–17712
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Naganuma S, Kuzuya N, Sakai K, Hasumi K, Endo A (1992) Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B₁ and destruxin E. Biochim Biophys Acta 1126: 41–48
11.

Zhang J, Feng Y, Forgac M (1994) Proton conduction and bafilomycin binding by the V₀ domain of the coated vesicle V-ATPase. J Biol Chem 269: 23518–23523
12.

Bowman EJ, Graham LA, Stevens TH, Bowman BJ (2004) The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae. J Biol Chem 279: 33131–33138

BI2536*

Keywords: [Plk1 inhibitor]

Structure

	BI 2536
Molecular formula	C₂₈H₃₉N₇O₃
Molecular weight	521.65
Solubility	DMSO, +++; MeOH, +++; H₂O, ?

Discovery/Isolation

BI2536 was screened out as a potent inhibitor of polo-like kinase 1 (Plk1) from a diverse library of organic compounds [1]. BI2536 is a compound inhibiting Plk1 at a concentration less than a nanomole in vitro (0.83 nM) and is active against human, mouse, and rat Plk1 equipotently [1]. It is over 1000-fold more selective to Plk1 when compared to other protein kinases, and some inhibitory activity against closely related kinases of polo-like kinase family members (Plk2 and Plk3) has been reported [2].

Biological Studies

BI2536 inhibits Plk1 at subnanomolar concentration, while it inhibits the activities of Plk2 and Plk3 to a slightly lesser extent with IC₅₀ values of 3.5 nM and 9.0 nM, respectively. BI2536 treatment (10–100 nM) leads to inhibition of mitotic properties such as the phosphorylation of Apc6 at mitotic centrosomes and recruitment of γ-tubulin. It also induces the inhibition of cohesin release from chromosome arms, and induction of monopolar spindles in HeLa cells. Treatment of BI2536 on HeLa cells leads to cell cycle arrest at the G2/M border, and subsequently increases cells with a sub-G1 DNA peak indicating the induction of apoptosis in these cells. Actually, accumulation of cleaved PARP p85 fragments was observed in the treated cells. It inhibits the proliferation of 32 human cancer cell lines, with EC₅₀ values of 2–25 nM. Because similar inhibitory effects have been observed in nontransformed, immortalized cells, BI2536 might affect the growth of all proliferating cells. Cells that are treated with BI 2536 arrest in M phase with monopolar mitotic spindles, a phenotype that is similar to that induced by an siRNA against Plk1, suggesting that Plk1 is the main cellular target. Plk1 inhibition by BI2536 reduces the growth and viability of anaplastic thyroid carcinoma cells such as OCUT-1, CAL62, SW1736, 8505C, and ACT-1 with EC₅₀ values of 1.4–5.6 nM [1, 3].

Biological Activity of BI2536

IC₅₀ values for Plk1, Plk2 and Plk3: 0.83 nM, 3.5 nM, and 9.0 nM, respectively [1]

References

1.

Steegmaier M, Hoffmann M, Baum A, Lenart P, Petronczki M, Krssak M, Gurtler U, Garin-Chesa P, Lieb S, Quant J, Grauert M, Adolf GR, Kraut N, Peters JM, Rettig WJ (2007) BI2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr Biol: 17:316–322
2.

Lenart P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffmann M, Rettig WJ, Kraut N, Peters JM (2007) The small-molecule inhibitor BI2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol: 17:304–315
3.

Nappi TC, Salerno P, Zitzelsberger H, Carlomagno F, Salvatore G, Santoro M (2009) Identification of Polo-like kinase 1 as a potential therapeutic target in anaplastic thyroid carcinoma. Cancer Res 69:1916–1923

Bisebromoamide

Keywords: [Actin stabilizer]

Structure

	Bisebromoamide
Molecular formula	C₅₁H₇₂BrN₇O₈S
Molecular weight	1023.13
Solubility	DMSO, ++; H₂O, ++; MeOH, ++

Discovery/Isolation

Bisebromoamide is a cytotoxic peptide isolated from the marine cyanobacterium Lyngbya sp. [1].

Biological Studies

Automated high-content image analysis revealed that bisebromoamide induced morphological changes in HeLa cells similar to those induced by known actin-stabilizing compounds [2]. Bisebromoamide stimulated actin polymerization and stabilized F-actin in vitro. Furthermore, a bisebromoamide–fluorescein conjugate localized at the actin filament in cells.

Bisebromoamide not only induced cell apoptosis, but inhibited the Raf/MEK/ERK and PI3K/Akt/mTOR pathways in advanced renal cell carcinomas [3].

Biological Activity of Bisebromoamide

IC₅₀ for cytotoxicity against HeLa: 0.04 μg/ml [1]

Inhibition of the phosphorylation of ERK in NRK cells by PDGF-stimulation: 0.1–10 μM [1]

F-actin stabilization in vitro: 10 nM [2]

IC₅₀ for two cell lines of advanced renal cell carcinoma: 1.5 and 2.1 μM [3]

References

1.

Teruya T, Sasaki H, Fukazawa H, Suenaga K (2009) Bisebromoamide, a potent cytotoxic peptide from the marine cyanobacterium Lyngbya sp.: isolation, stereostructure, and biological activity. Org Lett 11:5062–5065
2.

Sumiya E, Shimogawa H, Sasaki H, Tsutsumi M, Yoshita K, Ojika M, Suenaga K, Uesugi M (2011) Cell-morphology profiling of a natural product library identifies bisebromoamide and miuraenamide A as actin filament stabilizers. ACS Chem Biol 6:425–431
3.

Suzuki K, Mizuno R, Suenaga K, Teruya T, Tanaka N, Kosaka T, Oya M (2013) Bisebromoamide, an extract from Lyngbya species, induces apoptosis through ERK and mTOR inhibitions in renal cancer cells. Cancer Med 2:32–39

BMS-488516, BMS-509744

Keywords: [Itk kinase inhibitor] [immunosuppressive and inflammatory diseases]

Structure

	BMS-488516	BMS-509744
Molecular formula	C₃₃H₄₃N₅O₃S₂	C₃₂H₄₁N₅O₄S₂
Molecular weight	621.86	623.83
Solubility	DMSO, ++; H₂O, ? ; MeOH, ?	DMSO, ++; H₂O, ? ; MeOH, ?

Discovery/Isolation

BMS-488516 and BMS-509744 were developed from an aminothiazole compound, BMS-285047, which was isolated as an Itk kinase inhibitor by a homogeneous time-resolved fluorescence assay using GST-Itk kinase domain and GST-SLP-76 as a substrate [1].

Biological Studies

Both BMS-488516 and BMS-509744 potently and selectively inhibited Itk kinase activity in an ATP-competitive manner, and reduced TCR-induced functions in vitro and in cells [1]. MS-509744 also significantly diminished lung inflammation in an allergy/asthma model mouse. These results strongly suggest that Itk is a therapeutic target for immunosuppressive and inflammatory diseases. X-ray crystal structures of IL-2-inducible T-cell kinase in complex with BMS-509744 stabilized the activation loop in a substrate-blocking inactive conformation, indicating that structural change is involved in Itk kinase activity [2].

The HIV-1 Nef virulence factor interacts with the Src homology 3 (SH3) domain of Itk kinase, and induces constitutive Itk activation. BMS-509744 potently blocked HIV-1 infectivity and replication, but not that of a Nef-defective mutant, suggesting that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle [3].

Biological Activity of BMS-488516 and BMS-509744

IC₅₀ values for BMS-488516 and BMS-509744 on in vitro Itk kinase: 96 and 19 nM, respectively [1]

Inhibition of PLCγ phosphorylation by anti-CD3 antibody in Jurkat cells by BMS-509744: 10 μM [1]

IC₅₀ values for BMS-488516 and BMS-509744 on LPS-induced TNFR production from PBMCs: 11 and >25 μM, respectively [1]

IC₅₀ values for BMS-488516 and BMS-509744 on anti-CD3 antibody–induced IL-2 secretion in murine EL4 cells: 250 and 72 nM, respectively [1]

K_d for BMS-509744 on C477S/E614A/E617A Itk kinase variant measured by SPR: 0.8 nM [2]

IC₅₀ for BMS-509744 on HIV infectivity: ~3 μM [3]

References

1.

Lin TA, McIntyre KW, Das J, Liu C, O″Day KD, Penhallow B, Hung CY, Whitney GS, Shuster DJ, Yang X, Townsend R, Postelnek J, Spergel SH, Lin J, Moquin RV, Furch JA, Kamath AV, Zhang H, Marathe PH, Perez-Villar JJ, Doweyko A, Killar L, Dodd JH, Barrish JC, Wityak J, Kanner SB (2004) Selective Itk inhibitors block T-cell activation and murine lung inflammation. Biochemistry 43:11056–11062
2.

Kutach AK, Villasenor AG, Lam D, Belunis C, Janson C, Lok S, Hong LN, Liu CM, Deval J, Novak TJ, Barnett JW, Chu W, Shaw D, Kuglstatter A (2010) Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: Insights into rational drug design and activity regulation. Chem Biol Drug Des 76:154–163
3.

Tarafdar S, Poe JA, Smithgall TE (2014) The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem 289:15718–15728

BNS-22*

Keywords: [Antitumor] [Topoisomerase II]

Structure

	BNS-22
Molecular formula	C₂₄H₂₅NO₅
Molecular weight	407.47
Solubility	DMSO, ++; H₂O, −; MeOH, ++

Discovery/Isolation/Chemical Studies

BNS-22 is a synthesized derivative of the natural product GUT-70, which was isolated from the stem bark of Calophyllum brasiliense in Brazil as an antileukemic compound [1]. This compound has antiproliferative activity against human cancer cells. The target molecule of BNS-22 was suggested as DNA topoisomerase II (TOP2) by proteomic profiling as below. An affinity precipitation assay using purified human TOP2α showed that BNS-22 binds directly to TOP2α in a different mode from ICRF-193, a TOP2 catalytic inhibitor [2].

Biological Studies

By proteomic profiling analysis, it was suggested that the target molecule of BNS-22 is the same with ICRF-193 [2]. Furthermore, BNS-22 inhibits kinetoplast DNA decatenation that is mediated by human TOP2α and TOP2β. BNS-22 does not affect DNA damage and antagonizes TOP2 poison–mediated DNA damage, and induces mitotic abnormalities, characterized by impairments in chromosome alignment and segregation, thereby causing polyploidy in HeLa cells.

Biological Activity of BNS-22

IC₅₀ values for human TOP2α and TOP2β in vitro: 2.8 and 0.42 μM, respectively [2]

Perturbation of mitotic spindle formation and polyploidy induction in HeLa cells: 3 μM [2]

References

1.

Kimura S, Ito C, Jyoko N, Segawa H, Kuroda J, Okada M, Adachi S, Nakahata T, Yuasa T, Filho VC, Furukawa H, Maekawa T (2005) Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from Calophyllum brasiliense that acts by induction of apoptosis. Int J Cancer 113:158–165
2.

Kawatani M, Takayama H, Muroi M, Kimura S, Maekawa T, Osada H (2011) Identification of a small-molecule inhibitor of DNA topoisomerase II by proteomic profiling. Chem Biol 18:743–751

Brefeldin A

Keywords: [Antifungal] [Antiviral] [ARF GEF inhibitor] [Transport inhibitor]

Structure

	Brefeldin A
Molecular formula	C₁₆H₂₄O₄
Molecular weight	280.36
Solubility	DMSO, +++; H₂O, +; MeOH, ++

Discovery/Isolation

Brefeldin A (primarily named decumbin) was isolated as the chief component of the crystalline bodies produced from Penicillium decumbens [1]. Brefeldin A was reisolated as an antifungal antibiotic by Hayashi et al. [2].

Biological Studies

Brefeldin A is a unique agent, which impedes protein transport from the ER to the Golgi complex [3] by inducing the rapid and reversible dissociation of coat protein, β-COP, and small GTPase, ADP-ribosylation factor (ARF) [4, 5].

The inhibition step of brefeldin A was identified biochemically as guanine nucleotide exchange factor (GEF) activity on ARF in Golgi membranes [6, 7]. The first ARF GEF, Gea1, was identified using a genetic selection in yeast, and its GEF activity was inhibited by brefeldin A [8]. Gea1 contains a Sec7 domain that is sufficient for ARF GEF activity, and brefeldin A stabilizes the ARF-GDP-Sec7 domain complex as an inactive, abortive complex [9, 10]. This dead-end complex structure showed that brefeldin A binds at the protein–protein (ARF-GDP/Sec7 domain) interface and freezes the complex in an abortive conformation that cannot proceed to nucleotide dissociation [11, 12].

Brefeldin A is a good bioprobe for investigating the transport system, especially from the endoplasmic reticulum to the Golgi network. Nuchtern et al. showed that the influenza A matrix protein can be effectively presented to class II–restricted T cells by two pathways, one of which is chloroquine sensitive and BFA insensitive, the other being chloroquine insensitive and BFA sensitive [13]. Rubartelli et al. suggested a novel secretory pathway for interleukin-1β, a protein lacking a signal sequence [14]. Brefeldin A also induces an unfolded protein response, protein synthesis inhibition and apoptosis [15–17].

Biological Activity of Brefeldin A

Blockage of secretion in primary culture of rat hepatocyte: 1 μg/ml [3]

70% inhibition of protein synthesis in rat glioma C6 cells: 0.1–1 mg/ml [16]

50–60% inhibition of the membrane-dependent nucleotide-exchange reaction: 10 μM [6, 7]

Redistribution of S2P into the ER in HepG2 cells: 10 μg/ml [15]

References

1.

Singleton VL, Bohonos N, Ullstrup AJ (1958) Decumbin, a new compound from a species of Penicillium. Nature 181:1072–1073
2.

Hayashi T, Takatsuki A, Tamura G (1974) The action mechanism of brefeldin A. I. Growth recovery of Candida albicans by lipids from the action of brefeldin A. J Antibiot (Tokyo) 27:65–72
3.

Misumi Y, Miki K, Takatsuki A, Tamura G, Ikehara Y (1986) Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem 261:11398–11403
4.

Donaldson JG, Lippincott-Schwartz J, Bloom GS, Kreis TE, Klausner RD (1990) Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol 111:2295–2306
5.

Serafini T, Stenbeck G, Brecht A, Lottspeich F, Orci L, Rothman JE, Wieland FT (1991) A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein β-adaptin. Nature 349:215–220
6.

Donaldson JG, Finazzi D, Klausner RD (1992) Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360:350–352
7.

Helms JB, Rothman JE (1992) Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360:352–354
8.

Peyroche A, Paris S, Jackson CL (1996) Nucleotide exchange on ARF mediated by yeast Gea1 protein. Nature 384:479–481
9.

Peyroche A, Antonny B, Robineau S, Acker J, Cherfils J, Jackson CL (1999) Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. Mol Cell 3:275–285
10.

Jackson CL, Casanova JE (2000) Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol 10:60–67
11.

Mossessova E, Corpina RA, Goldberg J (2003) Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism. Mol Cell 12:1403–1411
12.

Renault L, Guibert B, Cherfils J (2003) Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426:525–530
13.

Nuchtern JG, Biddison WE, Klausner RD (1990) Class II MHC molecules can use the endogenous pathway of antigen presentation. Nature 343:74–76
14.

Rubartelli A, Cozzolino F, Talio M, Sitia R (1990) A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 9:1503–1510
15.

Citterio C, Vichi A, Pacheco-Rodriguez G, Aponte AM, Moss J, Vaughan M (2008) Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1. Proc Natl Acad Sci U S A 105:2877–2882
16.

Fishman PH, Curran PK (1992) Brefeldin A inhibits protein synthesis in cultured cells. FEBS Lett 314:371–374
17.

Shinjo S, Mizotani Y, Tashiro E, Imoto M (2013) Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds. Biosci Biotechnol Biochem 77:729–735

C646*

Keywords: [p300 inhibitor]

Structure

	C646
Molecular formula	C₂₄H₁₉N₃O₆
Molecular weight	445.43
Solubility	DMSO, +++; H₂O, ? ; MeOH, ?

Discovery/Isolation

C646 is a synthetic, pyrazolone-containing selective and competitive p300 inhibitor, which was identified by structure-based in silico screening [1].

Biological Studies

C646, but not its inactive analog, reduces histone acetylation levels in cells and inhibits proliferation of melanoma cells [1]. C646 exhibits an antiproliferative effect on acute myeloid leukemia cells with distinct alterations of transcription and histone acetylation associated with genomic integrity [2, 3] and increases radiosensitization of lung cancer cells by enhancing mitotic catastrophe [4]. In addition to the antitumor activity, inhibitory activity against influenza virus infection and antitauopathy activity were also reported [5, 6].

Biological Activity of C646

K_i for p300: 400 nM [1]

Inhibition of p300 enzymatic activity at 10 μM: 86% inhibition [1]

Inhibition of histone acetylation in mouse fibroblasts: 25 μM [1]

IC₅₀ for proliferation of melanoma cells: 10–20 μM [1]

References

1.

Bowers EM, Yan G, Mukherjee C, Orry A, Wang L, Holbert MA, Crump NT, Hazzalin CA, Liszczak G, Yuan H, Larocca C, Saldanha SA, Abagyan R, Sun Y, Meyers DJ, Marmorstein R, Mahadevan LC, Alani RM, Cole PA (2010) Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem Biol 17:471–482
2.

Gao XN, Lin J, Ning QY, Gao L, Yao YS, Zhou JH, Li YH, Wang LL, Yu L (2013) A histone acetyltransferase p300 inhibitor C646 induces cell cycle arrest and apoptosis selectively in AML1-ETO-positive AML cells. PLoS One 8:e55481
3.

Giotopoulos G, Chan WI, Horton SJ, Ruau D, Gallipoli P, Fowler A, Crawley C, Papaemmanuil E, Campbell PJ, Göttgens B, Van Deursen JM, Cole PA, Huntly BJ (2016) The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia. Oncogene 35:279–289
4.

Oike T, Komachi M, Ogiwara H, Amornwichet N, Saitoh Y, Torikai K, Kubo N, Nakano T, Kohno T (2014) C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe. Radiother Oncol 111:222–227
5.

Zhao D, Fukuyama S, Sakai-Tagawa Y, Takashita E, Shoemaker JE, Kawaoka Y (2015) C646, a novel p300/CREB-binding protein-specific inhibitor of histone acetyltransferase, attenuates influenza A virus infection. Antimicrob Agents Chemother 60:1902–1906
6.

Min SW, Cho SH, Zhou Y, Schroeder S, Haroutunian V, Seeley WW, Huang EJ, Shen Y, Masliah E, Mukherjee C, Meyers D, Cole PA, Ott M, Gan L. (2010) Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron 67:953–966

Celastrol

Keywords: [Anti-inflammatory] [Cysteine reactivity]

Structure

	Celastrol
Molecular formula	C₂₉H₃₈O₄
Molecular weight	450.62
Solubility	DMSO, +++; H₂O, −; MeOH, +

Discovery/Isolation/Chemical Studies

Celastrol is a quinone methide purified from traditional Chinese medicine Celastrus scandens (Thunder of God Vine) which is generally used for the treatment of inflammatory and autoimmune diseases [1, 2]. The structure was determined by Nakanishi et al. [3]. Celastrol is an electrophilic quinine methide, and sterospecifically reacts with biological sulfur nucleophiles by Michael additions [4].

Biological Studies

Celastrol has numerous pharmacological properties, inhibits the proliferation of a variety of tumor cells, and suppresses tumor initiation, promotion, and metastasis in various cancer models in vivo. Recently celastrol was identified as an inhibitor of androgen receptor–mediated signaling, and a gene expression–based analysis revealed that celastrol is classified as a HSP90 inhibitor [5]. Lin et al. found that a cancerous inhibitor of PP2A (CIP2A) is a target protein of celastrol using biotinylated derivative [6].

There are many reports that celastrol modulates several biological activities of NF-κB, proteasome, topoisomerase II, heat shock response, and so on [7]. Some of them are directly inhibited in vitro; therefore, celastrol probably has several molecular targets in cells.

Biological Activity of Celastrol

Inhibition of androgen receptor signaling in LNCaP cells stimulated with the synthetic androgen R1881: 1.25 μM [5]

CIP2A degradation in various cancer cell lines: 5 μM [6]

References

1.

Chou T, Mei P (1936) Study on Chinese herb Lei Gong Teng, Tripterygium wilfordii Hook f. I The coloring substance and the sugars. Chin J Phys 10:529–534
2.

Gisvold O (1939) The pigments contained in the bark of the root of Celastrus scandens. J Am Pharm Soc 28:440–443
3.

Nakanishi K, Kakisawa H, Hirata Y (1956) Structure of pristimerin and celastrol. Bull Chem Soc Jpn 29:7–15
4.

Klaic L, Trippier PC, Mishra RK, Morimoto RI, Silverman RB (2011) Remarkable stereospecific conjugate additions to the Hsp90 inhibitor celastrol. J Am Chem Soc 133:19634–19637
5.

Hieronymus H, Lamb J, Ross KN, Peng XP, Clement C, Rodina A, Nieto M, Du J, Stegmaier K, Raj SM, Maloney KN, Clardy J, Hahn WC, Chiosis G, Golub TR (2006) Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. Cancer Cell 10:321–330
6.

Liu Z, Ma L, Wen ZS, Hu Z, Wu FQ, Li W, Liu J, Zhou GB (2014) Cancerous inhibitor of PP2A is targeted by natural compound celastrol for degradation in non-small-cell lung cancer. Carcinogenesis 35:905–914
7.

Kannaiyan R, Shanmugam MK, Sethi G (2011) Molecular targets of celastrol derived from Thunder of God Vine: potential role in the treatment of inflammatory disorders and cancer. Cancer Lett 303:9–20

Cerulenin

Keywords: [Antifungal] [Lipid biosynthesis inhibitor]

Structure

	Cerulenin
Molecular formula	C₁₂H₁₇NO₃
Molecular weight	223.27
Solubility	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation

Cerulenin was isolated as an antifungal antibiotic from the culture broth of Cephalosporium caerulens [1].

Biological Studies

Cerulenin is a potent and noncompetitive inhibitor of fatty acid biosynthesis through irreversible inhibition of β-ketoacyl acylcarrier protein synthetase [2, 3]. The binding site of cerulenin in fatty acid synthetase has been determined as the highly reactive substrate binding Cys-204 using a combination of radiolabeling studies with [³H]cerulenin, proteolytic and chemical modification [4, 5]. The inhibition mechanism of cerulenin is suggested to be that cerulenin forms a hydroxylactam ring and reacts at its epoxide carbon with the SH group of the cysteine residue in the condensing reaction domain of fatty acid synthetase. The X-ray structure of the complex of cerulenin and β-ketoacyl carrier protein synthetase revealed that cerulenin is bound in a hydrophobic pocket formed at the dimer interface of β-ketoacyl carrier protein synthetases [6].

Cerulenin also shows several inhibitory activities on antigen processing in antigen-presenting cells [7], HIV-1 proteinase in vitro [8], protein palmitoylation, and insulin internalization [9].

Biological Activity of Cerulenin

IC₅₀ for fatty acid synthetase of E. coli: 30 μM [2]

IC₅₀ for β-ketoacyl-acyl carrier protein synthetase of E. coli: <5 μM [2]

IC₅₀ for HIV-1 protease in vitro: 2.5 mM [8]

References

1.

Sato Y, Nomura S, Kamio Y, Omura S, Hata T (1967) Studies on cerulenin, 3. Isolation and physico-chemical properties of cerulenin. J Antibiot (Tokyo) 20:344–348
2.

D”Agnolo G, Rosenfeld IS, Awaya J, Omura S, Vagelos PR (1973) Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of β-ketoacyl-acyl carrier protein synthetase. Biochim Biophys Acta 326:155–156
3.

Vance D, Goldberg I, Mitsuhashi O, Bloch K (1972) Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem Biophys Res Commun 48:649–656
4.

Child CJ, Shoolingin-Jordan PM (1998) Inactivation of the polyketide synthase, 6-methylsalicylic acid synthase, by the specific modification of Cys-204 of the β-ketoacyl synthase by the fungal mycotoxin cerulenin. Biochem J 330:933–937
5.

Funabashi H, Kawaguchi A, Tomoda H, Omura S, Okuda S, Iwasaki S (1989) Binding site of cerulenin in fatty acid synthetase. J Biochem 105:751–755
6.

Moche M, Schneider G, Edwards P, Dehesh K, Lindqvist Y (1999) Structure of the complex between the antibiotic cerulenin and its target, β-ketoacyl-acyl carrier protein synthase. J Biol Chem 274:6031–6034
7.

Falo LD, Jr., Benacerraf B, Rothstein L, Rock KL (1987) Cerulenin is a potent inhibitor of antigen processing by antigen-presenting cells. J Immunol 139:3918–3923
8.

Moelling K, Schulze T, Knoop MT, Kay J, Jupp R, Nicolaou G, Pearl LH (1990) In vitro inhibition of HIV-1 proteinase by cerulenin. FEBS Lett 261:373–377
9.

Jochen AL, Hays J, Mick G (1995) Inhibitory effects of cerulenin on protein palmitoylation and insulin internalization in rat adipocytes. Biochim Biophys Acta 1259:65–72

Chromeceptin*

Keywords: [Insulin-induced adipogenesis] [IGF2] [STAT6] [MFP-2] [ACC1]

Structure

	Chromeceptin
Molecular formula	C₁₉H₁₆F₃N₃O
Molecular weight	359.35
Solubility	DMSO, +++; H₂O, ±; MeOH, ++

Discovery/Isolation

Chromeceptin was identified as an inhibitor of insulin-induced adipogenesis of 3 T3-L1 cells by the morphology-based adipogenesis screen of a synthetic chemical library containing 10,000 divergent compounds [1].

Biological Studies

Chromeceptin exhibited cytotoxicity against IGF2-producing hepatocarcinoma cells with selectivity similar to that of a neutralizing antibody against IGF2 [1]. This compound blocks the autocrine loop of IGF2 in the hepatocarcinoma cells by activating the expression of IGFBP-1, a secreted IGF binding polypeptide, and STAT6 was identified as a chromeceptin-responsive element binding transcription factor in IGFBP-1 [1, 2]. Furthermore, chromoceptin induced the expression of SOCS-3 (suppressor of cytokine signaling-3) via STAT-6 activation. These results indicate that the expression of SOCS-3 and IGFBP-1, and perhaps that of the other genes that are controlled by STAT6, is up-regulated by chromeceptin and may work together to suppress the function of IGF2.

The chromeceptin binding protein was identified as MFP-2 (multifunctional protein-2) by affinity purification using biotinylated chromeceptin [2]. Furthermore, the chromeceptin-MFP-2 complex binds to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme important for the de novo synthesis of malonyl-CoA and fatty acids [3]. The formation of this ternary complex removes ACC1 from the cytosol and sequesters it in peroxisomes. As a result, chromeceptin impairs fatty acid synthesis.

Biological Activity of Chromeceptin

IC₅₀ for IGF2-producing hepatocellular carcinoma cells (Hep-G2): 29 nM [1]

IC₅₀ for hepatocellular carcinoma cells with low level of IGF2 (SK-Hep-1): ~1000 nM [1]

Inhibition of the promoter of IGF2 in hepatocellular carcinoma cells: 1 μM [1]

Induction of IGFBP-1 and SOCS-3: 1 μM [2]

Pex5p-dependent peroxisomal translocation of MPF1 and ACC1: 1 μM [3]

References

1.

Choi Y, Kawazoe Y, Murakami K, Misawa H, Uesugi M (2003) Identification of bioactive molecules by adipogenesis profiling of organic compounds. J Biol Chem 278:7320–7324
2.

Choi Y, Shimogawa H, Murakami K, Ramdas L, Zhang W, Qin J, Uesugi M (2006) Chemical genetic identification of the IGF-linked pathway that is mediated by STAT6 and MFP2. Chem Biol 13:241–249
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Jung D, Abu-Elheiga L, Ayuzawa R, Gu Z, Shirakawa T, Fujiki Y, Nakatsuji N, Wakil SJ, Uesugi M (2012) Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic small molecule. Biochem J 448:409–416

Compactin/ML-236B, Lovastatin

Keywords: [Hypolipidemic agent] [HMG-CoA reductase]

Structure

	Compactin	Lovastatin
Molecular formula	C₂₃H₃₄O₅	C₂₄H₃₆O₅
Molecular weight	390.51	404.54
Solubility	DMSO, +++; H₂O, ±; MeOH, ++	DMSO, +++; H₂O, ±; MeOH, ++

Discovery/Isolation

ML-236B was isolated from Penicillum citrinum as an inhibitor of cholesterol synthesis [1]. The same compound, compactin, was isolated as an antifungal metabolite from Penicillium brevicompactum [2]. Lovastatin (monacolin K, mevinolin) was isolated from Monascus sp. [3, 4] and Asperugillus terreus [5] as a hypocholesterolemic agents.

Biological Studies

Compactin, lovastatin, and other statins inhibit 3-hydroxy-3-methylglutaryl (HMG)–CoA reductase, the rate-limiting enzyme in the cholesterol synthetic pathway [6]. The inhibition is competitive with respect to the substrate HMG-CoA. The crystal structure of six statins with HMG-CoA reductase revealed that statins occupy a portion of the binding site of HMG-CoA, then blocking substrate access to the active site [7].

Statins not only decrease cholesterol content but also show several other phenotypes which are related or unrelated with HMG-CoA reductase inhibition. By HMG-CoA reductase inhibition, statins show antitumor effects on leukemia stem cells [8], antiviral activity by inhibiting the hepatitis C virus (HCV) RNA replication and assembly of the viral replication complex [9], and extracellular amyloid β-peptide degradation by microglia via stimulation of exosome-associated insulin-degrading enzyme secretion [10]. On the other hand, statins also show effects unrelated to HMG-CoA reductase inhibition, for example, cell cycle arrest at G1 phase by proteasome inhibition [11–13], and immunoresponse modulation via inhibition of LFA-1-mediated adhesion of leukocytes to ICAM-1 [14, 15] or via repression of MHC-II-mediated T-cell activation [16]. Other therapeutic effects on fragile X syndrome [17], Listeria monocytogenes infection [18], progressive multiple sclerosis [19], skeletal dysplasia [20], and Duchenne muscular dystrophy [21] have been reported.

Biological Activity of Compactin and Lovastatin

Compactin

IC₅₀ for cholesterol synthesis in ¹⁴C–acetate incorporation into digitonin-precipitable sterols in a rat liver enzyme system: 26 nM [1]

IC₅₀ for HMG-CoA reductase: 100 nM [6]

Lovastatin

Decrease of HCV RNA levels to 10–20% in Huh7-K2040 cells: 50 μM, 24-hour treatment [9]

IC₅₀ for LFA-1–ICAM-1 interaction: 2.1 ± 0.8 [15]

Inhibition of IFN-γ induced MHC-II expression on human endothelial cells and macrophages: 10 μM, 48-hour treatment [16]

References

1.

Endo A, Kuroda M, Tsujita Y (1976) ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinium. J Antibiot (Tokyo) 29:1346–1348
2.

Brown AG, Smale TC, King TJ, Hasenkamp R, Thompson RH (1976) Crystal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactum. J Chem Soc Perkin 1:1165–1170
3.

Endo A. (1979) Monacolin K, a new hypocholesterolemic agent produced by a Monascus species. J Antibiot (Tokyo) 32:852–854
4.

Endo A. (1980) Monacolin K, a new hypocholesterolemic agent that specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase. J Antibiot (Tokyo) 33:334–336
5.

Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, Springer J. (1980) Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci U S A 77:3957–3961
6.

Endo A, Kuroda M, Tanzawa K (1976) Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity. FEBS Lett 72:323–326
7.

Istvan ES, Deisenhofer J. (2001) Structural mechanism for statin inhibition of HMG-CoA reductase. Science 292:1160–1164
8.

Hartwell KA, Miller PG, Mukherjee S, Kahn AR, Stewart AL, Logan DJ, Negri JM, Duvet M, Järås M, Puram R, Dancik V, Al-Shahrour F, Kindler T, Tothova Z, Chattopadhyay S, Hasaka T, Narayan R, Dai M, Huang C, Shterental S, Chu LP, Haydu JE, Shieh JH, Steensma DP, Munoz B, Bittker JA, Shamji AF, Clemons PA, Tolliday NJ, Carpenter AE, Gilliland DG, Stern AM, Moore MA, Scadden DT, Schreiber SL, Ebert BL, Golub TR. (2013) Niche-based screening identifies small-molecule inhibitors of leukemia stem Cells. Nat Chem Biol 9:840–848
9.

Ye J, Wang C, Sumpter R Jr., Brown MS, Goldstein JL, Gale M Jr. (2003) Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci U S A 100:15865–15870
10.

Tamboli IY, Barth E, Christian L, Siepmann M, Kumar S, Singh S, Tolksdorf K, Heneka MT, Lütjohann D, Wunderlich P, Walter J. (2010) Statins promote the degradation of extracellular amyloid β-peptide by microglia via stimulation of exosome-associated insulin-degrading enzyme secretion. J Biol Chem 285:37405–37414
11.

Cornell RB, Horwitz AF. (1980) Apparent coordination of the biosynthesis of lipids in cultured cells: its relationship to the regulation of the membrane sterol:phospholipid ratio and cell cycling. J Cell Biol 86:810–819
12.

Habenicht AJ, Glomset JA, Ross R. (1980) Relation of cholesterol and mevalonic acid to the cell cycle in smooth muscle and Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 255:5134–5140
13.

Rao S, Porter DC, Chen X, Herliczek T, Lowe M, Keyomarsi K. (1999) Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase. Proc Natl Acad Sci U S A 96:7797–7802
14.

Kallen J, Welzenbach K, Ramage P, Geyl D, Kriwacki R, Legge G, Cottens S, Weitz-Schmidt G, Hommel U. (1999) Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain.J Mol Biol 292:1–9
15.

Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, Cottens S, Takada Y, Hommel U. (2001) Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 7:687–692
16.

Kwak B, Mulhaupt F, Myit S, Mach F. (2000) Statins as a newly recognized type of immunomodulator. Nat Med 6:1399–402
17.

Osterweil EK, Chuang SC, Chubykin AA, Sidorov M, Bianchi R, Wong RK, Bear MF. (2013) Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron 77:243–250
18.

Parihar SP, Guler R, Lang DM, Suzuki H, Marais AD, Brombacher F. (2013) Simvastatin enhances protection against Listeria monocytogenes infection in mice by counteracting Listeria-induced phagosomal escape. PLoS One 8:e75490
19.

Palace J, Robertson N. (2014) Modifying disability in progressive multiple sclerosis. Lancet 383:2189–2191
20.

Yamashita A, Morioka M, Kishi H, Kimura T, Yahara Y, Okada M, Fujita K, Sawai H, Ikegawa S, Tsumaki N. (2014) Statin treatment rescues FGFR3 skeletal dysplasia phenotypes. Nature 513:507–511
21.

Whitehead NP, Kim MJ, Bible KL, Adams ME, Froehner SC. (2015) A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy. Proc Natl Acad Sci U S A 112:12864–12869

Concanamycin A

Keywords: [Antifungal] [V-ATPase inhibitor]

Structure

	Concanamycin A
Molecular formula	C₄₆H₇₅NO₁₄
Molecular weight	866.10
Solubility	DMSO, +++; H₂O, ±; MeOH, +++

Discovery/Isolation/Chemical Studies

The first concanamycin to be isolated was folimycin which shows antifungal activities [1]. Concanamycins A, B, and C were isolated from Streptomyces diastatochromogenes S-45 as effective inhibitors of the proliferation of mouse splenic lymphocytes stimulated by concanavalin A [2, 3]. Folimycin is identical to concanamycin A [3]. Concanamycins D, E, and F were also isolated from Streptomyces sp. [4]. The structures were determined by several spectroscopic analyses and X-ray crystallography [2, 5].

Biological Studies

Concanamycins were reisolated as inhibitors of the acidification of endosome/lysosomes and of oxidized LDL-induced accumulation of lipid droplets [4, 6]. These results and other features indicate that concanamycins are V-ATPase inhibitors.

Concanamycins block intracellular protein secretion before arrival at the Golgi apparatus [7, 8], perforin-/Fas-based cytolytic activity of CTL [9, 10], and bone resorption by osteoclasts [11] via V-ATPase inhibition.

Biological Activity of Concanamycins

MIC for Sacchromyces cerevisiae: <0.39 μg/ml [3]

IC₅₀ for lysosome acidification of concanamycin A: 0.061 nM [4]

95% inhibition of the production of infectous VSV production: >10 ng/ml [8]

ID₅₀ for cytolysis activity of CTL clone OE4: 1.2 ng/ml [10]

References

1.

Yamamoto H, Nakazawa K, Horii S, Miyake A (1960) Studies on agricultural antibiotic. Folimycin, a new antibiotic produced by Streptomyces neyagawaensis nov. sp. Nippon Nogeikagaku Kaishi 34: 268–272
2.

Kinashi H, Sakaguchi K, Higashijima T, Miyazawa T (1982) Structures of concanamycins B and C. J Antibiot (Tokyo) 35: 1618–1620
3.

Kinashi H, Someno K, Sakaguchi K (1984) Isolation and characterization of concanamycins A, B and C. J Antibiot (Tokyo) 37: 1333–1343
4.

Woo JT, Shinohara C, Sakai K, Hasumi K, Endo A (1992) Isolation, characterization and biological activities of concanamycins as inhibitors of lysosomal acidification. J Antibiot (Tokyo) 45: 1108–1116
5.

Westley JW, Liu CM, Sello LH, Evans RH, Troupe N, Blount JF, Chiu AM, Todaro LJ, Miller PA (1984) The structure and absolute configuration of the 18-membered macrolide lactone antibiotic X-4357B (concanamycin A). J Antibiot (Tokyo) 37: 1738–1740
6.

Woo JT, Shinohara C, Sakai K, Hasumi K, Endo A (1992) Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774. Eur J Biochem 207: 383–389
7.

Muroi M, Shiragami N, Nagao K, Yamasaki M, Takatsuki A (1993) Folimycin (concanamycin A), a specific inhibitor of V-ATPase, blocks intracellular translocation of the glycoprotein of vesicular stomatitis virus before arrival to the Golgi apparatus. Cell Struct Funct 18: 139–149
8.

Muroi M, Takasu A, Yamasaki M, Takatsuki A (1993) Folimycin (concanamycin A), an inhibitor of V-type H⁺-ATPase, blocks cell-surface expression of virus-envelope glycoproteins. Biochem Biophys Res Commun 193: 999–1005
9.

Kataoka T, Shinohara N, Takayama H, Takaku K, Kondo S, Yonehara S, Nagai K (1996) Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J Immunol 156: 3678–3686
10.

Kataoka T, Takaku K, Magae J, Shinohara N, Takayama H, Kondo S, Nagai K (1994) Acidification is essential for maintaining the structure and function of lytic granules of CTL. Effect of concanamycin A, an inhibitor of vacuolar type H⁺-ATPase, on CTL-mediated cytotoxicity. J Immunol 153: 3938–3947
11.

Woo JT, Ohba Y, Tagami K, Sumitani K, Yamaguchi K, Tsuji T (1996) Concanamycin B, a vacuolar H⁺-ATPase specific inhibitor suppresses bone resorption in vitro. Biol Pharm Bull 19: 297–299

Cyclosporine A

Keywords: [Immunosuppressant] [cyclophilin] [calcineurin]

Structure

	Cyclosporine A
Molecular formula	C₆₂H₁₁₁N₁₁O₁₂
Molecular weight	1202.64
Solubility	DMSO, ++; H₂O, ±; MeOH, +++

Discovery/Isolation

Cyclosporine A, a cyclic undecapeptide, was found by researchers at Sandoz Ltd. as an antifungal antibiotic from a fungus (originally identified as Cylindrocarpum lucidum), collected in Wisconsin in the USA [1]. Later, the same compound was isolated from Trichoderma inflatum which was isolated from a soil sample from Hardangervidda in Norway [2].

Biological Studies

Cyclosporine A was originally isolated as an antifungal antibiotic; however, its remarkable immunosuppressive activity in several pharmacological models was discovered by Sandoz Ltd. in the early 1970s [2, 3]. Cyclosporine A inhibits DNA binding activity of NF-AT and the production of interleukin 2 (IL-2), and prevents the expression of IL-2 receptor, suggesting that cyclosporine A inhibits the initial steps of T-lymphocyte activation [4, 5].

A cyclosporine A binding protein, named cyclophlin, was isolated using tritium-labeled cyclosporine A [6], and identified as the peptidyl-prolyl cis-trans isomerase (PPIase) [7, 8]. Cyclosporine A specifically binds to cyclophilin and inhibits its enzyme activity; however, the inhibition of PPI activity is not sufficient for immunosuppressive activity [9]. Liu et al. found that the complex of cyclosporine A and cyclophilin binds to calcineurin and inhibits its phosphatase activity [10]. The inhibition of calcineurin is sufficient for the inhibition of T-cell activation, and it was revealed that NF-AT dephosphoryation by calcineurin is required for T-cell activation [11].

Because cyclosporine A binds to cyclophilin D which is a component of mitochondrial permeability pore complex, cyclosporine A is also used as a bioprobe for investigating the function of cyclophilin D on mitochondria-mediated apoptosis [12–14].

Biological Activity of Cyclosporine A

IC₉₀ of NF-AT binding in Jurkat nuclear extract: 10 ng/ml [5]

IC₅₀ for swelling of mitochondria: 25–50 pmole/mg protein [12]

Inhibition between cyclophilin D and Bcl-2: 1 μM [14]

References

1.

Svarstad H, Bugge HC, Dhillion SS (2000) From Norway to Novartis: cyclosporin from Tolypocladium inflatum in an open access bioprospecting regime. Biodivers Conserv 9:1521–1541
2.

Stahelin HF (1996) The history of cyclosporin A (Sandimmune) revisited: another point of view. Experientia 52:5–13
3.

Ruegger A, Kuhn M, Lichti H, Loosli HR, Huguenin R, Quiquerez C, Wartburg AV (1976) Cyclosporin-A, a peptide metabolite from Trichoderma-Polysporum (Link Ex Pers) Rifai, with a remarkable immunosuppressive activity. Helv Chim Acta 59:1075–1092
4.

Elliott JF, Lin Y, Mizel SB, Bleackley RC, Harnish DG, Paetkau V (1984) Induction of interleukin 2 messenger RNA inhibited by cyclosporin A. Science 226:1439–1441
5.

Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree GR (1989) Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science 246:1617–1620
6.

Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW (1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:544–547
7.

Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX (1989) Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337:476–478
8.

Takahashi N, Hayano T, Suzuki M (1989) Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature 337:473–475
9.

Clipstone NA, Crabtree GR (1992) Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357:695–697
10.

Liu J, Farmer JD, Jr., Lane WS, Friedman J, Weissman I, Schreiber SL (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:807–815
11.

McCaffrey PG, Perrino BA, Soderling TR, Rao A (1993) NF-ATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs. J Biol Chem 268:3747–3752
12.

Broekemeier KM, Dempsey ME, Pfeiffer DR (1989) Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 264:7826–7830
13.

Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031
14.

Eliseev RA, Malecki J, Lester T, Zhang Y, Humphrey J, Gunter TE (2009) Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect. J Biol Chem 284:9692–9699

Cytotrienin A

Keywords: [Apoptosis inducer] [Protein translation inhibitor]

Structure

	Cytotrienin A
Molecular formula	C₃₇H₄₈N₂O₈
Molecular weight	648.80
Solubility	DMSO, +++; H₂O, ±; MeOH, +++

Discovery/Isolation/Chemical Studies

Cytotrienin A was isolated as an apoptosis inducer from Streptomyces sp. [1, 2]. This compound is the first ansamycins with a unique 1-aminocyclopropane-1-carboxylic acid unit.

Biological Studies

Cytotrienin A induces apoptosis in HL-60 cells accompanied with the activation of a 36 kDa protein kinase (p36 MBP (myelin basic protein) kinase) which phosphorylates MBP in a gel kinase assay [3]. The 36 kDa MBP kinase was identified as the active proteolytic fragment of MST1/Krs2 and MST2/Krs1 which was activated in response to anticancer drugs including camptothecin and etoposide.

During the course of a high-throughput screen to identify translation inhibitors, cytotrienin A was identified as an inhibitor of both cap- and IRES-dependent translation in vitro [4]. Detailed investigation shows that cytotrienin A inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eEF1A function. Cytotrienin A also triggers ribotoxic stress response and selectively inhibits TNF-α-induced ICAM-1 expression by inducing ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase [5].

Biological Activity of Cytotrienin A

IC₅₀ values for HL-60 and WI-38 cells: 5 and >1000 nM, respectively [2]

IC₅₀ for protein synthesis in A549 cells: ~0.1 μM [5]

References

1.

Zhang HP, Kakeya H, Osada H (1997) Novel triene-ansamycins, cytotrienins A and B, inducing apoptosis on human leukemia HL-60 cells. Tetrahedron Lett 38:1789–1792
2.

Kakeya H, Zhang HP, Kobinata K, Onose R, Onozawa C, Kudo T, Osada H (1997) Cytotrienin A, a novel apoptosis inducer in human leukemia HL-60 cells. J Antibiot (Tokyo) 50:370–372
3.

Kakeya H, Onose R, Osada H (1998) Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis. Cancer Res 58:4888–4894
4.

Lindqvist L, Robert F, Merrick W, Kakeya H, Fraser C, Osada H, Pelletier J (2010) Inhibition of translation by cytotrienin A--a member of the ansamycin family. RNA 16:2404–2413
5.

Yamada Y, Taketani S, Osada H, Kataoka T (2011) Cytotrienin A, a translation inhibitor that induces ectodomain shedding of TNF receptor 1 via activation of ERK and p38 MAP kinase. Eur J Pharmacol 667:113–119

15-Deoxyspergualin

Keywords: [Immunosuppressant] [Antiangiogenesis]

Structure

	15-Deoxyspergualin
Molecular formula	C₁₇H₃₇N₇O₃
Molecular weight	387.52
Solubility	DMSO, ++; H₂O, +++; MeOH, ++

Discovery/Isolation/Chemical Studies

15-Deoxyspergualin (DSG) is a synthetic derivative of spergualin, an antitumor natural product isolated from Bacillus laterosporus [1–3]. DSG has a spermidine moiety and a guanidine moiety in its structure, and the spermidine moiety has cell binding activity and the guanidine moiety has cytotoxic activity [4].

Biological Studies

DSG not only inhibits growth of various tumor cell lines in a xenograft model [5], but also has a potent immunosuppressive effect [6, 7]. Several biological activities of DSG have been reported, for example the indirect inhibition of phosphatidylcholine (PC) synthesis [8, 9] and the inactivation of eIF5A [10].

Nadler et al. identified an intracellular binding protein of DSG by using 11-methoxy-DSG-sepharose as Hsc70, the constitutive or cognate member of the Hsp70 protein family [11]. The members of the Hsp70 family of heat shock proteins are important for many cellular processes, including immune responses, and this finding suggests that heat shock proteins may represent a class of immunosuppressant binding proteins, or immunophilins. The binding site was determined as the EEVD regulatory domain at the extreme C-terminal of Hsc70 by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)–cross-linking of [¹⁴C]-DSG [12]. Because the members of the Hsp70 family are important for many cellular processes including immune responses, these findings suggest that the Hsp70/Hsc70 are a class of immunosuppressant binding proteins.

Biological Activity of 15-Deoxysperugualin

In vivo antitumor activity in L-1210: 6.25 mg/kg/day, intraperitoneally [1]

K_d values for DSK binding to Hsc70 and Hsp90: 4 and 5 μM, respectively [13]

References

1.

Takeuchi T, Iinuma H, Kunimoto S, Masuda T, Ishizuka M, Takeuchi M, Hamada M, Naganawa H, Kondo S, Umezawa H (1981) A new antitumor antibiotic, spergualin: isolation and antitumor activity. J Antibiot (Tokyo) 34:1619–1621
2.

Umezawa H, Kondo S, Iinuma H, Kunimoto S, Ikeda Y, Iwasawa H, Ikeda D, Takeuchi T (1981) Structure of an antitumor antibiotic, spergualin. J Antibiot (Tokyo) 34:1622–1624
3.

Iwasawa H, Kondo S, Ikeda D, Takeuchi T, Umezawa H (1982) Synthesis of (−)-15-deoxyspergualin and (−)-spergualin-15-phosphate. J Antibiot (Tokyo) 35:1665–1669
4.

Umeda Y, Moriguchi M, Kuroda H, Nakamura T, Fujii A, Iinuma H, Takeuchi T, Umezawa H (1987) Synthesis and antitumor activity of spergualin analogues. II. Chemical modification of the spermidine moiety. J Antibiot (Tokyo) 40:1303–1315
5.

Plowman J, Harrison SD, Jr., Trader MW, Griswold DP, Jr., Chadwick M, McComish MF, Silveira DM, Zaharko D (1987) Preclinical antitumor activity and pharmacological properties of deoxyspergualin. Cancer Res 47:685–689
6.

Masuda T, Mizutani S, Iijima M, Odai H, Suda H, Ishizuka M, Takeuchi T, Umezawa H (1987) Immunosuppressive activity of 15-deoxyspergualin and its effect on skin allografts in rats. J Antibiot (Tokyo) 40:1612–1618
7.

Nemoto K, Hayashi M, Abe F, Nakamura T, Ishizuka M, Umezawa H (1987) Immunosuppressive activities of 15-deoxyspergualin in animals. J Antibiot (Tokyo) 40:561–562
8.

Kawada M, Masuda T, Ishizuka M, Takeuchi T (2002) 15-Deoxyspergualin inhibits Akt kinase activation and phosphatidylcholine synthesis. J Biol Chem 277:27765–27771
9.

Kawada M, Ishizuka M (2003) Inhibition of CTP: phosphocholine cytidylyltransferase activity by 15-deoxyspergualin. J Antibiot (Tokyo) 56:725–726
10.

Nishimura K, Ohki Y, Fukuchi-Shimogori T, Sakata K, Saiga K, Beppu T, Shirahata A, Kashiwagi K, Igarashi K (2002) Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin. Biochem J 363:761–768
11.

Nadler SG, Tepper MA, Schacter B, Mazzucco CE (1992) Interaction of the immunosuppressant deoxyspergualin with a member of the Hsp70 family of heat shock proteins. Science 258:484–486
12.

Nadler SG, Dischino DD, Malacko AR, Cleaveland JS, Fujihara SM, Marquardt H (1998) Identification of a binding site on Hsc70 for the immunosuppressant 15-deoxyspergualin. Biochem Biophys Res Commun 253:176–180
13.

Nadeau K, Nadler SG, Saulnier M, Tepper MA, Walsh CT (1994) Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90. Biochemistry 33:2561–2567

7-Desacetoxy-6,7-Dehydrogedunin

Keywords: [Antimalarial] [PKR inhibitor] [HSF1-dependent stress response]

Structure

	7-Desacetoxy-6,7-dehydrogedunin
Molecular formula	C₂₆H₃₀O₅
Molecular weight	422.52
Solubility	DMSO, ++; H₂O, ±; MeOH, ?

Discovery/Isolation/Chemical Studies

7-Desacetoxy-6,7-dehydrogedunin is a derivative of gedunin, an antimalarial compound isolated from Melia azedarach [1] and Azadirachta indica [2]. 7-Desacetoxy-6,7-dehydrogedunin and gedunin contain an epoxide and α,β-unsaturated ketone.

Biological Studies

7-Desacetoxy-6,7-dehydrogedunin was identified as a compound that protects macrophages from death induced by anthrax lethal toxin [3]. SAR analysis revealed that epoxide and α,β-unsaturated ketone are not essential for the macrophage protection. Eleven interacting proteins were identified by biotinylated analog, suggesting that this compound is moderately selective. Further analysis using siRNA showed that PKR is needed for macrophage cell death and the phenotype of PKR knockdown resembles that of treatment with 7-desacetoxy-6,7-dehydrogedunin. Different from the other PKR inhibitors, 2-aminopurine and C16,7-desacetoxy-6,7-dehydrogedunin dose not bind the ATP catalytic domain and inhibit kinase activity of PKR, but prevents ASC assembly. Because the lethal toxin induces inflammasome formation and caspase-1 activation, these results suggest that PKR interactions, rather than PKR kinase activity, are involved in the broad mechanisms of inflammasome-mediated caspase-1 activation.

It was also reported that 7-desacetoxy-6,7-dehydrogedunin activates the heat shock factor (HSF) 1-dependent stress response [4].

Biological Activity of 7-Desacetoxy-6,7-Dehydrogedunin

IC₅₀ for J774 macrophage protection from anthrax lethal toxin: 5 μM [3]

References

1.

Khalid SA, Farouk A, Geary TG, Jensen JB (1986) Potential antimalarial candidates from African plants: and in vitro approach using Plasmodium falciparum. J Ethnopharmacol 15:201–209
2.

Khalid SA, Duddeck H, Gonzalez-Sierra M (1989) Isolation and characterization of an antimalarial agent of the neem tree Azadirachta indica. J Nat Prod 52:922–926
3.

Hett EC, Slater LH, Mark KG, Kawate T, Monks BG, Stutz A, Latz E, Hung DT (2013) Chemical genetics reveals a kinase-independent role for protein kinase R in pyroptosis. Nat Chem Biol 9:398–405
4.

Santagata S, Xu YM, Wijeratne EM, Kontnik R, Rooney C, Perley CC, Kwon H, Clardy J, Kesari S, Whitesell L, Lindquist S, Gunatilaka AA (2012) Using the heat-shock response to discover anticancer compounds that target protein homeostasis. ACS Chem Biol 7:340–349

Destruxin E

Keywords: [V-ATPase inhibitor]

Structure

	Destruxin E
Molecular formula	C₂₉H₄₇N₅O₈
Molecular weight	593.72
Solubility	DMSO, +++; H₂O, +; MeOH, +++

Discovery/Isolation/Chemical Studies

Destruxin E was isolated from the culture medium of Metarhizium anisopliae, which is a fungi known to cause diseases in several kinds of insects [1]. The absolute structure was determined by total synthesis [2].

Biological Studies

Destruxin E shows cytotoxicity against murine lymphocytes and L1210 leukemia cells [3]. In the course of screening of microbial metabolites that inhibit the accumulation of cholesteryl ester in macrophages incubated with oxidized LDL, destruxin E was reisolated as an active compound [4]. Detailed biochemical investigations revealed that destruxin E inhibits V-ATPase-depedent acidification of endosomes and lysosomes [4]. Destruxin E inhibited perforin-dependent cytotoxicity mediated by CD8⁺ CTL [5] and bone resorption by osteoclasts [6] through V-ATPase inhibition. Antitumor and antiangiogenic acitivities were also reported [7].

Biological Activity of Destruxin E

Inhibition of ATP-depedent acidification of endosomes and lysosomes in macrophage: 1 μM [4]

IC₅₀ for perforin-dependent cytotoxicity mediated by CD⁸⁺ CTL: 4.0 μM [5]

Induction of morphological changes of osteoclast-like multinucleated cells: 10–50 nM [6]

References

1.

Pais M, Das BC, Ferron P (1981) Depsipeptides from Metarhizium anisopliae. Phytochemistry 20:715–723
2.

Yoshida M, Takeuchi H, Ishida Y, Yashiroda Y, Takagi M, Shin-ya K, Doi T (2010) Synthesis, structure determination, and biological evaluation of destruxin E. Org Lett 12:3792–3795
3.

Morel E, Pais M, Turpin M, Guyot M (1983) Cytotoxicity of cyclodepsipeptides on murine lymphocytes and on L1210 leukemia cells. Biomed Pharmacother 37:184–185
4.

Naganuma S, Kuzuya N, Sakai K, Hasumi K, Endo A (1992) Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E. Biochim Biophys Acta 1126:41–48
5.

Togashi K, Kataoka T, Nagai K (1997) Characterization of a series of vacuolar type H⁺ −ATPase inhibitors on CTL-mediated cytotoxicity. Immunol Lett 55:139–144
6.

Nakagawa H, Takami M, Udagawa N, Sawae Y, Suda K, Sasaki T, Takahashi N, Wachi M, Nagai K, Woo JT (2003) Destruxins, cyclodepsipeptides, block the formation of actin rings and prominent clear zones and ruffled borders in osteoclasts. Bone 33:443–455
7.

Dornetshuber-Fleiss R, Heffeter P, Mohr T, Hazemi P, Kryeziu K, Seger C, Berger W, Lemmens-Gruber R (2013) Destruxins: fungal-derived cyclohexadepsipeptides with multifaceted anticancer and antiangiogenic activities. Biochem Pharmacol 86:361–377

Diazonamide A

Keywords: [Mitotic inhibitor] [Ornithine δ-aminotransferase]

Structure

	Diazonamide A
Molecular formula	C₄₀H₃₄Cl₂N₆O₆
Molecular weight	765.65
Solubility	DMSO, ? ; H₂O, ? ; MeOH, ++

Discovery/Isolation/Chemical Studies

Diazonamide A was isolated as a cytotoxic compound from the colonial ascidian Diazona chinensis (order Phlebobranchia) collected from the ceilings of small caves along the northwest coast of Siquijor Island, Philippines [1]. The structure was determined except for the stereochemistry of C-37 by total synthesis [2, 3].

Biological Studies

Diazonamide A causes cells to arrest in mitosis, and treated cells lose both interphase and spindle microtubules [4]. This compound inhibits microtubule assembly by inhibition of tubulin-dependent GTP hydrolysis. Because diazonamide A does not inhibit the binding of [³H]vinblastine, [³H]dolastatin 10, or [8-¹⁴C]GTP to tubulin, it was suggested that the binding site of diazonamide A on tubulin is different from that of the vinca alkaloid and dolastatin 10.

Wang et al. purified a mitochondria matrix protein, ornithine δ-aminotransferase (OAT), as a diazonamide A binding protein using biotinylated diazonamide A [5]. They showed that RanGTP disrupts the interaction between diazonamide A and OAT, and that OAT knockdown blocks cell division and causes cell death in cancer cell lines. These results revealed the new function of OAT in mitotic regulation.

Biological Activity of Diazonamide A

IC₅₀ for HCT116 human colon carcinoma and B16 murine melanoma cancer cell lines: 15 ng/ml [1]

Inhibition of cell cycle progression of human ovarian carcinoma 2008 cells in M phase: 40–100 nM [4]

IC₅₀ for in vitro microtubule polymerization stimulated by MAP or glutamate: 0.3 and 0.75 μM [4]

References

1.

Lindquist N, Fenical W, Vanduyne GD, Clardy J (1991) Isolation and structure determination of diazonamide-A and diazonamide-B, unusual cytotoxic metabolites from the marine Ascidian Diazona chinensis. J Am Chem Soc 113:2303–2304
2.

Nicolaou KC, Hao JL, Reddy MV, Rao PB, Rassias G, Snyder SA, Huang XH, Chen DYK, Brenzovich WE, Giuseppone N, Giannakakou P, O″Brate A (2004) Chemistry and biology of diazonamide A: second total synthesis and biological investigations. J Am Chem Soc 126:12897–12906
3.

Nicolaou KC, Chen DYK, Huang XH, Ling TT, Bella M, Snyder SA (2004) Chemistry and biology of diazonamide A: first total synthesis and confirmation of the true structure. J Am Chem Soc 126:12888–12896
4.

Cruz-Monserrate Z, Vervoort HC, Bai R, Newman DJ, Howell SB, Los G, Mullaney JT, Williams MD, Pettit GR, Fenical W, Hamel E (2003) Diazonamide A and a synthetic structural analog: disruptive effects on mitosis and cellular microtubules and analysis of their interactions with tubulin. Mol Pharmacol 63:1273–1280
5.

Wang G, Shang L, Burgett AW, Harran PG, Wang X (2007) Diazonamide toxins reveal an unexpected function for ornithine δ-amino transferase in mitotic cell division. Proc Natl Acad Sci U S A 104:2068–2073

Didemnin B

Keywords: [Antitumor] [Antiviral] [EF-1α]

Structure

	Didemnin B
Molecular formula	C₅₇H₈₉N₇O₁₅
Molecular weight	1112.37
Solubility	DMSO, ++; H₂O, ? ; MeOH, ?

Discovery/Isolation

Cyclic depsipeptide didemnin B was isolated as a highly active antiviral and antitumor compound from Caribbean tunicate Trididemnum solidum [1].

Biological Studies

In addition to antiviral and antitumor activity, didemnin B inhibits polypeptide synthesis in both cellular and cell-free assay, and cell cycle progression in G1 phase [2]. Crews et al. purified the elongation factor 1α (EF-1α) as a target protein of didemnin by using biotinylated didemnin A [3]. EF-1α is an abundant guanine nucleotide binding protein that transports aminoacyl-tRNAs to the ribosomal A site in a GTP-dependent manner. [³H] N-acetyl didemnin A bound to EF-1α in vitro in a GTP-dependent manner. Didemnin B stimulates EF-1α-dependent aminoacyl-tRNA binding to ribosome and inhibits translocation by stabilizing aminoacyl-tRNA bound to the ribosomal A site without prevention of peptide bond formation [4].

Biological Activity of Didemnin B

Antiviral activity against herpes simplex virus type I and II: 0.05 μM [1]

IC₅₀ for L1210 cell line: 0.001 μg/ml [1]

IC₅₀ for in vitro protein synthesis in rabbit reticulocyte lysates: 3 μM [4]

References

1.

Rinehart KL, Jr., Gloer JB, Hughes RG, Jr., Renis HE, McGovren JP, Swynenberg EB, Stringfellow DA, Kuentzel SL, Li LH (1981) Didemnins: antiviral and antitumor depsipeptides from a caribbean tunicate. Science 212:933–935
2.

Crampton SL, Adams EG, Kuentzel SL, Li LH, Badiner G, Bhuyan BK (1984) Biochemical and cellular effects of didemnins A and B. Cancer Res 44:1796–1801
3.

Crews CM, Collins JL, Lane WS, Snapper ML, Schreiber SL (1994) GTP-dependent binding of the antiproliferative agent didemnin to elongation factor 1α. J Biol Chem 269:15411–15414
4.

SirDeshpande BV, Toogood PL (1995) Mechanism of protein synthesis inhibition by didemnin B in vitro. Biochemistry 34:9177–9184

DX-52-1* (Quinocarcin/Quinocarmycin Derivative)

Keywords: [Antitumor] [Hypoxia-inducible factor 1α (HIF1α)] [Radixin] [Galectin-3]

Structure

	DX-52-1	Quinocarcin/Quinocarmycin
Molecular formula	C₁₉H₂₃N₃O₄	C₁₈H₂₂N₂O₄
Molecular weight	357.41	330.38
Solubility	DMSO, ? ; H₂O, +; MeOH, ?	DMSO, ? ; H₂O, +; MeOH, ?

Discovery/Isolation

DX-52-1 is an analog of quinocarcin/quinocarmycin which was isolated from Streptomyces melanovinaceus nov. sp. as an antitumor compound [1–3].

Biological Studies

DX-52-1 was identified as an inhibitor of HIF1α transcriptional activation pathway under both deferoxamine mesylate-treatment and hypoxia conditions [4, 5].

Kahsai et al. identified DX-52-1 as an inhibitor of epithelial cell migration in the course of screening for new small-molecule modulators of cell motility [6]. They found four major DX-52-1 binding proteins using biotinylated DX-52-1, and identified radixin [6] and galactin-3 [7] as the primary and secondary targets of DX-52-1, respectively. Radixin is a member of the ezrin/radixin/moesin (ERM) family of membrane-actin cytoskeleton linker protein, and DX-52-1 is the first inhibitor of radixin which interferes with the interaction between radixin and both actin and the cell adhesion molecule CD44. Galectin-3 is a multifunctional carbohydrate binding protein, and DX-52-1 binds galectin-3 outside its β-galactoside binding site. Furthermore, it was recently reported that DX-52-1 also inhibits moesin–CD44 interaction and reduces the proliferation of glioblastoma [8]. Because moesin is a member of the ERM family, these results suggest that DX-52-1 inhibits cell migration and proliferation by not only disrupting the interaction of ERM proteins with actin and CD44 but also inhibiting a carbohydrate binding–independent function of galectin-3.

Biological Activity of DX-52-1

IC₅₀ for HeLa S3 cell proliferation: 50 ng/ml [9]

Reduction of HIF1α protein level under hypoxia conditions: 20 μM [4]

IC₅₀ for inhibition of wound closure at 24 hours in MDCK cell monolayer: 140 nM [6]

Inhibition of moesin–CD44 interaction in LN299 cells: 200 nM [8]

References

1.

Saito H, Hirata T (1987) Synthetic Approach to quinocarcin. Tetrahedron Lett 28:4065–4068
2.

Takahashi K, Tomita F (1983) DC-52, a novel antitumor antibiotic. 2. Isolation, physico-chemical characteristics and structure determination. J Antibiot (Tokyo) 36:468–470
3.

Tomita F, Takahashi K, Shimizu K (1983) DC-52, a novel antitumor antibiotic. 1. Taxonomy, fermentation and biological activity. J Antibiot (Tokyo) 36:463–467
4.

Chau NM, Rogers P, Aherne W, Carroll V, Collins I, McDonald E, Workman P, Ashcroft M (2005) Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1α induction in response to hypoxic stress and growth factors. Cancer Res 65:4918–4928
5.

Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G (2002) Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 62:4316–4324
6.

Kahsai AW, Zhu S, Wardrop DJ, Lane WS, Fenteany G (2006) Quinocarmycin analog DX-52-1 inhibits cell migration and targets radixin, disrupting interactions of radixin with actin and CD44. Chem Biol 13:973–983
7.

Kahsai AW, Cui J, Kaniskan HU, Garner PP, Fenteany G (2008) Analogs of tetrahydroisoquinoline natural products that inhibit cell migration and target galectin-3 outside of its carbohydrate-binding site. J Biol Chem 283:24534–24545
8.

Zhu X, Morales FC, Agarwal NK, Dogruluk T, Gagea M, Georgescu MM (2013) Moesin is a glioma progression marker that induces proliferation and Wnt/β-catenin pathway activation via interaction with CD44. Cancer Res 73:1142–1155
9.

Saito H, Kobayashi S, Uosaki Y, Sato A, Fujimoto K, Miyoshi K, Ashizawa T, Morimoto M, Hirata T (1990) Synthesis and biological evaluation of quinocarcin derivatives. Chem Pharm Bull 38:1278–1285

Eeyarestatin I* and II*

Keywords: [ER-associated protein degradation (ERAD) inhibitor] [Sec61 complex inhibitor]

Structure

	Eeyarestatin I	Eeyarestatin II
Molecular formula	C₂₇H₂₅Cl₂N₇O₇	C₃₅H₃₁N₇O₇
Molecular weight	630.44	661.68
Solubility	DMSO, ++; H₂O, −; MeOH, +++	DMSO, ++; H₂O, −; MeOH, +++

Discovery/Isolation

Eeyarestatin I and II were found from a chemical library of 16,320 compounds by high-throughput screen of the inhibitor which prevents the turnover of EGFP-fused class I MHC heavy chain (EGFP-HC) [1].

Biological Studies

Eeyarestatin I and II inhibited the degradation of three substrates: EGFP-HC, wild-type class I HC, and T-cell receptor α, by retaining them in the ER [1]. Therefore, it is thought that these compounds inhibit ER-associated protein degradation (ERAD), which is the system eliminating the misfolded protein from the ER. Several biochemical analyses revealed that eeyarestatin I accumulated the polyubiquitinated proteins by inhibiting p97 ATPase complex–mediated deubiquitination [2]. p97 ATPase is an essential component of the ERAD machinery, and eeyarestatin I directly binds p97 via the nitrofuran-containing group [3]. Inhibition of ERAD by eeyarestatin I further evokes the ER stress response- and histone H2A–mediated up-regulation of BH3-only protein NOXA, leading to apoptosis in cancer cells [4].

It has also been reported that eeyarestatin I inhibits nascent polypeptide cotranslational translocation into the ER by targeting Sec61 complex which forms the ER translocon [5] and intercellular vesicle transport [6].

Biological Activity of Eeyarestatin I

Inhibition of class I HC degradation in U373: 8 μM [1]

Accumulation of polyubiquitinated proteins in A9 cells: 10 μM [2]

IC₅₀ values for cell growth of Jurkat, MINO, JEKO, KMS-12, BJAB cells: 6.43, 6.29, 4.1, 3.07, and 5.21 μM, respectively [4]

NOXA induction in cancer cells: 10 μM [4]

IC₅₀ for cotranslational translcation of the P2X2 purinergic receptor in vitro:70 μM [5]

References

1.

Fiebiger E, Hirsch C, Vyas JM, Gordon E, Ploegh HL, Tortorella D (2004) Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Mol Biol Cell 15:1635–1646
2.

Wang Q, Li L, Ye Y (2008) Inhibition of p97-dependent protein degradation by eeyarestatin I. J Biol Chem 283:7445–7454
3.

Wang Q, Shinkre BA, Lee JG, Weniger MA, Liu Y, Chen W, Wiestner A, Trenkle WC, Ye Y (2010) The ERAD inhibitor eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group. PLoS One 5:e15479
4.

Wang Q, Mora-Jensen H, Weniger MA, Perez-Galan P, Wolford C, Hai T, Ron D, Chen W, Trenkle W, Wiestner A, Ye Y (2009) ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells. Proc Natl Acad Sci U S A 106:2200–2205
5.

Cross BC, McKibbin C, Callan AC, Roboti P, Piacenti M, Rabu C, Wilson CM, Whitehead R, Flitsch SL, Pool MR, High S, Swanton E (2009) Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum. J Cell Sci 122:4393–4400
6.

Aletrari MO, McKibbin C, Williams H, Pawar V, Pietroni P, Lord JM, Flitsch SL, Whitehead R, Swanton E, High S, Spooner RA (2011) Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways. PLoS One 6:e22713

Epigallocatechin 3-Gallate (EGCG)

Keywords: [Tumor prevention] [Antiangiogenesis] [67-kDa laminin receptor]

Structure

	(−)-Epicallocatechin 3-gallate
Molecular formula	C₂₂H₁₈O₁₁
Molecular weight	458.38
Solubility	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation

Epigallochatechin gallate (EGCG) was isolated from green tea as a member of the catechins [1].

Biological Studies

EGCG, a bioactive constituent of green tea, shows cancer prevention, antimatrix metalloproteinase, and antiangiogenesis activities [2–4]. The binding protein of EGCG was identified as the 67 kDa laminin receptor by a subtraction cloning strategy from cells treated or untreated with all-trans-retinoic acid, which enhances the binding of EGCG to the cell surface of cancer cells [5]. Further analyses revealed that EGCG signaling is mediated by the 67 kDa laminin receptor, eukaryotic translation elongation factor 1A (eEF1A), and myosin phosphatase-targeting subunit (MYPT1), and inhibits tumor growth [6, 7]. Because the 67 kDa laminin receptor is expressed on a variety of tumor cells, and the expression level is strongly correlated with the risk of tumor invasion and metastasis, EGCG might be a useful chemopreventing agent for cancer.

Biological Activity of Epigallocatechin 3-Gallate

IC₅₀ values for MMP-2 and MMP-9: 20 and 50 μM, respectively [4]

K_d for binding to 67 kDa laminin receptor: 39.9 nM [5]

References

1.

Bradfield AE, Penney M (1948) 456. The catechins of green tea. Part II. J Chem Soc:2249–2254
2.

Yamane T, Takahashi T, Kuwata K, Oya K, Inagake M, Kitao Y, Suganuma M, Fujiki H (1995) Inhibition of N-methyl-N′-nitro-N-nitrosoguanidine-induced carcinogenesis by (−)-epigallocatechin gallate in the rat glandular stomach. Cancer Res 55:2081–2084
3.

Cao Y, Cao R (1999) Angiogenesis inhibited by drinking tea. Nature 398:381
4.

Garbisa S, Biggin S, Cavallarin N, Sartor L, Benelli R, Albini A (1999) Tumor invasion: molecular shears blunted by green tea. Nat Med 5:1216
5.

Tachibana H, Koga K, Fujimura Y, Yamada K (2004) A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol 11:380–381
6.

Umeda D, Yano S, Yamada K, Tachibana H (2008) Involvement of 67-kDa laminin receptor-mediated myosin phosphatase activation in antiproliferative effect of epigallocatechin-3-O-gallate at a physiological concentration on Caco-2 colon cancer cells. Biochem Biophys Res Commun 371:172–176
7.

Umeda D, Yano S, Yamada K, Tachibana H (2008) Green tea polyphenol epigallocatechin-3-gallate signaling pathway through 67-kDa laminin receptor. J Biol Chem 283:3050–3058

Epolactaene, ETB*

Keywords: [HSP60] [DNA polymerase] [Topoisomerase]

Structure

	Epolactaene	ETB
Molecular formula	C₂₁H₂₇NO₆	C₂₄H₃₃NO₆
Molecular weight	389.45	431.52
Solubility	DMSO, ++; H₂O, +; MeOH, ++	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation/Chemical Studies

Epolactaene was isolated from the fungal strain Penicillium sp. BM 1689-P as a neuritogenic compound which induced neurite outgrowth in the human neuroblastoma cell line SH-SY5Y that lacks significant TRK family mRNAs [1]. This compound possesses a highly functionalized α,β-epoxy-γ-lactam and a conjugated triene moiety in the side chain. Epolactaene is present in a 5:1 diasteromeric mixture derived from epimerization at the hemiaminal moiety.

Biological Studies

Epolactaene promoted neurite outgrowth in a neuroblastoma cell line [1] and induced apoptosis in a human leukemia B-cell line BALL-1 in a dose- and time-dependent manner [2]. At the molecular level, epolactaene inhibits DNA polymerases α and β, and DNA topoisomerase II [3].

Nagumo et al. found that the methyl ester moiety could be substituted by tertiary butyl ester, and synthesized ETB (epolactaene tertiary butyl ester) [4]. Using a biotinylated compound, human HSP60 was identified as a binding protein of epolactaene. Epolactaene inhibits chaperone activity of HSP60 by covalent binding on Cys442 [5]. The binding is reversible; therefore, it was speculated that epolactaene reacts with the α,β-unsaturated ketone via Michael addition. Recently, Sun et al. reported that ETB inhibits Mtf1-dependent mitochondrial transcription but not nuclear transcription by disrupting the binding between Hsp60 and Mtf1 in Schizossacharomyces pombe [6]. The specificity of ETB as an inhibitor of mitochondrial transcription suggests a novel chemical approach to the study of mitochondria.

Interestingly, it has been reported that epolactaene derivatives lacking α,β-unsaturated ketone also showed cytotoxicity [2, 4, 7]. Kuramochi et al. prepared a biotinylated derivative, Bio-Epo-C12, that lacks α,β-unsaturated ketone but possesses α,β-epoxy-γ-lactam moiety, and identified several binding proteins including HSP60 [8]. They further investigated the binding mechanism and proposed the model that epolactaene induces intramolecular or intermolecular disulfide formation between protein cysteines via retro-Claisen reaction [8, 9].

Biological Activity of Epolactaene and Its Derivatives

Epolactaene

Promotion of neurite outgrowth in SH-SY5Y: 2.5–10 μg/ml [1]

IC₅₀ for BALL-1 cells: 3.82 μM [2]

ETB

Inhibition of Mtf1-dependent mitochondrial transcription: 1 μM [6]

References

1.

Kakeya H, Takahashi I, Okada G, Isono K, Osada H (1995) Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, produced by a marine fungus. J Antibiot (Tokyo) 48:733–735
2.

Nakai J, Kawada K, Nagata S, Kuramochi K, Uchiro H, Kobayashi S, Ikekita M (2002) A novel lipid compound, epolactaene, induces apoptosis: its action is modulated by its side chain structure. Biochim Biophys Acta 1581:1–10
3.

Mizushina Y, Kobayashi S, Kuramochi K, Nagata S, Sugawara F, Sakaguchi K (2000) Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II. Biochem Biophys Res Commun 273:784–788
4.

Nagumo Y, Kakeya H, Yamaguchi J, Uno T, Shoji M, Hayashi Y, Osada H (2004) Structure-activity relationships of epolactaene derivatives: structural requirements for inhibition of Hsp60 chaperone activity. Bioorg Med Chem Lett 14:4425–4429
5.

Nagumo Y, Kakeya H, Shoji M, Hayashi Y, Dohmae N, Osada H (2005) Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity. Biochem J 387:835–840
6.

Sun W, Wang L, Jiang H, Chen D, Murchie AI (2012) Targeting mitochondrial transcription in fission yeast with ETB, an inhibitor of HSP60, the chaperone that binds to the mitochondrial transcription factor Mtf1. Genes Cells 17:122–131
7.

Kuramochi K, Matsui R, Matsubara Y, Nakai J, Sunoki T, Arai S, Nagata S, Nagahara Y, Mizushina Y, Ikekita M, Kobayashi S (2006) Apoptosis-inducing effect of epolactaene derivatives on BALL-1 cells. Bioorg Med Chem 14:2151–2161
8.

Kuramochi K, Yukizawa S, Ikeda S, Sunoki T, Arai S, Matsui R, Morita A, Mizushina Y, Sakaguchi K, Sugawara F, Ikekita M, Kobayashi S (2008) Syntheses and applications of fluorescent and biotinylated epolactaene derivatives: epolactaene and its derivative induce disulfide formation. Bioorg Med Chem 16:5039–5049
9.

Kuramochi K, Sunoki T, Tsubaki K, Mizushina Y, Sakaguchi K, Sugawara F, Ikekita M, Kobayashi S (2011) Transformation of thiols to disulfides by epolactaene and its derivatives. Bioorg Med Chem 19:4162–4172

Epoxomicin

Keywords: [Proteasome inhibitor] [Chymotrypsin]

Structure

	Epoxomicin
Molecular formula	C₂₈H₅₀N₄O₇
Molecular weight	554.73
Solubility	DMSO, ++; H₂O, −; MeOH, ++

Discovery/Isolation/Chemical Studies

In the course of screening for antitumor agents showing specific activity against B16 murine melanoma, epoxomicin was isolated from an unidentified actinomycete strain No. Q996-17 [1]. Epoxomicin has an epoxy-β-aminoketone moiety in the structure, suggesting that this compound covalently binds to its target protein.

Biological Studies

Epoxomicin exhibits strong in vitro cytotoxicities against various tumor cell lines, and showed strong in vivo inhibitory activity against B16 melanoma but moderate activity against P388 mouse leukemia [1]. Meng et al. identified the target protein of epoxomicin by using a biotinylated epoxomicin analog [2]. They found that the epoxomicin probe covalently binds to two major (23 and 30 kDa) and one minor (28 kDa) proteins. All these proteins are proteasome components, and it was found that epoxomicin inhibits proteolytic activity, especially chymotrypsin-like activity, of 20S proteasome. Furthermore, epoxomicin induces accumulation of ubiquitinated proteins and inhibits activation of NFκB DNA binding activity by preventing IκB degradation.

Biological Activity of Epoxomicin

IC₅₀ for several tumor cells: 2–44 ng/ml [1]

Accumulation of p53 in HUVECs: 100 nM [2]

Accumulation of ubiquitinated proteins in HeLa cells: 10 μM [2]

Inhibition of NFκB activation in HeLa cells: 10 μM [2]

References

1.

Hanada M, Sugawara K, Kaneta K, Toda S, Nishiyama Y, Tomita K, Yamamoto H, Konishi M, Oki T (1992) Epoxomicin, a new antitumor agent of microbial origin. J Antibiot (Tokyo) 45:1746–1752
2.

Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, Crews CM (1999) Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl Acad Sci U S A 96:10403–10408

EPZ004777*

Keywords: [Methyltransferase] [DOT1L]

Structure

	EPZ004777
Molecular formula	C₂₈H₄₁N₇O₄
Molecular weight	539.68
Solubility	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

EPZ004777 was designed as a potent and highly selective DOT1L inhibitor based on the structures of S-adenosylmethionine and S-adenosylhomocysteine, which are a DOT1L substrate and product, respectively, and the crystal structure of the DOT1L active site [1].

Biological Studies

DOT1L catalyzes the methylation of lysine 79 of histone H3 (H3K79), a chromatin modification associated with actively transcribed genes. EPZ004777 decreases the cellular methylation level of H3K79 [1]. DOT1L mislocation by MLL (mixed-lineage leukemia)–fusion protein is assumed to be a driver for leukemogenesis in MLL. EPZ004777 blocks MLL-fusion target gene expression and selectively inhibits proliferation of MLL-rearranged cells. Furthermore, leukemic transformation by MLL-fusion oncoprotein is inhibited by EPZ004777, suggesting that MLL patients may benefit from therapeutic agents targeting aberrant H3K79 methylation [2, 3].

Biological Activity of EPZ004777

IC₅₀ for DOT1L in vitro: 400 ± 100 pM [1]

Inhibition of cellular H3K79 methylation in MLL- and non-MLL cell lines: 3 μM [1]

Apoptosis induction in MLL-rearranged cells: 3 μM [1]

References

1.

Daigle SR, Olhava EJ, Therkelsen CA, Majer CR, Sneeringer CJ, Song J, Johnston LD, Scott MP, Smith JJ, Xiao Y, Jin L, Kuntz KW, Chesworth R, Moyer MP, Bernt KM, Tseng JC, Kung AL, Armstrong SA, Copeland RA, Richon VM, Pollock RM (2011) Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell 20:53–65
2.

Chen L, Deshpande AJ, Banka D, Bernt KM, Dias S, Buske C, Olhava EJ, Daigle SR, Richon VM, Pollock RM, Armstrong SA (2013) Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia 27:813–822
3.

Deshpande AJ, Chen L, Fazio M, Sinha AU, Bernt KM, Banka D, Dias S, Chang J, Olhava EJ, Daigle SR, Richon VM, Pollock RM, Armstrong SA (2013) Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood 121:2533–2541

EX-527*

Keywords: [SIRT1 inhibitor]

Structure

	EX-527
Molecular formula	C₁₃H₁₃ClN₂O
Molecular weight	248.71
Solubility	DMSO, +++; H₂O, ? ; MeOH, ?

Discovery/Isolation

EX-527 is an indole-containing potent and selective synthetic SIRT1 inhibitor, which was identified by high-throughput screening [1].

Biological Studies

Treatment with EX-527 increases acetylation of a number of substrates of SIRT1 including p53 [2] and tau [3]. EX-527 suppresses pathology of Huntington’s disease in both mammalian cells and animal models [4]. Administration of EX-527 either intraperitoneally or directly into the brain in mice decreases food intake through the melanocortin system [5]. These reports indicate that EX-527 is an effective SIRT1 inhibitor in vivo.

Biological Activity of EX-527

In vitro IC₅₀ for SIRT1: 98 nM [1]

In vitro IC₅₀ values for SIRT2 and SIRT3: 19.6 μM and 48.7 μM, respectively [1]

Increase of p53 acetylation in response to DNA damage in cells: 1 μM [2]

Increase of tau acetylation in cells: 10–50 μM [3]

References

1.

Napper AD, Hixon J, McDonagh T, Keavey K, Pons JF, Barker J, Yau WT, Amouzegh P, Flegg A, Hamelin E, Thomas RJ, Kates M, Jones S, Navia MA, Saunders JO, DiStefano PS, Curtis R (2005) Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem 48:8045–8054
2.

Solomon JM, Pasupuleti R, Xu L, McDonagh T, Curtis R, DiStefano PS, Huber LJ (2006) Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage. Mol Cell Biol 26:28–38
3.

Min SW, Cho SH, Zhou Y, Schroeder S, Haroutunian V, Seeley WW, Huang EJ, Shen Y, Masliah E, Mukherjee C, Meyers D, Cole PA, Ott M, Gan L. (2010) Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron 6:953–966
4.

Smith MR, Syed A, Lukacsovich T, Purcell J, Barbaro BA, Worthge SA, Wei SR, Pollio G, Magnoni L, Scali C, Massai L, Franceschini D, Camarri M, Gianfriddo M, Diodato E, Thomas R, Gokce O, Tabrizi SJ, Caricasole A, Landwehrmeyer B, Menalled L, Murphy C, Ramboz S, Luthi-Carter R, Westerberg G, Marsh JL (2014) A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington’s disease. Hum Mol Genet 23:2995–3007
5.

Dietrich MO, Antunes C, Geliang G, Liu ZW, Borok E, Nie Y, Xu AW, Souza DO, Gao Q, Diano S, Gao XB, Horvath TL (2010) Agrp neurons mediate Sirt1’s action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity. J Neurosci 30:11815–11825

FK228 (Romidepsin, FR901228)

Keywords: [Antitumor] [Histone deacetylase (HDAC) inhibitor]

Structure

	FK228
Molecular formula	C₂₄H₃₆N₄O₆S₂
Molecular weight	540.69
Solubility	DMSO, ++; H₂O, −; MeOH, −

Discovery/Isolation/Chemical Studies

FK228 (romidepsin, FR901228) was isolated from a broth culture of Chromobacterium violaceum as an antitumor bicyclic depsipeptide [1].

Biological Studies

FK228 reversed the transformed morphology of Ha-ras transformant, Ras-1 cells, and inhibited their cell cycle progression at G0/G1 phases [2]. FK228 also reduced c-myc and Fas ligand expression, then effectively blocked activation-induced apoptosis in T-cell hybridomas [3].

FK228 was reisolated as a transcriptional activator of the SV40 promoter. This compound caused cell cycle arrest at both G1 and G2/M phases, induction of internucleosomal breakdown of chromatin, and inhibition of intracellular histone deacetylase activity [4].

Biological Activity of FK228

Antitumor activity against human tumor A549 implanted under the kidney capsule of immunosuppressed BDF1 mice: 0.56–3.2 mg/kg [1]

Morphological reversion of Ras1, Ha-ras-transformed NIH/3T3: 2.5 ng/ml [1]

Cell cycle arrest at G1 and G2/M phases in M-8 cells: 10 ng/ml [4]

IC₅₀ for histone deacetylase activity in vitro: 1.1 nM [4]

References

1.

Ueda H, Nakajima H, Hori Y, Fujita T, Nishimura M, Goto T, Okuhara M (1994) FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties, and antitumor activity. J Antibiot (Tokyo) 47:301–310
2.

Shigematsu N, Ueda H, Takase S, Tanaka H, Yamamoto K, Tada T (1994) FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum No. 968. II. Structure determination. J Antibiot (Tokyo) 47:311–314
3.

Wang R, Brunner T, Zhang L, Shi Y (1998) Fungal metabolite FR901228 inhibits c-Myc and Fas ligand expression. Oncogene 17:1503–1508
4.

Nakajima H, Kim YB, Terano H, Yoshida M, Horinouchi S (1998) FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor. Exp Cell Res 241:126–133

FK506 (Tacrolimus)

Keywords: [Immunosuppressant]

Structure

	FK506
Molecular formula	C₄₄H₆₉NO₁₂
Molecular weight	804.02
Solubility	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation

FK506 was isolated in the course of the search for an immunosuppressant from Streptomyces tsukubaensis [1–3].

Biological Studies

FK506 inhibited mixed lymphocyte reaction, cytotoxic T-cell generation, and expression of early T-cell activation genes [4]. An FK506 binding protein named FKBP12 was isolated by an affinity purification method [5] and was identified as peptidyl-prolyl cis-trans isomerase (PPIase) [6, 7]. Therefore, it was speculated that FKBP regulates T-cell activation by the recognition of proline-containing epitopes in target proteins. However, Bierer et al. found that the inhibition of peptidyl-prolyl isomerase activity is insufficient for immunosuppressive effects by using an unnatural immunophilin ligand, 506BD, which contains only the common structural element of FK506 and rapamycin [8]. Liu et al. finally identified calcineurin as the target protein of the FKBP-FK506 complex [9]. They showed that the FKBP-FK506 complex, but not FKBP-506BD, competitively binds to and inhibits phosphatase activity of calcineurin. Inhibition of calcineurin phosphatase activity is strongly correlated with the inhibition of transcriptional activation by NF-AT, a T-cell-specific transcription factor that regulates IL-2 gene synthesis in human T cells [10].

Biological Activity of FK506

IC₅₀ for mouse mixed lymphocyte reaction in vitro: 0.32 nM [1]

ED₅₀ for humoral and cellular immunity in mice: 4.4 mg/kg and 14 mg/kg [1]

IC₅₀ values for production of IL-2, IL-3, IFN-γ, and expression of IL-2 receptor in MLR in vitro: 0.1, 0.3, 1, and 10 nM, respectively [2]

K_d for FKBP12 by Scatchard analysis: 0.4 nM [7]

References

1.

Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H (1987) FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot (Tokyo) 40:1249–1255
2.

Kino T, Hatanaka H, Miyata S, Inamura N, Nishiyama M, Yajima T, Goto T, Okuhara M, Kohsaka M, Aoki H, et al. (1987) FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiot (Tokyo) 40:1256–1265
3.

Tanaka H, Kuroda A, Marusawa H, Hashimoto M, Hatanaka H, Kino T, Goto T, Okuhara M (1987) Physicochemical properties of FK-506, a novel immunosuppressant isolated from Streptomyces tsukubaensis. Transplant Proc 19:11–16
4.

Kino T, Inamura N, Sakai F, Nakahara K, Goto T, Okuhara M, Kohsaka M, Aoki H, Ochiai T (1987) Effect of FK-506 on human mixed lymphocyte reaction in vitro. Transplant Proc 19:36–39
5.

Harding MW, Galat A, Uehling DE, Schreiber SL (1989) A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 34:758–760
6.

Rosen MK, Standaert RF, Galat A, Nakatsuka M, Schreiber SL (1990) Inhibition of FKBP rotamase activity by immunosuppressant FK506: twisted amide surrogate. Science 248:863–866
7.

Siekierka JJ, Hung SH, Poe M, Lin CS, Sigal NH (1989) A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341:755–757
8.

Bierer BE, Somers PK, Wandless TJ, Burakoff SJ, Schreiber SL (1990) Probing immunosuppressant action with a nonnatural immunophilin ligand. Science 250:556–559
9.

Liu J, Farmer JD, Jr., Lane WS, Friedman J, Weissman I, Schreiber SL (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:807–815
10.

Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ, Cohen P, MacKintosh C, Klee CB, Schreiber SL (1992) Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry 31:3896–3901

FR177391

Keywords: [Anti-hyperlipidemic] [Protein phosphatase 2A (PP2A)]

Structure

	FR177391
Molecular formula	C₂₃H₃₁ClO₈
Molecular weight	470.94
Solubility	DMSO, ++; H₂O, +; MeOH, +++

Discovery/Isolation

In the course of the screening for new antihyperlipidemia drugs, FR177391 was isolated from Serratia liquefaciens [1].

Biological Studies

FR177391 potently enhanced the differentiation of mouse 3Y3-L1 fibroblasts to mature adipocytes with oil droplets. The oral administration of FR177391 to C57BL/KsJ-db/db non-insulin-dependent diabetes mellitus (NIDDM) model mice also reduced the blood levels of triglyceride [1].

The target protein for FR177391 was identified to be protein phosphatase 2A (PP2A) by affinity chromatography using biotinylated FR177391 [2, 3]. FR177391 potently inhibited PP2A activity and shared its binding pocket with okadaic acid [3].

In the FR177391-treated 3 T3-L1 cells, the phosphorylation/activation of ERK was prolonged by the inhibition of its dephosphorylation by PP2A, suggesting that PP2A inhibition plays an important role in adipocyte maturation and regulation of blood levels of lipids [3].

Biological Activity of FR177391

Minimum effective concentration for adipogenesis-enhancing effect: 0.1 μM [1]

Enhancement of ERK1/2 phosphorylation in 3 T3-L1 adipocyte: 10 μM [3]

K_i values for PP2A and PP1: 2.9 and 7853 nM, respectively [3]

References

1.

Sato B, Nakajima H, Fujita T, Takase S, Yoshimura S, Kinoshita T, Terano H (2005) FR177391, a new anti-hyperlipidemic agent from Serratia. I. Taxonomy, fermentation, isolation, physico-chemical properties, structure elucidation and biological activities. J Antibiot (Tokyo) 58:634–639
2.

Kobayashi M, Sato K, Yoshimura S, Yamaoka M, Takase S, Ohkubo M, Fujii T, Nakajima H (2005) FR177391, a new anti-hyperlipidemic agent from Serratia. III. Microbial conversion of FR177391 and synthesis of FR177391 derivatives for its target protein screening by chemical genetic approaches. J Antibiot (Tokyo) 58:648–653
3.

Yamaoka M, Sato K, Kobayashi M, Nishio N, Ohkubo M, Fujii T, Nakajima H (2005) FR177391, a new anti-hyperlipidemic agent from Serratia. IV. Target identification and validation by chemical genetic approaches. J Antibiot (Tokyo) 58:654–662

Fumagillin

Keywords: [Antiangiogenesis] [Methionine aminopeptidase inhibitor]

Structure

	Fumagillin
Molecular formula	C₂₆H₃₄O₇
Molecular weight	458.55
Solubility	DMSO, +++; H₂O, −; MeOH, +

Discovery/Isolation/Chemical Studies

Fumagillin was isolated from a culture broth of Aspergillus sp. as active concentrates that were capable of inhibiting Staphylococcus aureus 209 bacteriophage [1]. Total synthesis was done by Corey and Snider [2].

Biological Studies

Ingber et al. reisolated fumagillin from Aspergillus fumigatus, as a potent inhibitor of angiogenesis [3]. The compound completely inhibited endothelial cell proliferation and suppressed tumor-induced neovascularization in the mouse dorsal air sac. However, severe side effects limited the in vivo effectiveness.

Fumagillin contains two epoxides, suggesting that this compound covalently binds its target protein. Sin et al. [4]. and Griffith et al. [5] synthesized biotinylated fumagillin or fumagillin derivative, and showed that fumagillin covalently binds and inhibits type II methionine aminopeptidase (MetAP2). Fumagillin covalently modified histidine231 in the active site of MetAP2, and irreversibly inhibited the aminopeptidase activity of MetAP2 [6–8].

The reason why MetAP2 inhibition by fumagillin resulted in antiangiogenic activity is largely unknown. It was reported that MetAP2 binds and down-regulates ERK1/2 in a fumagillin-dependent manner, resulting in the inhibition of cell proliferation [9]. On the other hand, Kim et al. reported that depletion of MetAP2 did not change the fumagillin sensitivity of endothelial cells, suggesting that MetAP2 function is not required for endothelial cell proliferation [10]. Further investigation is required for elucidation of fumagillin functions.

Biological Activity of Fumagillin

Inhibition of tumor-induced angiogenesis in the subcutaneous dorsal air sac: 100 mg/kg [3]

Growth inhibition of MetAP-1 deleted Sacchromyces cerevisiae: 0.5 pmol/filter disk [4]

IC₅₀ for cell growth of C2C12 myoblasts: 2.6 nM [9]

References

1.

Hanson FR, Eble TE (1949) An antiphage agent Isolated from Aspergillus sp. J Bacteriol 58:527–529
2.

Corey EJ, Snider BB (1972) Total synthesis of (+/−)-fumagillin. J Am Chem Soc 94:2549–255
3.

Ingber D, Fujita T, Kishimoto S, Sudo K, Kanamaru T, Brem H, Folkman J (1990) Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 348:555–557
4.

Sin N, Meng LH, Wang MQW, Wen JJ, Bornmann WG, Crews CM (1997) The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. Proc Natl Acad Sci U S A 94:6099–6103
5.

Griffith EC, Su Z, Turk BE, Chen S, Chang YH, Wu Z, Biemann K, Liu JO (1997) Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin. Chem Biol 4:461–471
6.

Griffith EC, Su Z, Niwayama S, Ramsay CA, Chang YH, Liu JO (1998) Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2. Proc Natl Acad Sci U S A 95:15183–15188
7.

Liu SP, Widom J, Kemp CW, Crews CM, Clardy J (1998) Structure of human methionine aminopeptidase-2 complexed with fumagillin. Science 282:1324–1327
8.

Lowther WT, McMillen DA, Orville AM, Matthews BW (1998) The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase. Proc Natl Acad Sci U S A 95:12153–12157
9.

Datta B, Majumdar A, Datta R, Balusu R (2004) Treatment of cells with the angiogenic inhibitor fumagillin results in increased stability of eukaryotic initiation factor 2-associated glycoprotein, p67, and reduced phosphorylation of extracellular signal-regulated kinases. Biochemistry 43:14821–14831
10.

Kim S, LaMontagne K, Sabio M, Sharma S, Versace RW, Yusuff N, Phillips PE (2004) Depletion of methionine aminopeptidase 2 does not alter cell response to fumagillin or bengamides. Cancer Res 64:2984–2987

Fusicoccin, Cotylenin A

Keywords: [14-3-3] [Plasma membrane H⁺-ATPase] [Protein-protein interaction inhibitor]

Structure

	Fusicoccin	Cotylenin A
Molecular formula	C₃₆H₅₆O₁₂	C₃₃H₅₀O₁₁
Molecular weight	680.83	622.75
Solubility	DMSO, ++; H₂O, ±; MeOH, +++	DMSO, ++; H₂O, ±; MeOH, +++

Discovery/Isolation

Fusicoccin was isolated as the main phytotoxin produced by the fungus Fusicoccum amygdali Del [1]. Cotylenin A, the major metabolite of novel fusicocane-diterpene glycosides from Cladosporium sp. 501-7 W, was isolated as a potent plant growth stimulant possessing phytohormone-like activity [2].

Biological Studies

Fusicoccin stimulated stomatal opening of Commelina communis [3] and rapid cell elongation in Avena coleoptiles [4], which were accompanied by potassium entry and proton excretion, respectively. Further in vitro biochemical investigations suggested that the ultimate target of fusicoccin action is H⁺-APTase [5], but the molecular mechanism of this activation remained to be revealed.

The two fusicoccin binding proteins were purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose from Avena sativa [6] or by photoaffinity labeling from Commelina communis [7], and these proteins were identified as 14-3-3 family proteins [8–10]. In vitro binding assay revealed that 14-3-3 protein binds to H⁺-ATPase, and that fusicoccin strengthens the interaction between 14-3-3 protein and H⁺-ATPase [11]. Furthermore, fusicoccin activates plant plasma membrane H⁺-APTase through the formation of a ternary complex with plant 14-3-3 [12, 13], suggesting that fusicoccin is a protein–protein interaction stabilizer.

Different from fusicoccin, studies of cotylenin A has been focused on its antitumor activities [14]. Because 14-3-3 family proteins play a critical role in serine/threonine kinase–dependent signaling pathways through protein–protein interactions with multiple phosphorylated ligands, fusicoccin and cotylenin A derivatives have been developed as antitumor agents [15, 16].

Biological Activity of Fusicoccin and Cotylenin A

Fusicoccin

Enhancement of NaHCO₃ uptake in Avena sativa: 20 μM [17]

Cotylenin A

Morphological differentiation in M1 and HL60 cells: 20 μM [14]

EC₅₀ for 14-3-3/C-RAFpS233pS259 interaction in vitro: 60 μM [15]

References

1.

Ballio A, Chain EB, De Leo P, Erlanger BF, Mauri M, Tonolo A (1964) Fusicoccin: a new wilting toxin produced by Fusicoccum amygdali Del. Nature 203:297
2.

Sassa T (1971) Cotylenins, leaf growth substances produced by a fungus: isolation and characterization of cotylenins A and B. Agric Biol Chem 35:1415–1418
3.

Squire GR, Mansfield TA (1972) Studies of the mechanism of action of fusicoccin, the fungal toxin that induces wilting, and its interaction with abscisic acid. Planta 105:71–78
4.

Cleland RE (1976) Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: relation to auxin responses. Planta 128:201–206
5.

Rasi-Caldogno F, De Michelis MI, Pugliarello MC, Marre E (1986) H⁺-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin. Plant Physiol 82:121–125
6.

de Boer AH, Watson BA, Cleland RE (1989) Purification and identification of the fusicoccin binding protein from oat root plasma membrane. Plant Physiol 89:250–259
7.

Oecking C, Weiler EW (1991) Characterization and purification of the fusicoccin-binding complex from plasma membranes of Commelina communis. Eur J Biochem 199:685–689
8.

Korthout HA, de Boer AH (1994) A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs. Plant Cell 6: 1681–1692
9.

Marra M, Fullone MR, Fogliano V, Pen J, Mattei M, Masi S, Aducci P (1994) The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein. Plant Physiol 106:1497–1501
10.

Oecking C, Eckerskorn C, Weiler EW (1994) The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins. FEBS Lett 352:163–166
11.

Fullone MR, Visconti S, Marra M, Fogliano V, Aducci P (1998) Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H⁺ −ATPase. J Biol Chem 273:7698–7702
12.

Fuglsang AT, Visconti S, Drumm K, Jahn T, Stensballe A, Mattei B, Jensen ON, Aducci P, Palmgren MG (1999) Binding of 14-3-3 protein to the plasma membrane H⁺-ATPase AHA2 involves the three C-terminal residues Tyr946-Thr-Val and requires phosphorylation of Thr947. J Biol Chem 274:36774–36780
13.

Svennelid F, Olsson A, Piotrowski M, Rosenquist M, Ottman C, Larsson C, Oecking C, Sommarin M (1999) Phosphorylation of Thr-948 at the C terminus of the plasma membrane H⁺-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11:2379–2391
14.

Asahi K, Honma Y, Hazeki K, Sassa T, Kubohara Y, Sakurai A, Takahashi N (1997) Cotylenin A, a plant-growth regulator, induces the differentiation in murine and human myeloid leukemia cells. Biochem Biophys Res Commun 238:758–763
15.

Molzan M, Kasper S, Röglin L, Skwarczynska M, Sassa T, Inoue T, Breitenbuecher F, Ohkanda J, Kato N, Schuler M, Ottmann C (2013) Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers. ACS Chem Biol 8:1869–1875
16.

Takahashi M, Kawamura A, Kato N, Nishi T, Hamachi I, Ohkanda J (2012) Phosphopeptide-dependent labeling of 14-3-3ζ proteins by fusicoccin-based fluorescent probes. Angew Chem Int Ed Eng 51:509–512
17.

Johnson KD, Rayle DL (1976) Enhancement of CO₂ uptake in Avena coleoptiles by fusicoccin. Plant Physiol 57:806–811

Geldanamycin

Keywords: [HSP90 inhibitor]

Structure

	Geldanamycin
Molecular formula	C₂₉H₄₀N₂O₉
Molecular weight	560.64
Solubility	DMSO, +++; H₂O, −; MeOH, −

Discovery/Isolation

Geldanamycin was isolated from culture broth of Streptomyces hygroscopicus as a compound showing moderate inhibitory activity against protozoa, bacteria, fungi, and tumor cells [1].

Biological Studies

Uehara et al. found that geldanamycin and other benzoquinonoid ansamycins reverse the morphology of src-transformed cells from the transformed to the normal phenotype by inhibiting the autophosphorylation of p60^src [2]. Because these compound did not inhibit the autophosphorylation of src kinase in vitro, it was thought that geldanamycins are not kinase inhibitors. The target protein of geldanamycin was determined as HSP90 by using affinity resin which immobilizes the diamine derivative of geldanamycin [3].

Geldanamycin binds the N-terminal ATP/ADP binding domain of HSP90 [4], and the inhibition of ATP/ADP binding leads the inhibition of the Hsp90-mediated conformational maturation/refolding reaction, and the degradation of HSP90 substrates [5]. Because HSP90 client proteins include several oncogenic proteins, such as mutated p53, Bcr-Abl, Raf-1, Akt, ErbB2, and HIF-1α, geldanamycin is a promising antitumor compound in the preclinical model system [6].

Biological Activity of Geldanamycin

Morphological changes from the transformed to the normal phenotype: 0.1 μg/ml [2]

Disruption of multimolecular complexes containing Raf-1: 2 μM [7]

References

1.

DeBoer C, Meulman PA, Wnuk RJ, Peterson DH (1970) Geldanamycin, a new antibiotic. J Antibiot (Tokyo) 23:442–447
2.

Uehara Y, Hori M, Takeuchi T, Umezawa H (1986) Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60^src in rat kidney cells infected with Rous sarcoma virus. Mol Cell Biol 6:2198–2206
3.

Whitesell L, Mimnaugh EG, De Costa B, Myers CE, Neckers LM (1994) Inhibition of heat shock protein HSP90-pp60^v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci U S A 91:8324–8328
4.

Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP (1997) Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89:239–250
5.

Schneider C, Sepp-Lorenzino L, Nimmesgern E, Ouerfelli O, Danishefsky S, Rosen N, Hartl FU (1996) Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc Natl Acad Sci U S A 93:14536–14541
6.

Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8:S55–61
7.

Schulte TW, Blagosklonny MV, Ingui C, Neckers L (1995) Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association. J Biol Chem 270:24585–24588

Gerfelin, Methyl Gerfelin*

Keywords: [Geranylgeranyl diphosphate (GGPP) synthase inhibitor] [Osteoclastogenesis inhibitor] [Glyoxalase inhibitor]

Structure

	Gerfelin	Methyl gerfelin
Molecular formula	C₁₅H₁₄O₆	C₁₆H₁₆O₆
Molecular weight	209.27	304.29
Solubility	DMSO, +++; H₂O, ? ; MeOH, ++	DMSO, +++; H₂O, ±; MeOH, ++

Discovery/Isolation/Chemical Studies

Gerfelin was isolated from Beauveria felina QN22047 as an inhibitor of geranylgeranyl diphosphate (GGPP) synthase [1, 2]. Structure–activity relationship studies using synthetic gerfelin derivatives revealed that the 2-hydroxybenzoic acid moiety and the catechol moiety are important for GGPP synthase inhibition [3]. Methyl gerfelin, a methyl ester of gerfelin, was identified as an osteoclastogenesis inhibitor in cellular phenotype–based screening of osteoclast inhibitors [4].

Biological Studies

Gerfelin inhibits GGPP synthase noncompetitively against isopentenyl diphosphate and uncompetitively against farnesyl diphosphate [1].

Gerfelin and methyl gerfelin suppressed the formation of tartrate-resistant acid phosphatase–positive multinucleated osteoclasts induced by RANKL and M-CSF [4]. However, this osteoclastogenesis inhibition was not dependent on the GGPP synthase inhibition of methyl gerfelin, suggesting that this compound could interact with another protein. Kawatani et al. made a methyl gerfelin affinity matrix and isolated glyoxalase I (GLO1) as a binding protein [4]. Further analysis revealed that GLO1 activity is required for osteoclastogenesis. SAR analysis using methyl gerfelin derivatives, each having an amine-terminated tri(ethylene glycol) linker at the peripheral position, revealed that derivatization at the C6-methyl group would be suitable for the further development of selective GLO1 inhibitors [5].

Methyl gerfelin also inhibited Plasmodium falciparum GLO1 with a complicated mechanism [6]. Because it has been reported that glycolytic flux increases in malaria parasites [7], methyl gerfelin might be a good lead compound for developing antimalarial medicines that increase harmful methylglyoxal concentrations in P. falciparum.

Biological Activity of Gerfelin

IC₅₀ for GGPP synthase in vitro: 3.5 μg/ml [1]

K_i for GGPP synthase: 5.5 μg/ml [1] and 19.0 μM [4]

IC₅₀ for osteoclastogenesis: 61 μM [4]

Biological Activity of Methyl Gerfelin

IC₅₀ for osteoclastogenesis: 2.8 μM [4]

IC₅₀ for GGPP synthase in vitro: 2.5 μM [4]

K_i for GLO1 activity in vitro: 0.23 μM, competitively [4]

References

1.

Zenitani S, Tashiro S, Shindo K, Nagai K, Suzuki K, Imoto M (2003) Gerfelin, a novel inhibitor of geranylgeranyl diphosphate synthase from Beauveria felina QN22047. I. Taxonomy, fermentation, isolation, and biological activities. J Antibiot (Tokyo) 56:617–621
2.

Zenitani S, Shindo K, Tashiro S, Sekiguchi M, Nishimori M, Suzuki K, Imoto M (2003) Gerfelin, a novel inhibitor of geranylgeranyl diphosphate synthase from Beauveria felina QN22047. II. Structural elucidation. J Antibiot (Tokyo) 56:658–660
3.

Islam MS, Kitagawa M, Imoto M, Kitahara T, Watanabe H. (2006) Synthesis of gerfelin and related analogous compounds. Biosci Biotechnol Biochem 70:2523–2528
4.

Kawatani M, Okumura H, Honda K, Kanoh N, Muroi M, Dohmae N, Takami M, Kitagawa M, Futamura Y, Imoto M, Osada H (2008) The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I. Proc Natl Acad Sci U S A 105:11691–11696
5.

Kanoh N, Suzuki T, Kawatani M, Katou Y, Osada H, Iwabuchi Y. (2013) Dual structure-activity relationship of osteoclastogenesis inhibitor methyl gerfelin based on TEG scanning. Bioconjug Chem 24:44–52
6.

Urscher M, Przyborski JM, Imoto M, Deponte M. (2010) Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases. Mol Microbiol 76:92–103
7.

Akoachere M, Iozef R, Rahlfs S, Deponte M, Mannervik B, Creighton DJ, Schirmer H, Becker K. (2005) Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts. Biol Chem 386:41–52

Glaziovianin A, Gatastatin*

Keywords: [Tubulin binder] [Microtubule dynamics inhibitor] [γ-tubulin inhibitor]

Structure

	Glaziovianin A	Gatastatin
Molecular formula	C₂₀H₁₈O₈	C₂₆H₂₂O₈
Molecular weight	386.36	462.45
Solubility	DMSO, +++; H₂O, −; MeOH, +	DMSO, +++; H₂O, −; MeOH, +

Discovery/Isolation/Chemical Studies

Glaziovianin A was isolated from the leaves of Ateleia glazioviana (Legminosae) as a cytotoxic compound against human leukemia cell line HL-60 [1]. SAR studies using synthetic glaziovianin A derivatives revealed that O⁷-alkylated glaziovianin A analogs have strong cytotoxicity against HeLa cells [2, 3]. Gatastatin, O⁷-demethyl benzyl glaziovianin A, was identified as a γ-tubulin-specific inhibitor in the course of screening the γ-tubulin binder [4].

Biological Studies

Glaziovianin A induced a multipole spindle strucuture in 3Y1 cells, suggesting that this compound targets microtubules [1]. Detailed biochemical investigations revealed that glaziovianin A and O⁷-demethyl propargyl glaziovianin A bind the colchicine binding site of tubulin and suppresses microtubule dynamics in vitro and in cells [5]. The microtubule dynamic inhibition causes the delay of endosome maturation and the down-regulation of growth factor–dependent signaling.

Gatastatin inhibited γ-tubulin-dependent microtubule nucleation and minus end capping of microtubules by a γ-tubulin complex in vitro [4]. Gatastatin also inhibited microtubule nucleation from the centrosome, and prolonged not only metaphase but also anaphase duration by affecting anaphase spindle structure. These results indicate that the microtubule nucleation activity of γ-tubulin is continuously required throughout mitosis.

Biological Activity of Glaziovianin A

IC₅₀ for glaziovianin A on cell growth of HL-60 cells: 0.29 μM [1]

Inhibition of microtubule dynamics in PtK2 cells: 1 μM [2]

Inhibition of endosome transport and maturation in HeLa cells: 2 μM [2]

Biological Activity of Gatastatin

K_d for γ-tubulin determined by tryptophan fluorescence–based drug binding assay: 3.6 μM [4]

References

1.

Yokosuka A, Haraguchi M, Usui T, Kazami S, Osada H, Yamori T, Mimaki Y (2007) Glaziovianin A, a new isoflavone, from the leaves of Ateleia glazioviana and its cytotoxic activity against human cancer cells. Bioorg Med Chem Lett 17: 3091–3094
2.

Ikedo A, Hayakawa I, Usui T, Kazami S, Osada H, Kigoshi H. (2010) Structure-activity relationship study of glaziovianin A against cell cycle progression and spindle formation of HeLa S3 cells. Bioorg Med Chem Lett 20:5402–5404
3.

Hayakawa I, Ikedo A, Chinen T, Usui T, Kigoshi H. (2012) Design, synthesis, and biological evaluation of the analogues of glaziovianin A, a potent antitumor isoflavone. Bioorg Med Chem 20:5745–5756
4.

Chinen T, Liu P, Shioda S, Pagel J, Cerikan B, Lin T, Gruss O, Hayashi Y, Takeno H, Shima T, Okada T, Hayakawa I, Hayashi Y, Kigoshi H, Usui T, Schiebel E. (2015) The γ-tubulin specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle. Nat Commun 6:8722
5.

Chinen T, Kazami S, Nagumo Y, Hayakawa I, Ikedo A, Takagi M, Yokosuka A, Imamoto N, Mimaki Y, Kigoshi H, Osada H, Usui T (2013) Glaziovianin A prevents endosome maturation via inhibiting microtubule dynamics. ACS Chem Biol 8: 884–889

Gleevec*

Keywords: [Antitumor] [Bcr-Abl inhibitor]

Structure

	Gleevec
Molecular formula	C₂₉H₃₁N₇O
Molecular weight	493.60 (589.71: mesylate)
Solubility	DMSO, +; H₂O, +++ (mesilate); MeOH, +

Discovery/Isolation/Chemical Studies

As the protein kinase activity of Bcr-Abl is essential for its transformation-inducing activity, a research program began to develop low molecular weight inhibitors of Bcr-Abl. The high-throughput screening study identified 2-phenylaminopyrimidine as a lead compound of the inhibitor of Bcr-Abl. To improve the binding properties of this molecule to Bcr-Abl, methyl and benzamide groups were introduced and, as a result, a drug named Gleevec (CGP-57148, imatinib mesylate, STI-571, and Glivec) emerged [1]. This compound inhibits the Abl protein tyrosine kinases from several sources with an IC₅₀ of 0.025–0.038 μM in vitro. This is a very selective inhibitor of Abl kinase and no significant inhibition of other protein kinases except c-kit and PDGF-R has been reported.

Biological Studies

In Philadelphia chromosome (Ph)–positive CML cells, a tyrosine kinase, Bcr-Abl, is kept active, and it is shown that this causes the CML properties. Gleevec inhibits this Bcr-Abl kinase by binding to the catalytic cleft between the two major structural lobes of the kinase domain of Bcr-Abl. As a result, these cells stop growing and even die by a process of apoptosis. Because the fusion gene product from Ph, Bcr-Abl tyrosine kinase, exists only in leukemia cells and not in normal cells, Gleevec kills cancer cells selectively. Gleevec was one of the first cancer treatments inhibiting a specific target molecule and is often cited as a paradigm for research in cancer therapeutics [2].

Biological Activity of Gleevec

IC₅₀ values for v-Abl, Bcr-Abl, and c-Abl protein kinases; 0.038, 0.025, and 0.025 μM, respectively [1]

Growth of cells with expression of Bcr-Abl (MO7p210); 1 μM [1].

References

1.

Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566
2.

Goldman JM, Melo JV (2003) Chronic myeloid leukemia–advances in biology and new approaches to treatment. N Engl J Med 349:1451–1464

Glucopiericidin A

Keywords: [Filopodia protrusion inhibitor] [Glucose transporter inhibitor]

Structure

	Glucopiericidin A
Molecular formula	C₃₁H₄₇NO₉
Molecular weight	577.71
Solubility	DMSO, ++; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

Glucopiericidin A was isolated as a new piericidin group antibiotic from a culture broth of Streptomyces pactum S48727 (FERM P-8117) [1]. Because glucopiericidin showed more potent inhibition of antibody formation in mouse spleen cell cultures and antimicrobial activities than piericidin A1 in vitro [1], it was suggested that D-glucose in glucopiericidin is important in modulating their physiological activities.

Biological Studies

Glucopiericidin A inhibited platelet-derived growth factor (PDGF)–induced activation of phospholipase γ1 (PLC-γ1) by reducing the tyrosine kinase activity of the PDGF receptor and PI turnover induced by PDGF [2].

Recently, in the course of screening microbial samples for a filopodia protrusion inhibitor, glucopiericidin A was reisolated [3]. Interestingly, glucopiericidin A alone did not inhibit filopodia protrusion, but synergistically inhibited protrusion with piericidin A, the mitochondria respiration inhibitor, suggesting that glucopiericidin A might be a glycolysis inhibitor. Furthermore, metabolomic analysis using capillary electrophoresis TOF-MS strongly suggested that glucopiericidin targets glucose transporters.

Biological Activity of Glucopiericidin

Inhibition of antibody formation: 0.1 ng/ml [1]

Inhibition of filopodia protrusion in combination with piericidin A (0.68 nM): 17 nM [3]

References

1.

Matsumoto M, Mogi K, Nagaoka K, Ishizeki S, Kawahara R, Nakashima T (1987) New piericidin glucosides, glucopiericidins A and B. J Antibiot (Tokyo) 40:149–156
2.

Ahn SC, Kim BY, Park CS, Lee HS, Suh PG, Ryu SH, Rho HM, Rhee JS, Mheen TI, Ahn JS (1995) Inhibition of PDGF-induced phosphoinositide-turnover by glucopiericidin A. Biochem Mol Biol Int 37:125–132
3.

Kitagawa M, Ikeda S, Tashiro E, Soga T, Imoto M (2010) Metabolomic identification of the target of the filopodia protrusion inhibitor glucopiericidin A. Chem Biol 17:989–998

Gossypol

Keywords: [Apoptosis inducer] [Bcl-2 family inhibitor] [BH3 mimetics]

Structure

	Gossypol
Molecular formula	C₃₀H₃₀O₈
Molecular weight	518.56
Solubility	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation/Chemical Studies

Gossypol was isolated from crude cottonseed oil [1]. Withers and Carruth found that gossypol is quickly oxidized in an alcoholic solution of sodium hydroxide, suggesting that gossypol is a toxic substance but its oxidation by an alcoholic alkali renders its toxicity [2]. Chemical reaction with free ε–amino groups of lysine in proteins has been reported [3].

Biological Studies

Gossypol shows antitumor activities in vivo [4] and in vitro [5]. Gossypol induced cell cycle arrest in G0/G1 phase by up-regulation of p21, and apoptosis by both down-regulation of antiapoptosis Bcl-2 family proteins and up-regulation of the proapoptosis Bcl-2 member in HT-29 human colon carcinoma cells [6]. Furthermore, gossypol inhibited the binding of the BH3 domain of Bak to Bcl-xL in a dose-dependent manner. Detailed NMR analyses revealed that gossypol binds on the BH3 binding pocket of Bcl-xL. These results strongly suggest that gossypol is a BH3 mimetic and functions as a Bcl-2/Bcl-xL antagonist [7].

Biological Activity of Gossypol

IC₅₀ for cell proliferation against several cancer cell lines: 5–35 μM [5]

IC₅₀ for BH3 peptide binding to Bcl-X_L and Bcl-2: 0.4 and 10 μM [6]

References

1.

Marchlewski L (1899) Gossypol, ein Bestandtheil der Baumwollsamen. J Prakt Chem 60:84–90
2.

Withers WA, Carruth FE (1915) Gossypol–a toxic substance in cottonseed. A preliminary note. Science 41:324
3.

Conkerton EJ, Frampton VL (1959) Reaction of gossypol with free epsilon-amino groups of lysine in proteins. Arch Biochem Biophys 81:130–134
4.

Vermel EM, Krugliak SA (1963) Antineoplastic activity of gossypol in experimental transplanted tumors. Vopr Onkol 21:39–43
5.

Tuszynski GP, Cossu G (1984) Differential cytotoxic effect of gossypol on human melanoma, colon carcinoma, and other tissue culture cell lines. Cancer Res 44:768–771
6.

Zhang M, Liu H, Guo R, Ling Y, Wu X, Li B, Roller PP, Wang S, Yang D (2003) Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells. Biochem Pharmacol 66:93–103
7.

Kitada S, Leone M, Sareth S, Zhai D, Reed JC, Pellecchia M (2003) Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. J Med Chem 46:4259–4264

GSK126, EPZ005687

Keywords: [Enhancer of zeste 2 (EZH2) inhibitor] [Methylation of histone H3 on lysine 27]

Structure

	GSK126	EPZ005687
Molecular formula	C₃₁H₃₈N₆O₂	C₃₂H₃₇N₅O₃
Molecular weight	526.67	539.68
Solubility	DMSO, ++; H₂O, ±; MeOH, ±	DMSO, ++; H₂O, ±; MeOH, ±

Discovery/Isolation/Chemical Studies

Methylation of histone H3 lysine 27 is catalyzed by polycomb repressive complex 2 (PRC2), containing the enzymatic subunit EZH2 or EZH1. GSK126 [1, 2] and EPZ005687 [3] were synthesized by optimization of the EZH2 inhibitor which was previously identified by a high-throughput biochemical screen with PRC2 protein.

Biological Studies

GSK126 and EPZ005687 are potent and selective inhibitors of PRC2 activity in a competitive manner against PRC2 substrate S-adenosylmethionine [1, 3].

GSK126 is more than 1000-fold more selective for EZH2 than 20 other human methyltransferases. Furthermore, this compound is a slow dissociation inhibitor from the activated enzyme and has a significantly longer residence time of inhibition on the activated form of EZH2 as compared to unactivated EZH2, suggesting that GSK126 may be beneficial in vivo and result in durable target inhibition after drug systemic clearance [4].

EPZ005687 shows ~50-fold selectivity for EZH2 over EZH1-containing PRC2. This compound decreased the methylation level of H3K27 in lymphoma cell line OCI-LY19 (wild-type EZH2) in a dose-dependent manner [3]. Similar results were obtained in EZH2 Tyr641 and Ala677 mutant lymphoma cell lines as well as cell lines of other cancer types. Furthermore, EPZ005687 decreases proliferation in mutant but not wild-type EZH2 lymphoma cells by inhibiting the cell cycle progression in G1 phase.

Biological Activity of GSK126 and EPZ004777: GSK126

Apparent K_i values for EZH2 and EZH1 in vitro: 0.5–3 and 89 nM, respectively [1]

IC₅₀ for EZH2 mutant DLBCL cell lines: 28–861 nM [1]

IC₅₀ for wild-type EZH2 cell line (HT cells): 516 nM [1]

EPZ004777

IC₅₀ for PRC2 in vitro: 54 ± 5 nM [3]

IC₅₀ for the level of methylated H3K27 in OCI-LY19 cells: 80 ± 30 nM [3]

References

1.

McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Della Pietra A, 3rd, Diaz E, LaFrance LV, Mellinger M, Duquenne C, Tian X, Kruger RG, McHugh CF, Brandt M, Miller WH, Dhanak D, Verma SK, Tummino PJ, Creasy CL (2012) EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492:108–112
2.

Diaz E, Machutta CA, Chen S, Jiang Y, Nixon C, Hofmann G, Key D, Sweitzer S, Patel M, Wu Z, Creasy CL, Kruger RG, LaFrance L, Verma SK, Pappalardi MB, Le B, Van Aller GS, McCabe MT, Tummino PJ, Pope AJ, Thrall SH, Schwartz B, Brandt M (2012) Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens. J Biomol Screen 17:1279–1292
3.

Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, Scott MP, Jin L, Smith JJ, Olhava EJ, Chesworth R, Moyer MP, Richon VM, Copeland RA, Keilhack H, Pollock RM, Kuntz KW (2012) A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 8:890–896
4.

Van Aller GS, Pappalardi MB, Ott HM, Diaz E, Brandt M, Schwartz BJ, Miller WH, Dhanak D, McCabe MT, Verma SK, Creasy CL, Tummino PJ, Kruger RG (2014) Long residence time inhibition of EZH2 in activated polycomb repressive complex 2. ACS Chem Biol 9:622–629

GSK-J4*

Keywords: [JMJD3/UTX inhibitor]

Structure

	GSK-J4
Molecular formula	C₂₄H₂₇N₅O₂
Molecular weight	417.50
Solubility	DMSO, +++; H₂O, ? ; MeOH, ?

Discovery/Isolation

GSK-J4 is the ethyl ester analog of GSK-J1, a potent and selective inhibitor of Jumonji domain–containing histone H3K27 demethylases including JMJD3 and UTX. GSK-J1 was identified by optimizing weakly active hit compounds discovered by high-throughput screening [1].

Biological Studies

GSK-J4 is a cell permeable prodrug which is rapidly hydrolyzed in cells by esterases to GSK-J1, a potent selective JMJD3/UTX inhibitor [1]. GSK-J4 inhibits TNF-α production in human primary macrophages accompanied by an increase in histone H3K27 trimethylation [1]. GSK-J4 reduces proliferation of T cells in acute lymphoblastic leukemia [2] and glioma cells harboring a histone K27M mutation in both in vitro and in vivo xenografts [3]. An inhibitory effect of GSK-J4 on reactivation of herpes simplex virus 1 in sensory neurons was also reported [4].

Biological Activity of GSK-J4

In vitro IC₅₀ for GSK-J1 against JMJD3: 60 nM [1]

Increase in histone H3K27 trimetylation: 10–50 μM [1]

IC₅₀ for proliferation of T cells in acute lymphoblastic leukemia: 2 μM [2]

IC₅₀ for proliferation of K27 M-expressing glioma cells: 1.3–3.0 2 μM [3]

References

1.

Kruidenier L, Chung CW, Cheng Z, Liddle J, Che K, Joberty G, Bantscheff M, Bountra C, Bridges A, Diallo H, Eberhard D, Hutchinson S, Jones E, Katso R, Leveridge M, Mander PK, Mosley J, Ramirez-Molina C, Rowland P, Schofield CJ, Sheppard RJ, Smith JE, Swales C, Tanner R, Thomas P, Tumber A, Drewes G, Oppermann U, Patel DJ, Lee K, Wilson DM (2012) A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488:404–408
2.

Ntziachristos P, Tsirigos A, Welstead GG, Trimarchi T, Bakogianni S, Xu L, Loizou E, Holmfeldt L, Strikoudis A, King B, Mullenders J, Becksfort J, Nedjic J, Paietta E, Tallman MS, Rowe JM, Tonon G, Satoh T, Kruidenier L, Prinjha R, Akira S, Van Vlierberghe P, Ferrando AA, Jaenisch R, Mullighan CG, Aifantis I (2014) Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia. Nature 514:513–517
3.

Hashizume R, Andor N, Ihara Y, Lerner R, Gan H, Chen X, Fang D, Huang X, Tom MW, Ngo V, Solomon D, Mueller S, Paris PL, Zhang Z, Petritsch C, Gupta N, Waldman TA, James CD (2014) Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat Med 20:1394–1396
4.

Messer HG, Jacobs D, Dhummakupt A, Bloom DC (2015) Inhibition of H3K27me3-specific histone demethylases JMJD3 and UTX blocks reactivation of herpes simplex virus 1 in trigeminal ganglion neurons. J Virol 89:3417–3420

Halichondrin B, Eribulin*

Keywords: [Antitumor] [Tubulin polymerization inhibitor]

Structure

	Halichondrin B	Eribulin
Molecular formula	C₆₀H₈₆O₁₉	C₄₀H₅₉NO₁₁
Molecular weight	1111.33	729.91
Solubility	DMSO, ++; H₂O, ±; MeOH, +++	DMSO, ++; H₂O, ++; MeOH, +++

Discovery/Isolation

Halichondrin B was isolated as an antitumor compound from the marine sponge Halichondria okadai Kadota [1].

Biological Studies

Halichondrin B showed potent antitumor activity in vitro and in vivo [2, 1]. Data generated in an NCI drug evaluation program (60 human cell line panel) suggested that halichondrin B is a antimitotic agent [2]. Halichondrin B inhibited the cell cycle progression in mitosis and the polymerization of purified tubulin in vitro. Halichondrin B inhibited vinblastine and dolastatin 15 binding to tubulin [2, 3].

Based on the structure of halichondrin B, eribulin was synthesized and has been used as an antitumor agent clinically [4].

Biological Activity of Halichondrin B

IC₅₀ for cytotoxicity against B16 melanoma in vitro: 0.093 ng/ml [1]

Antitumor activity against B16 melanoma in vivo: 2.5 μg/kg intraperitoneally, daily administration [1]

Apparent K_i for binding of vinblastine to tubulin: 5 μM, noncompetitive manner [2]

References

1.

Hirata Y, Uemura D (1986) Halichondrins – antitumor polyether macrolides from a marine sponge. Pure Appl Chem 58:701–710
2.

Bai RL, Paull KD, Herald CL, Malspeis L, Pettit GR, Hamel E (1991) Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin-based mechanism of action by analysis of differential cytotoxicity data. J Biol Chem 266:15882–15889
3.

Cruz-Monserrate Z, Mullaney JT, Harran PG, Pettit GR, Hamel E (2003) Dolastatin 15 binds in the vinca domain of tubulin as demonstrated by Hummel-Dreyer chromatography. Eur J Biochem 270:3822–3828
4.

Towle MJ, Salvato KA, Budrow J, Wels BF, Kuznetsov G, Aalfs KK, Welsh S, Zheng W, Seletsky BM, Palme MH, Habgood GJ, Singer LA, Dipietro LV, Wang Y, Chen JJ, Quincy DA, Davis A, Yoshimatsu K, Kishi Y, Yu MJ, Littlefield BA (2001) In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res 61:1013–1021

Hydrazinobenzoylcurcumin* (HBC, CTK7A)

Keywords: [Antitumor] [Ca²⁺/calmodulin ] [Histone acetyltransferase]

Structure

	HBC	CTK7A
Molecular formula	C₂₈H₂₄N₂O₆	C₂₈H₂₃N₂NaO₆
Molecular weight	484.51	506.49
Solubility	DMSO, +++; H₂O, ±; MeOH, +	DMSO, +++; H₂O, +; MeOH, +

Discovery/Isolation

HBC was synthesized as an antitumor compound from curcumin, a natural chemopreventive agent found in the rhizome of Curcuma longa [1]. CTK7A is a water-soluble sodium salt of HBC [2].

Biological Studies

HBC induced prolonged phosphorylation of ERK1/2 and activated p21WAF1 expression, resulting in the induction of G0/G1 cell cycle arrest in HCT15 colon cancer cells [3]. HBC also blocked angiogenesis by inhibiting the expression of HIF1α at the translational level [4].

The target molecule was identified as Ca²⁺/calmodulin by phage-display biopanning [3]. More recently, it was reported that CTK7A, a water-soluble derivative of HBC, is a histone acetyltransferase inhibitor [2]. Therefore, antiproliferative activity of HBC/CTK7A might be dependent on the inhibition of both Ca²⁺/calmodulin and histone acetlytransferase.

Biological Activity of HBC and CTK7A: HBC

IC₅₀ for the proliferation of HCT15: 20 μM [3]

IC₅₀ for the proliferation of HUVECs: 20 μM [4]

Inhibition of HIF1α expression: 10 μM [4]

CTK7A

K_i values for acetyl-CoA and core histone in histone acetyltransfease assay: 13.8 and 18.6 μM, respectively [2]

Inhibition of wound-healing, induction of polyploidal cells, and senescence-like growth arrest: 100 μM [2]

References

1.

Shim JS, Kim DH, Jung HJ, Kim JH, Lim D, Lee SK, Kim KW, Ahn JW, Yoo JS, Rho JR, Shin J, Kwon HJ (2002) Hydrazinocurcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation. Bioorg Med Chem 10:2987–2992
2.

Arif M, Vedamurthy BM, Choudhari R, Ostwal YB, Mantelingu K, Kodaganur GS, Kundu TK (2010) Nitric oxide-mediated histone hyperacetylation in oral cancer: target for a water-soluble HAT inhibitor, CTK7A. Chem Biol 17:903–913
3.

Shim JS, Lee J, Park HJ, Park SJ, Kwon HJ (2004) A new curcumin derivative, HBC, interferes with the cell cycle progression of colon cancer cells via antagonization of the Ca²⁺/calmodulin function. Chem Biol 11:1455–1463
4.

Jung HJ, Kim JH, Shim JS, Kwon HJ (2010) A novel Ca²⁺/calmodulin antagonist HBC inhibits angiogenesis and down-regulates hypoxia-inducible factor. J Biol Chem 285:25867–25874

Iejimalides A–D

Keywords: [Antitumor] [V-ATPase inhibitor]

Structure

	Iejimalide A	Iejimalide B
Molecular formula	C₄₀H₅₈N₂O₇	C₄₁H₆₀N₂O₇
Molecular weight	678.90	692.94
Solubility	DMSO, ++; H₂O, ±; MeOH, +	DMSO, ++; H₂O, ±; MeOH, +

	Iejimalide C	Iejimalide D
Molecular formula	C₄₀H₅₇N₂NaO₁₀S	C₄₁H₅₉N₂NaO₁₀S
Molecular weight	780.95	794.98
Solubility	DMSO, ++; H₂O, +; MeOH, +	DMSO, ++; H₂O, +; MeOH, +

Discovery/Isolation/Chemical Studies

Iejimalides A–D, isolated from a marine tunicate Eudistoma cf. rigida, exhibit potent cytotoxic activity in vitro [1, 2] and antitumor activity in vivo [3]. The absolute structures were identified on the basis of detailed analysis of NMR data and chemical means [3]. Total synthesis was reported by Fürstner et al. [4, 5].

Biological Studies

Iejimalides A–D exerted antitumor and antiosteoporotic activity. The phenotypic assay revealed that these compounds inhibit vacuolar H⁺-ATPases (V-ATPases) by binding with the c subunit in V_o domain of V-ATPases [6, 7]. It was also reported that iejimalide B and its analogs showed cytotoxicity via disorganization of the actin cytoskeleton [5]. However, different from actin inhibitors latrunculin A and jasplakinolide which inhibit cytokinesis, iejimalide B induces cell cycle arrest G0/G1 followed by S-phase arrest [8]. Furthermore, it has been confirmed that iejimalides inhibit V-ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome-initiated cell death process [9].

Biological Activity of Iejimalides A–D

IC₅₀ values for cytotoxicity of iejimalides A and B against L1210: 62 and 32 ng/ml, respectively [1]

IC₅₀ values for cytotoxicity of iejimalides C and D against L1210: 10 and 0.58 μg/ml, respectively [2]

IC₅₀ values for cytotoxicity of iejimalides A and B against TRAP-positive multinucleated cells: 5 and 6 nM, respectively [6]

K_i values for iejimalides A and B against yeast V-ATPase in vitro: 6.7 and 8.7 nM, respectively [6].

References

1.

Kobayashi J, Cheng J-f, Ohta T, Nakamura H, Nozoe S, Hirata Y, Ohizumi Y, Sasaki T (1988) Iejimalides A and B, novel 24-membered macrolides with potent antileukemic activity from the Okinawan tunicate Eudistoma cf. rigida. J Org Chem 53:6147–6150
2.

Kikuchi Y, Ishibashi M, Sasaki T, Kobayashi J (1991) Iejimalides C and D, new antineoplastic 24-membered macrolide sulfates from the Okinawan marine tunicate Eudistoma cf. rigida. Tetrahedron Lett 32:797–798
3.

Nozawa K, Tsuda M, Ishiyama H, Sasaki T, Tsuruo T, Kobayashi J (2006) Absolute stereochemistry and antitumor activity of iejimalides. Bioorg Med Chem 14:1063–1067
4.

Fürstner A, Nevado C, Tremblay M, Chevrier C, Teplý F, Aïssa C, Waser M (2006) Total synthesis of iejimalide B. Angew Chem Int Ed Eng 45:5837–5842
5.

Fürstner A, Nevado C, Waser M, Tremblay M, Chevrier C, Teplý F, Aïssa C, Moulin E, Müller O (2007) Total synthesis of iejimalide A-D and assessment of the remarkable actin-depolymerizing capacity of these polyene macrolides. J Am Chem Soc 129:9150–9161
6.

Kazami S, Muroi M, Kawatani M, Kubota T, Usui T, Kobayashi J, Osada H (2006) Iejimalides show anti-osteoclast activity via V-ATPase inhibition. Biosci Biotechnol Biochem 70:1364–1370
7.

Kazami S, Takaine M, Itoh H, Kubota T, Kobayashi J, and Usui T. (2014) Iejimalide C is a potent V-ATPase inhibitor, and induces actin disorganization. Biol Pharm Bull, 37: 1944–1947
8.

Wang WW, McHenry P, Jeffrey R, Schweitzer D, Helquist P, Tenniswood M (2008) Effects of Iejimalide B, a marine macrolide, on growth and apoptosis in prostate cancer cell lines. J Cell Biochem 105:998–1007
9.

McHenry P, Wang WW, Devitt E, Kluesner N, Davisson VJ, McKee E, Schweitzer D, Helquist P, Tenniswood M (2010) Iejimalides A and B inhibit lysosomal vacuolar H⁺ −ATPase (V-ATPase) activity and induce S-phase arrest and apoptosis in MCF-7 cells. J Cell Biochem 109:634–642

(+)-JQ1*

Keywords: [Bromodomain] [Signal transduction]

Structure

	(+)-JQ1
Molecular formula	C₂₃H₂₅ClN₄O₂S
Molecular weight	456.99
Solubility	DMSO, ++; H₂O, −; MeOH, ++

Discovery/Isolation/Chemical Studies

JQ1 was synthesized based on the patent that simple theinodiazepines possessed binding activity for BRD4, one of the BET (bromodomain and extraterminal) family, and the structure–activity relationship (SAR) data derived from molecular modeling of candidate ligands within the binding pocket of the apo crystal structure of the bromodomain of BRD4 [1].

Biological Studies

JQ1 increased the thermal stability of all bromodomains of the BET family in an (+)-enantiomer-specific manner [1]. Furthermore, (+)-JQ1 strongly inhibited the binding of a tetraacetlyated histone H4 peptide to BRD4. The determined cocrystal structure showed that (+)-JQ1 bound directly to the acetyl-lysine binding site of BRD4. Fluorescence recovery after photobleaching experiments using GFP-BRD4 showed that (+)-JQ1 displaced BRD4 from nuclear chromatin in cells.

(+)-JQ1 induced the differentiation and growth arrest of NMC (nuclear protein in testis (NUT) midline carcinoma) cells expressing BRD4-NUT fusion oncoprotein, and showed antitumor efficacy in an NMC xenograft model [1].

BET inhibition by JQ1 down-regulates MYC transcription, followed by genome-wide down-regulation of Myc-dependent target genes. In experimental models of multiple myeloma, an Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell cycle arrest and cellular senescence [2].

JQ1 reduced murine leukemia virus (MLV) DNA integration into the transcription start site of the host genome by inhibiting the interactions of BET proteins with modified histone sites. Therefore, JQ1 or the other BET inhibitors will facilitate the development of safer MLV-based vectors for human gene therapy by minimizing the risks of insertional activation of protooncogenes [3].

Biological Activity of (+)-JQ1

IC₅₀ values for binding of a tetraacetylated histone H4 peptide to BRD4 for the first and second bromodomain: 77 nM and 33 nM, respectively [1]

Induction of a differentiation phenotype in a 797 NMC cell line: 500 nM (48 hours) [1]

G1 arrest of 797 NMC cells: 250 nM (48-hours) [1]

Antitumor activity in NMC 797 xenograft: 50 mg/kg daily intraperitoneal injection [1]

Down-regulation of MYC and cancer-related gene expression in MM cells: 500 nM [2]

IC₅₀ for TGF-β1-mediated secretion of IL-6: 3.2 nM for JQ1 and 220.8 nM for iBET [4]

References

1.

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue N, French CA, Wiest O, Kung AL, Knapp S, Bradner JE (2010) Selective inhibition of BET bromodomains. Nature 468:1067–1073
2.

Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, Kastritis E, Gilpatrick T, Paranal RM, Qi J, Chesi M, Schinzel AC, McKeown MR, Heffernan TP, Vakoc CR, Bergsagel PL, Ghobrial IM, Richardson PG, Young RA, Hahn WC, Anderson KC, Kung AL, Bradner JE, Mitsiades CS (2011) BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146: 904–917
3.

Sharma A, Larue RC, Plumb MR, Malani N, Male F, Slaughter A, Kessl JJ, Shkriabai N, Coward E, Aiyer SS, Green PL, Wu L, Roth MJ, Bushman FD, Kvaratskhelia M (2013) BET proteins promote efficient murine leukemia virus integration at transcription start sites. Proc Natl Acad Sci U S A 110:12036–12041
4.

Tang X, Peng R, Ren Y, Apparsundaram S, Deguzman J, Bauer CM, Hoffman AF, Hamilton S, Liang Z, Zeng H, Fuentes ME, Demartino JA, Kitson C, Stevenson CS, Budd DC (2013) BET bromodomain proteins mediate downstream signaling events following growth factor stimulation in human lung fibroblasts and are involved in bleomycin-induced pulmonary fibrosis. Mol Pharmacol 83: 283–293

KU0058684, KU0058948

Keywords: [Poly (ADP-ribose) polymerase (PARP) inhibitor]

Structure

	KU0058684	KU0058948
Molecular formula	C₁₉H₁₄FN₃O₃	C₂₁H₂₁FN₄O₂
Molecular weight	351.34	380.42
Solubility	DMSO, +++; H₂O, −; MeOH, ++	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation

Several 4-aryl-2H-phthalazinones were identified as poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors by high-throughput screening of a chemical library using a FlashPlate scintillation proximity assay. Based on these structures, both KU0058684 and KU0058948 were synthesized as specific and very potent PARP inhibitors [1, 2].

Biological Studies

KU0058684 and KU0058948 potently and irreversibly inhibit the cell proliferation of BRCA1- or BRCA2-deficient cells compared with heterogygous mutant or wild-type cells [3]. These compounds induce chromosomal instability, cell cycle arrest, and subsequent apoptosis. It is known that PARPs are required for efficient repair of DNA single-strand breaks and both BRCA1 and BRCA2 is involved in RAD51-dependent homologous recombination. Therefore, PARP inhibition results in the lack of a DNA repair pathway in BRCA1- or BRCA2-deficient cells, and would be a specific and less toxic therapy for cancer.

Biological Activity of KU0058684 and KU0058948

IC₅₀ values for KU0058684 against PAR activity of PARP-1, -2, -3, and tankylase: 3.2, 1.5, 30, and 1600 nM, respectively [3]

IC₅₀ values for KU0058948 against PAR activity of PARP-1, -2, -3, and tankylase: 3.4, 1.5, 40, and >10,000 nM, respectively [3]

IC₅₀ values for KU0058684 on cell proliferation of BRCA1-, BRCA2-deficient and wild-type cells: 35, 15, and ~2000 nM, respectively [3]

IC₅₀ values for KU0058684 and KU0058948 on cell proliferation of CAPAN-1. a BRCA2 mutant cell line: 3.2, and 3.4 nM, respectively [4]

References

1.

Loh VM, Jr., Cockcroft XL, Dillon KJ, Dixon L, Drzewiecki J, Eversley PJ, Gomez S, Hoare J, Kerrigan F, Matthews IT, Menear KA, Martin NM, Newton RF, Paul J, Smith GC, Vile J, Whittle AJ (2005) Phthalazinones. Part 1: the design and synthesis of a novel series of potent inhibitors of poly(ADP-ribose)polymerase. Bioorg Med Chem Lett 15:2235–2238
2.

Cockcroft XL, Dillon KJ, Dixon L, Drzewiecki J, Kerrigan F, Loh VM, Jr., Martin NM, Menear KA, Smith GC (2006) Phthalazinones 2: optimisation and synthesis of novel potent inhibitors of poly(ADP-ribose)polymerase. Bioorg Med Chem Lett 16:1040–1044
3.

Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A (2005) Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434:917–921
4.

McCabe N, Lord CJ, Tutt AN, Martin NM, Smith GC, Ashworth A (2005) BRCA2-deficient CAPAN-1 cells are extremely sensitive to the inhibition of Poly (ADP-Ribose) polymerase: an issue of potency. Cancer Biol Ther 4:934–936

Lactacystin

Keywords: [Neurite outgrowth inducer] [Proteasome inhibitor]

Structure

	Lactacystin
Molecular formula	C₁₅H₂₄N₂O₇S
Molecular weight	376.43
Solubility	DMSO, ++; H₂O, −, MeOH, ++

Discovery/Isolation

Lactacystin was isolated as the differentiation inducer of the mouse neuroblastoma Neuro 2A from the culture broth of a Streptomyces sp. OM-6519 [1, 2].

Biological Studies

Lactacystin induced neurite-like structure generation, increased the intracellular cAMP level transiently [2], and arrested cell cycle progression at both G0/G1 and G2 phases in Neuro 2A cells [3, 4].

[³H]-labeled lactacystin covalently modified the highly conserved amino-terminal threonine of the mammalian proteasome subunit X, a component of 20S proteasome [5]. The ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome, strongly suggesting that 20S proteasome is a lactacystin-specific cellular target.

Because the ubiquitin–proteasome system plays important roles in several cellular processes including down-regulation of signal transduction by degradation of ligand–receptor complex, denaturated proteins, and apoptosis, there are many reports investigating the cellular function using lactacystin [6].

Biological Activity of Lactacystin

Neurite outgrowth in Neuro 2A: 1.3 μM [2]

Inhibition of cell cycle progression at both G0/G1 and G2 phases: 1.3 μM [4]

Accumulation of ubiquitinated proteins and apoptosis induction: 5 μM [6]

References

1.

Omura S, Matsuzaki K, Fujimoto T, Kosuge K, Furuya T, Fujita S, Nakagawa A (1991) Structure of lactacystin, a new microbial metabolite which induces differentiation of neuroblastoma cells. J Antibiot (Tokyo) 44:117–118
2.

Omura S, Fujimoto T, Otoguro K, Matsuzaki K, Moriguchi R, Tanaka H, Sasaki Y (1991) Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J Antibiot (Tokyo) 44:113–116
3.

Fenteany G, Standaert RF, Reichard GA, Corey EJ, Schreiber SL (1994) A β-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line. Proc Natl Acad Sci U S A 91:3358–3362
4.

Katagiri M, Hayashi M, Matsuzaki K, Tanaka H, Omura S (1995) The neuritogenesis inducer lactacystin arrests cell cycle at both G0/G1 and G2 phases in Neuro2A cells. J Antibiot (Tokyo) 48:344–346
5.

Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL (1995) Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268:726–731
6.

Soldatenkov VA, Dritschilo A (1997) Apoptosis of Ewing’s sarcoma cells is accompanied by accumulation of ubiquitinated proteins. Cancer Res 57:3881–3885

Leptomycin B

Keywords: [Antitumor] [Nuclear export inhibitor]

	Leptomycin B
Molecular formula	C₃₃H₄₈O₆
Molecular weight	540.74
Solubility	DMSO, +; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

Leptomycin B was isolated from Streptomyces sp. ATS1287 as a strong antifungal antibiotic, which causes inhibition of cell division, elongated cells with morphologically altered nuclei, and several cell plates in Schizosaccharomyces pombe [1, 2]. There are several related compounds, anguinomycins A–D [3, 4], kazusamycin A and B [5, 6], leptolstatin [7, 8], and leptofuranins A–D [9, 10]. All these compounds have the structure characteristics of an unsaturated, branched-chain fatty acid with a terminal δ-lactone ring.

Biological Studies

Leptomycin B reversibly blocked cell cycle progression at both G1 and G2 phases in mammalian cells and S. pombe [11]. Interestingly, after removal of leptomycin B, G2-arrested cells started DNA replication without passage through mitosis, then proliferative tetraploid cells were produced. The crm1⁺ gene, which was previously identified as the gene required for maintenance of higher-order chromosome structures, was identified as the molecular target of leptomycin B by genetic analyses using a leptomycin B–specific resistant mutant of S. pombe [12]. At first, the function of crm1⁺ was unknown, but the discovery of leptomycin B in the course of screening of the compounds inhibiting the nuclear export of HIV-1 protein Rev revealed that leptomycin B is a potent and specific inhibitor of protein export from nuclei. Leptomycin B covalently binds with CRM1 and inhibits the binding of CRM1 to proteins containing nuclear export signal (NES) [13–17]. Mutation analysis of leptomycin B strongly suggests that leptomycin B binds covalently via its α,β-unsaturated δ-lactone to the sulfhydryl group of Cys529 in the central conserved region. Now leptomycin B has been widely used for investigation of biological function mediated by nuclear protein export.

The other leptomycins also shows antitumor activity, cell cycle proliferation inhibition, and nuclear export inhibition [3–6, 8, 10].

Biological Activity of Leptomycin B

Minimum inhibitory concentrations against S. pombe and Mucor sp.: 12–250 ng/ml [2]

Reversible cell cycle arrest at G1 and G2 phases: 1–200 ng/ml [11]

Inhibition of nuclear export in vitro: 40 nM [13]

References

1.

Hamamoto T, Gunji S, Tsuji H, Beppu T (1983) Leptomycins A and B, new antifungal antibiotics. I. Taxonomy of the producing strain and their fermentation, purification and characterization. J Antibiot (Tokyo) 36:639–645
2.

Hamamoto T, Seto H, Beppu T (1983) Leptomycins A and B, new antifungal antibiotics II Structure elucidation. J Antibiot (Tokyo) 36:646–650
3.

Hayakawa Y, Adachi K, Komeshima N (1987) New antitumor antibiotics, anguinomycins A and B. J Antibiot (Tokyo) 40:1349–1352
4.

Hayakawa Y, Sohda KY, Shin-Ya K, Hidaka T, Seto H (1995) Anguinomycins C and D, new antitumor antibiotics with selective cytotoxicity against transformed cells. J Antibiot (Tokyo) 48:954–961
5.

Umezawa I, Komiyama K, Oka H, Okada K, Tomisaka S, Miyano T, Takano S (1984) A new antitumor antibiotic, kazusamycin. J Antibiot (Tokyo) 37:706–711
6.

Funaishi K, Kawamura K, Sugiura Y, Nakahori N, Yoshida E, Okanishi M, Umezawa I, Funayama S, Komiyama K (1987) Kazusamycin B, a novel antitumor antibiotic. J Antibiot (Tokyo) 40:778–785
7.

Abe K, Yoshida M, Horinouchi S, Beppu T (1993) Leptolstatin from Streptomyces sp. SAM1595, a new gap phase-specific inhibitor of the mammalian cell cycle. I. Screening, taxonomy, purification and biological activities. J Antibiot (Tokyo) 46:728–734
8.

Abe K, Yoshida M, Naoki H, Horinouchi S, Beppu T (1993) Leptolstatin from Streptomyces sp. SAM1595, a new gap phase-specific inhibitor of the mammalian cell cycle. II. Physico-chemical properties and structure. J Antibiot (Tokyo) 46:735–740
9.

Hayakawa Y, Sohda K, Seto H (1996) Studies on new antitumor antibiotics, leptofuranins A, B, C and D II Physiocochemical properties and structure elucidation. J Antibiot (Tokyo) 49:980–984
10.

Hayakawa Y, Sohda K, Furihata K, Kuzuyama T, Shin-ya K, Seto H (1996) Studies on new antitumor antibiotics, leptofuranins A, B, C and D.I. Taxonomy, fermentation, isolation and biological activities. J Antibiot (Tokyo) 49:974–979
11.

Yoshida M, Nishikawa M, Nishi K, Abe K, Horinouchi S, Beppu T (1990) Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells. Exp Cell Res 187:150–156
12.

Nishi K, Yoshida M, Fujiwara D, Nishikawa M, Horinouchi S, Beppu T (1994) Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J Biol Chem 269:6320–6324
13.

Fornerod M, Ohno M, Yoshida M, Mattaj IW (1997) CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051–1060
14.

Fukuda M, Asano S, Nakamura T, Adachi M, Yoshida M, Yanagida M, Nishida E (1997) CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390:308–311
15.

Kudo N, Khochbin S, Nishi K, Kitano K, Yanagida M, Yoshida M, Horinouchi S (1997) Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins. J Biol Chem 272:29742–29751
16.

Ossareh-Nazari B, Bachelerie F, Dargemont C (1997) Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science 278:141–144
17.

Watanabe M, Fukuda M, Yoshida M, Yanagida M, Nishida E (1999) Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast. Genes Cells 4:291–297

Locostatin* (UIC-1005)

Keywords: [Antitumor] [Protein-protein interaction inhibitor] [RKIP inhibitor]

Structure

	Locostatin
Molecular formula	C₁₄H₁₅NO₃
Molecular weight	245.27
Solubility	DMSO, +; H₂O,-; MeOH, +++

Discovery/Isolation/Chemical Studies

Locostatin (former name: UIC-1005) was a 3,4-disubstituted oxazolidinone identified by a high-throughput assay for screening chemical libraries for compounds that affect cell sheet migration during wound closure in epithelial cell monolayers [1]. This compound bears an electrophilic α,β-unsaturated N-acyl group required for activity.

Biological Studies

Locostatin inhibited not only cell sheet migration during wound closure but also cell growth of epithelial cells [1]. Zhu et al. found that Raf kinase inhibitor protein (RKIP) is the relevant cellular target of locostatin using a [³H]-labeled compound [2]. RKIP is an endogenous inhibitor of cell signaling by binding with multiple kinases, and locostatin covalently binds with RKIP and abrogates the binding of RKIP to and inhibition of kinase activity of Raf-1 kinase [2]. Locostatin also inhibits the binding of RKIP to G-protein coupled receptor kinase 2 (GRK2), but not to IKKα and TAK1, implying that there are at least two regions of RKIP interacting with different proteins [3]. The binding site of locostatin was identified as the residue alkylated by locostatin as His86, a highly conserved residue in RKIP’s ligand binding pocket [3]. A detailed binding process between RKIP and locostatin was proposed [4].

Overexpression of RKIP converts epithelial cells into a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin, suggesting that RKIP is therefore the compound’s valid target and a key regulator of cell motility [2].

Biological Activity of Locostatin

IC₅₀ for wound closure in MDCK cells: 17.9 μM [2]

References

1.

Mc Henry KT, Ankala SV, Ghosh AK, Fenteany G (2002) A non-antibacterial oxazolidinone derivative that inhibits epithelial cell sheet migration. ChemBioChem 3:1105–1111
2.

Zhu S, Mc Henry KT, Lane WS, Fenteany G (2005) A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration. Chem Biol 12:981–991
3.

Beshir AB, Argueta CE, Menikarachchi LC, Gascon JA, Fenteany G (2011) Locostatin disrupts association of raf kinase inhibitor protein with binding proteins by modifying a conserved histidine residue in the ligand-binding pocket. For Immunopathol Dis Therap 2:47–58
4.

Rudnitskaya AN, Eddy NA, Fenteany G, Gascon JA (2012) Recognition and reactivity in the binding between Raf kinase inhibitor protein and its small-molecule inhibitor locostatin. J Phys Chem B 116:10176–10181

Miuraenamide A

Keywords: [Actin stabilizer] [NADH oxidase inhibitor]

Structure

	Miuraenamide A
Molecular formula	C₃₄H₄₂BrN₃O₇
Molecular weight	684.63
Solubility	DMSO, ? ; H₂O, ? ; MeOH, ++

Discovery/Isolation

Miuraenamide A was produced by a slightly halophilic myxobacterial strain, SMH-27-4, which was a new myxobacterium genus isolated from nearshore soil [1].

Biological Studies

Miuraenamide A exhibited potent and selective inhibition against a phytopathogenic microorganism, Phytophthora sp., and moderate inhibition against some fungi and yeasts, but was ineffective against bacteria [1]. This compound inhibited NADH oxidase, suggesting the electron transfer system of the mitochondrial respiratory chain as the cellular target.

By cell morphological profiling of a natural product library using automated high-content image analysis, it was found that miuraenamide A induces morphological changes in HeLa cells similar to those induced by known actin-stabilizing compounds [2]. Miuraenamide A stabilized actin filaments in vitro, suggesting that this compound is an actin filament stabilizer.

Biological Activity of Miuraenamide A

IC₅₀ for NADH oxidase: 50 μM [1]

Stabilization of actin in HeLa cell: 100 nM [2]

Stabilization of actin filament in vitro: 10 nM [2]

References

1.

Iizuka T, Fudou R, Jojima Y, Ogawa S, Yamanaka S, Inukai Y, Ojika M (2006) Miuraenamides A and B, novel antimicrobial cyclic depsipeptides from a new slightly halophilic myxobacterium: taxonomy, production, and biological properties. J Antibiot (Tokyo) 59:385–391
2.

Sumiya E, Shimogawa H, Sasaki H, Tsutsumi M, Yoshita K, Ojika M, Suenaga K, Uesugi M (2011) Cell-morphology profiling of a natural product library identifies bisebromoamide and miuraenamide A as actin filament stabilizers. ACS Chem Biol 6:425–431

MJE3*

Keywords: [Antitumor] [Phosphoglycerate mutase 1 inhibitor]

Structure

	MJE3
Molecular formula	C₃₅H₄₀N₂O₇
Molecular weight	600.70
Solubility	DMSO, ++; H₂O, ? ; MeOH, ?

Discovery/Isolation/Chemical Studies

MJE3 was found to block proliferation of invasive human breast cancer cell line MDA-MB-231 from a ~50-member probe library [1]. An analog of MJE3 in which the spiroepoxide was replaced by an exocyclic alkene did not exhibit antiproliferative effects, indicating that the reactivity of MJE3 was essential for biological activity.

Biological Studies

To identify the molecular target of MJE3, Evans et al. compared the in situ proteome reactivity profiles of library members, and found that MJE3 covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) [1]. Furthermore, treatment with MJE3, but not other derivatives, resulted in the inhibition of PGAM1 activity in situ. Tandem orthogonal proteolysis analysis suggested that MJE3 is hydrolyzed in situ to produce an acid product that covalently modifies the PGAM1 active site, resulting in enzyme inhibition. The precise binding site was determined as Lys100, a conserved active-site residue [2]. Structural features of MJE3 important for PGAM1 labeling have also been reported.

Biological Activity of MJE3

IC₅₀ for cell growth: 19 μM [1]

IC₅₀ for PGAM1 in situ: 33 μM [1]

References

1.

Evans MJ, Saghatelian A, Sorensen EJ, Cravatt BF (2005) Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat Biotechnol 23:1303–1307
2.

Evans MJ, Morris GM, Wu J, Olson AJ, Sorensen EJ, Cravatt BF (2007) Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxides. Mol BioSyst 3:495–506

MKT-077*

Keywords: [Antitumor] [HSP70 inhibitor] [Telomerase inhibitor] [F-actin binder]

Structure

	MKT-077
Molecular formula	C₂₁H₂₂ClN₃OS₂
Molecular weight	432.00
Solubility	DMSO, +++; H₂O, +++; MeOH, +++

Discovery/Isolation/Chemical Studies

Rhodacyanine dyes form one group of delocalized lipophilic cations which are accumulated by mitochondria in response to plasma and mitochondrial membrane potentials. MKT-077 was synthesized as a rhodacyanine dye exhibiting marked anticarcinoma activity with relatively low toxicity [1]. This compound is a highly water-soluble (>200 mg/ml) lipophilic compound.

Biological Studies

MKT-077 showed antitumor activity in situ and in vivo, and localization in the mitochondria selectively [2]. This compound disorganized the structure and function of mitochondria in a carcinoma-specific manner [3]. At a pharmacologically toxic dose, MKT-077 significantly but reversibly decreased respiratory rates in rat liver mitochondria in vivo [4].

Two MKT-077 binding proteins were purified by affinity chromatography, actin, and Hsc70 [5, 6]. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and suppresses ras transformation by blocking membrane ruffling [6]. MKT-077 also binds to mortalin, an Hsp70 family member, and abrogates the interaction with p53. This inhibition of protein–protein interaction (PPI) results in transcriptional activation of p53 and apoptosis induction in cancer cells [7]. Furthermore, the inhibition of mortalin by MKT-077 induced cell senescence in human tumor cell lines [8]. The MKT-077 binding site has been identified as the nucleotide binding domain of mortalin by a MKT-077 pull-down assay [7], NMR analyses [9], and crystallography [10]. This allosteric binding causes the tertiary structural changes of mortalin and inactivates its chaperone function [8].

In vitro telomerase inhibitory activity of MKT-077 was also reported, but there was no detectable effect on telomerase activity in vivo [11, 12].

Biological Activity of MKT-077

IC₅₀ for several cancer cells: 0.15–0.5 μg/ml [2]

Abnormalities in the mitochondria structure in CRK1420 cells: 2 μg/ml [3]

References

1.

Kawakami M, Koya K, Ukai T, Tatsuta N, Ikegawa A, Ogawa K, Shishido T, Chen LB (1998) Structure-activity of novel rhodacyanine dyes as antitumor agents. J Med Chem 41:130–142
2.

Koya K, Li Y, Wang H, Ukai T, Tatsuta N, Kawakami M, Shishido, Chen LB (1996) MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation. Cancer Res 56:538–543
3.

Modica-Napolitano JS, Koya K, Weisberg E, Brunelli BT, Li Y, Chen LB (1996) Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077. Cancer Res 56:544–550
4.

Weisberg EL, Koya K, Modica-Napolitano J, Li Y, Chen LB (1996) In vivo administration of MKT-077 causes partial yet reversible impairment of mitochondrial function. Cancer Res 56:551–555
5.

Maruta H, Tikoo A, Shakri R, Shishido T (1999) The anti-RAS cancer drug MKT-077 is an F-actin cross-linker. Ann N Y Acad Sci 886:283–284
6.

Tikoo A, Shakri R, Connolly L, Hirokawa Y, Shishido T, Bowers B, Ye LH, Kohama K, Simpson RJ, Maruta H (2000) Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077. Cancer J 6:162–168
7.

Wadhwa R, Sugihara T, Yoshida A, Nomura H, Reddel RR, Simpson R, Maruta H, Kaul SC (2000) Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function. Cancer Res 60:6818–6821
8.

Deocaris CC, Widodo N, Shrestha BG, Kaur K, Ohtaka M, Yamasaki K, Kaul SC, Wadhwa R (2007) Mortalin sensitizes human cancer cells to MKT-077-induced senescence. Cancer Lett 252:259–269
9.

Rousaki A, Miyata Y, Jinwal UK, Dickey CA, Gestwicki JE, Zuiderweg ER (2011) Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones. J Mol Biol 411:614–632
10.

Amick J, Schlanger SE, Wachnowsky C, Moseng MA, Emerson CC, Dare M, Luo WI, Ithychanda SS, Nix JC, Cowan JA, Page RC, Misra S (2014) Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone. Protein Sci 23:833–842
11.

Naasani I, Seimiya H, Yamori T, Tsuruo T (1999) FJ5002: a potent telomerase inhibitor identified by exploiting the disease-oriented screening program with COMPARE analysis. Cancer Res 59:4004–4011
12.

Wadhwa R, Colgin L, Yaguchi T, Taira K, Reddel RR, Kaul SC (2002) Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo. Cancer Res 62:4434–4438

Myriocin/ISP-1, FTY720*

Keywords: [Immunosuppressant] [Serine palmitoyltransferase inhibitor] [Sphingolipid metabolism]

Structure

	Myriosin	FTY720
Molecular formula	C₂₁H₃₉NO₆	C₁₉H₃₃NO₂
Molecular weight	401.54	307.47
Solubility	DMSO, +++; H₂O, −; MeOH, ±	DMSO, ++; H₂O, ? ; MeOH, ?

Discovery/Isolation

Myriocin was originally isolated as an antifungal compound from Myriococcum albomyces [1], and reisolated as a potent immunosuppressant [2]. FTY720 is a synthetic immunosuppressant led by modification of ISP-I, and displayed potent immunosuppressive activity both in vitro and in vivo [3].

Biological Studies

Myriocin is structurally related to sphingosine, and inhibited serine palmitoyltransferase (SPT) which catalyzes the first step of sphingolipid biosynthesis [4]. The growth inhibition of CTLL-2 cells induced by myriocin was completely abolished by the addition of sphingosines or sphingosine-1-phosphate, but not by sphingomyelin or glycosphingolipids, suggesting that myriocin suppresses T-cell proliferation by the modulation of sphinogolipid metabolism. The myriocin binding proteins were purified by an affinity purification method and identified to be LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis [5]. It has been revealed that SPT consists of three proteins, LCB1/TSC2, LCB2/TSC1, and TSC3 [6–8]; therefore, these results strongly suggest that myriocin is a direct SPT inhibitor. Myriocin has been used not only for the effect of de novo synthesis of ceramide but also for the biological function of several sphingosine-regulated processes.

Compared with myriocin, FTY720 does not inhibit SPT [9]. The structure similarity of FTY720 with sphingosine led to the discovery that FTY720 is phosphorylated by sphingosine kinases and that phosphorylated FTY720 functions as an agonist at sphingosine-1-phosphate (S1P) receptors [10]. Furthermore, phosphorylated FTY720 induced the accumulation of lymphocytes at lymphatic endothelial barriers [11]. Because egress of lymphocytes from lymph nodes requires lymphocytic S1P₁ receptors [12], these results suggest that the inhibition of lymphocyte recirculation by activation of S1P receptors results in therapeutically useful immunosuppression.

Biological Activity of Myriocin

MIC for Candida albicans: 0.32–25 μg/ml [1]

IC₅₀ for mouse allogenic mixed lymphocyte reaction: 10–18 nM [2]

IC₅₀ for CTLL-2 cell growth: 15 nM [4]

IC₅₀ for SPT: 0.28 nM [4]

Biological Activity of FTY720

IC₅₀ for mouse allogenic mixed lymphocyte reaction: 6.1 nM [3]

EC₅₀ for agonist activity against S1P₁, S1P₄, and S1P₅ receptors: 0.3–0.6 nM [10]

Lymphopenia in blood and thoracic duct in rats: 2.5 mg/kg, oral administration [11]

References

1.

Kluepfel D, Bagli J, Baker H, Charest MP, Kudelski A, Sehgal SN, Vézina C (1972) Myriocin, a new antifungal antibiotic from Myriococcum albomyces. J Antibiot (Tokyo) 25:109–115
2.

Fujita T, Inoue K, Yamamoto S, Ikumoto T, Sasaki S, Toyama R, Chiba K, Hoshino Y, Okumoto T (1994) Fungal metabolites. Part 11. A potent immunosuppressive activity found in Isaria sinclairii metabolite. J Antibiot (Tokyo) 47:208–215
3.

Adachi K, Kohara T, Nakao N, Arita M, Chiba K, Mishina T, Sasaki S, Fujita T (1995) Design, synthesis, and structure-activity relationships of 2-substituted-2-amino-1,3-propanediols: discovery of a novel immunosuppressant, FTY720. Bioorg Med Chem Lett 5:853–856
4.

Miyake Y, Kozutsumi Y, Nakamura S, Fujita T, Kawasaki T (1995) Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin. Biochem Biophys Res Commun 211:396–403
5.

Chen JK, Lane WS, Schreiber SL (1999) The identification of myriocin-binding proteins. Chem Biol 6:221–235
6.

Buede R, Rinker-Schaffer C, Pinto WJ, Lester RL, Dickson RC (1991) Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids. J Bacteriol 173:4325–4332
7.

Gable K, Slife H, Bacikova D, Monaghan E, Dunn TM (2000) Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity. J Biol Chem 275:7597–7603
8.

Nagiec MM, Baltisberger JA, Wells GB, Lester RL, Dickson RC (1994) The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc Natl Acad Sci U S A 91:7899–7902
9.

Fujita T, Hirose R, Hamamichi N, Kitao Y, Sasaki S, Yoneta M, Chiba K. (1995) 2-Substituted 2-aminoethanol: minimum essential structure for immunosuppressive activity of ISP-I (Myriocin). Bioorg Med Chem Lett 5:1857–1860
10.

Brinkmann V, Davis MD, Heise CE, Albert R, Cottens S, Hof R, Bruns C, Prieschl E, Baumruker T, Hiestand P, Foster CA, Zollinger M, Lynch KR. (2002) The immune modulator FTY720 targets sphingosine 1-phosphate receptors. J Biol Chem 277:21453–21457
11.

Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, Thornton R, Shei GJ, Card D, Keohane C, Rosenbach M, Hale J, Lynch CL, Rupprecht K, Parsons W, Rosen H.(2002) Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science 296:346–349
12.

Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG. (2004) Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427:355–360

N-89, N-251

Keywords: [Antimalarial] [Endoplasmic reticulum-resident calcium binding protein (ERC) inhibitor]

Structure

	N-89	N-251
Molecular formula	C₁₅H₂₈O₄	C₂₁H₄₀O₅
Molecular weight	272.38	372.54
Solubility	DMSO, +; H₂O, ? ; MeOH, ?	DMSO, +; H₂O, ? ; MeOH, ?

Discovery/Isolation/Chemical Studies

N-89 and N-251 are synthetic compounds containing endoperoxide [1, 2].

Biological Studies

Synthetic endoperoxides N-89 and its derivative with hydroxyl group, N-251, had high antimalarial activities both in vivo and in vitro [1, 3]. The target molecules in artemisinin-resitant Plasmodium falciparum were surveyed by a chemical proteomics approach using the affinity spin column, and several candidate proteins were determined. One of the candidate proteins, PfERC, a member of the CREC (Cab45, reticulocalbin, ERC-45, calumenin) protein family, had high affinity to the endoperoxide structure and this interaction was competitively inhibited by N-89 or N-251, suggesting that PfERC is a target protein of endoperoxide antimalarial compounds.

Biological Activity of N-89 and N-251

EC₅₀ values for N-89 against P. falciparum and FM3A in vitro: 25 nM and 8.0 μM, respectively [1]

EC₅₀ values for N-251 against against P. falciparum and FM3A in vitro: 23 nM and 8.0 μM, respectively [3]

K_d values for N-89 and N-251 to recombinant PfERC in HEPES running buffer: 3.8 and 0.16 mM, respectively [4]

References

1.

Kim HS, Nagai Y, Ono K, Begum K, Wataya Y, Hamada Y, Tsuchiya K, Masuyama A, Nojima M, McCullough KJ (2001) Synthesis and antimalarial activity of novel medium-sized 1,2,4,5-tetraoxacycloalkanes. J Med Chem 44:2357–2361
2.

Sato A, Kawai S, Hiramoto A, Morita M, Tanigawa N, Nakase Y, Komichi Y, Matsumoto M, Hiraoka O, Hiramoto K, Tokuhara H, Masuyama A, Nojima M, Higaki K, Hayatsu H, Wataya Y, Kim HS (2011) Antimalarial activity of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) and its carboxylic acid derivatives. Parasitol Int 60:488–492
3.

Sato A, Hiramoto A, Morita M, Matsumoto M, Komich Y, Nakase Y, Tanigawa N, Hiraoka O, Hiramoto K, Hayatsu H, Higaki K, Kawai S, Masuyama A, Nojima M, Wataya Y, Kim HS (2011) Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol. Parasitol Int 60:270–273
4.

Morita M, Sanai H, Hiramoto A, Sato A, Hiraoka O, Sakura T, Kaneko O, Masuyama A, Nojima M, Wataya Y, Kim HS (2012) Plasmodium falciparum endoplasmic reticulum-resident calcium binding protein is a possible target of synthetic antimalarial endoperoxides, N-89 and N-251. J Proteome Res 11:5704–5711

Necrostatin-1*

Keywords: [Necroptosis inducer] [RIP1 kinase inhibitor]

Structure

	Necrostatin-1
Molecular formula	C₁₃H₁₃N₃OS
Molecular weight	259.33
Solubility	DMSO, +++; H₂O, −; MeOH, −

Discovery/Isolation

Necroptosis is characterized by necrotic cell death morphology and activation of autophagy when undergoing death domain receptor–induced cell death without caspase activation. Necrostatin-1 was identified by screening of inhibitors of the necroptosis of human monocytic U937 cells induced by tumor necrosis factor-α (TNFα) and z-VAD-fmk from a chemical library of ~15,000 chemicals [1].

Biological Studies

Necrostatin-1 inhibited TNFα-stimulated necrosis, but not FasL-cycloheximide-induced apoptosis of FADD-deficinent Jurkat cells [1]. Interestingly, necrostatin-1 failed to inhibit oxidative stress-induced necrosis, suggesting that this compound is a specific inhibitor of necroptosis, and does not act as an antioxidant. An in vitro kinase assay revealed that necrostatin-1 directly inhibits receptor interacting Ser/Thr protein kinase 1 (RIP1) [2]. The crystal structure of necrostatin-1 and RIP1 showed that necrostatin-1 stabilizes RIP1 in an inactive comformation by binding to the activation loop [3].

Necrostatin-1 has been widely used for investigations of necroptosis and RIP kinase–mediated cellular processes [4–7].

Biological Activity of Necrostatin-1

EC₅₀ for necroptosis against FADD-deficient Jurkat cells: 494 ± 125 nM [1]

EC₅₀ for endogenous RIP1 kinase from Jurkat cells: 182 nM [2]

References

1.

Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1:112–119
2.

Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4:313–321
3.

Xie T, Peng W, Liu Y, Yan C, Maki J, Degterev A, Yuan J, Shi Y (2013) Structural basis of RIP1 inhibition by necrostatins. Structure 21:493–499
4.

Trichonas G, Murakami Y, Thanos A, Morizane Y, Kayama M, Debouck CM, Hisatomi T, Miller JW, Vavvas DG (2010) Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis. Proc Natl Acad Sci U S A 107:21695–21700
5.

Motani K, Kushiyama H, Imamura R, Kinoshita T, Nishiuchi T, Suda T (2011) Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity. J Biol Chem 286:33963–33972
6.

Linkermann A, Brasen JH, Darding M, Jin MK, Sanz AB, Heller JO, De Zen F, Weinlich R, Ortiz A, Walczak H, Weinberg JM, Green DR, Kunzendorf U, Krautwald S (2013) Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury. Proc Natl Acad Sci U S A 110:12024–12029
7.

Nomura M, Ueno A, Saga K, Fukuzawa M, Kaneda Y (2014) Accumulation of cytosolic calcium induces necroptotic cell death in human neuroblastoma. Cancer Res 74:1056–1066

NITD609* (Spiroindolone)

Keywords: [Antimalarial] [PfATP4 inhibitor]

Structure

	NITD609
Molecular formula	C₁₉H₁₄Cl₂FN₃O
Molecular weight	390.24
Solubility	DMSO, ++; H₂O, ? ; MeOH, ++

Discovery/Isolation/Chemical Study

NITD609 is a synthetic spiroindolone compound optimized from a spiroazepineindole class compound showing antimalarial activity against Plasmodium falciparum [1, 2]. NITD609 has good drug-like attributes and physicochemical properties.

Biological Studies

NITD609 showed antimalarial blood-stage activity with no evidence of diminished potency against drug-resistant strains [1]. All stages (rings, trophozoites, and schizonts) were similarly sensitive to NITD609, suggesting that the target molecule is present at all asexual blood stages. Furthermore NITD609 inhibited protein synthesis in P. falciparum, suggesting that the mechanism of action is different from those of artemisinin and mefloquine. Mutational analysis, which shows NITD609 resistance, revealed that P-type cation-transporter ATPase4 (PfATP4) mediates the spiroindolone resistance. However, PfATP4 has a high level homology with Saccharomyces cerevisiae PMR1, a P-type ATPase required for high-affinity Ca²⁺ and Mn²⁺ transport; this protein of the intraerythrocytic stage actively extrudes Na⁺ against an inward gradient [3]. Therefore, spiroindolones causes a profound disruption of Na⁺ homeostasis in P. falciparum.

Biological Activity of NITD609

IC₅₀ for antimalarial acitivty against blood-stage P. falciparum: 0.5–1.4 nM [1]

References

1.

Rottmann M, McNamara C, Yeung BK, Lee MC, Zou B, Russell B, Seitz P, Plouffe DM, Dharia NV, Tan J, Cohen SB, Spencer KR, Gonzalez-Paez GE, Lakshminarayana SB, Goh A, Suwanarusk R, Jegla T, Schmitt EK, Beck HP, Brun R, Nosten F, Renia L, Dartois V, Keller TH, Fidock DA, Winzeler EA, Diagana TT (2010) Spiroindolones, a potent compound class for the treatment of malaria. Science 329:1175–1180
2.

Yeung BK, Zou B, Rottmann M, Lakshminarayana SB, Ang SH, Leong SY, Tan J, Wong J, Keller-Maerki S, Fischli C, Goh A, Schmitt EK, Krastel P, Francotte E, Kuhen K, Plouffe D, Henson K, Wagner T, Winzeler EA, Petersen F, Brun R, Dartois V, Diagana TT, Keller TH (2010) Spirotetrahydro β-carbolines (spiroindolones): a new class of potent and orally efficacious compounds for the treatment of malaria. J Med Chem 53:5155–5164
3.

Spillman NJ, Allen RJ, McNamara CW, Yeung BK, Winzeler EA, Diagana TT, Kirk K (2013) Na⁺ regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials. Cell Host Microbe 13:227–237

Papuamide A and B

Keywords: [Phosphatidylserine binder] [HIV-1 entry inhibitor]

Structure

	Papuamide A	Papuamide B
Molecular formula	C₆₆H₁₀₅N₁₃O₂₁	C₆₅H₁₀₃N₁₃O₂₁
Molecular weight	1416.61	1402.59
Solubility	DMSO, ++; H₂O, ? ; MeOH, +++	DMSO, ++; H₂O, ? ; MeOH, +++

Discovery/Isolation/Chemical Studies

Papuamides A and B are cyclic depsipeptides isolated from a Papua New Guinea collection of the sponges Theonella mirabilis and Theonella swinhoei, as HIV-inhibitory and cytotoxic compounds [1]. The structures were determined by a combination of spectroscopic analysis, chemical degradation, and derivatization studies.

Biological Studies

Pupuamide A inhibited the infection of human T-lymphoblastoid cells by HIV-1 in vitro [1] by inhibiting HIV-1 entry into host cells [2]. Yeast chemical–genetic profiling, resistant mutant analysis, and an in vitro marker release experiment using liposomes suggested that papuamide B targets phosphatidylserine [3]. Therefore, papuamide B has been used as a probe for investigating the function of phosphatidylserine in a plasma membrane [4, 5].

Biological Activity of Papuamide A

EC₅₀ for HIV-1 infection to human T-lymphoblastoid cells: 4 ng/ml [1]

Inhibition of HIV-1 entry (approximately 80% inhibition): 710 nM [2]

References

1.

Ford PW, Gustafson KR, McKee TC, Shigematsu N, Maurizi LK, Pannell LK, Williams DE, de Silva ED, Lassota P, Allen TM, Van Soest R, Andersen RJ, Boyd MR (1999) Papuamides A-D, HIV-inhibitory and cytotoxic depsipeptides from the sponges Theonella mirabilis and Theonella swinhoei collected in Papua New Guinea. J Am Chem Soc 121:5899–5909
2.

Andjelic CD, Planelles V, Barrows LR (2008) Characterizing the anti-HIV activity of papuamide A. Mar Drugs 6:528–549
3.

Parsons AB, Lopez A, Givoni IE, Williams DE, Gray CA, Porter J, Chua G, Sopko R, Brost RL, Ho CH, Wang JY, Ketela T, Brenner C, Brill JA, Fernandez GE, Lorenz TC, Payne GS, Ishihara S, Ohya Y, Andrews B, Hughes TR, Frey BJ, Graham TR, Andersen RJ, Boone C (2006) Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126:611–625
4.

Chen S, Wang J, Muthusamy BP, Liu K, Zare S, Andersen RJ, Graham TR (2006) Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane. Traffic 7:1503–151
5.

Georgiev AG, Johansen J, Ramanathan VD, Sere YY, Beh CT, Menon AK (2013) Arv1 regulates pm and er membrane structure and homeostasis but is dispensable for intracellular sterol transport. Traffic 14:912–921

Pateamine A

Keywords: [Eukaryotic initiation factor 4A (eIF4A) activator] [Nonsense-mediated mRNA decay (NMD) inhibitior]

Structure

	Pateamine A
Molecular formula	C₃₁H₄₅N₃O₄S
Molecular weight	555.78
Solubility	DMSO, ? ; H₂O, ? ; MeOH, ++

Discovery/Isolation/Chemical Studies

Pateamine A, a sulfur-containing heteroaromatic macrolide, was isolated as an antifungal and cytotoxic compound from the sponge Mycale sp. [1].

Biological Studies

Pateamine A was reidentified as an inhibitor of eukaryotic translation initiation by high-throughput screening [2]. The pateamine A binding protein was identified to be eIF4AI by an affinity purification method using a matrix containing immobilized pateamine A [2] or biotinylated pateamine A [3]. eIF4A is an RNA- and ATP-dependent helicase that unwinds a local secondary structure in mRNA to allow binding to the 43S ribosomal complex to form the 48S ribosomal complex. Pateamine A bound to and enhanced the ATPase activity of eIF4A [2, 3]. Furthermore, pateamine A inhibited the interaction between eIF4A and eIF4G, then stabilized the ternary complex between eIF4A and eIF4B [4, 3]. These results suggest that pateamine A prevents eIF4A from participating in the recruitment of ribosomes.

Using recombinant protein, it was shown that pateamine A also binds to eIF4AIII which involves a nonsense-mediated mRNA decay (NMD) pathway [2]. Indeed, pateamine A inhibited NMD through direct interaction with eIF4AIII, which is independent of its inhibitory activity of translation initiation [5].

In cells, pateamine A induces stress granules, which are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA binding proteins, by different mechanisms of arsenite [6]. Recently, it has been reported that pateamine A prevents cachexia-induced muscle wasting in a nontoxic dose in mice [7]. Interestingly, a low dose of pateamine A selectively inhibits the translation of procachectic factors such as iNOS by recruitment of iNOS mRNA to stress granules. These results suggest that pateamine A could be a useful medicine for treatment of cachexia.

Biological Activity of Pateamine A

IC₅₀ for P388 murine cells: 0.15 ng/ml [1]

IC₅₀ for BSC cells: ~300 ng/ml [1]

Stress granule formation in HeLa cells: 50 nM [6]

Muscle differentiation of C2C12 cells: 12.5 nM [7]

Suppression of tumor-induced muscle wasting in BALB/c mice injected with C26 adenocarcinoma cells: 20 μg/kg [7]

References

1.

Northcote PT, Blunt JW, Munro MHG (1991) Pateamine – a potent cytotoxin from the new-zealand marine sponge, Mycale sp. Tetrahedron Lett 32:6411–6414
2.

Bordeleau ME, Matthews J, Wojnar JM, Lindqvist L, Novac O, Jankowsky E, Sonenberg N, Northcote P, Teesdale-Spittle P, Pelletier J (2005) Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. Proc Natl Acad Sci U S A 102:10460–10465
3.

Low WK, Dang Y, Schneider-Poetsch T, Shi Z, Choi NS, Merrick WC, Romo D, Liu JO (2005) Inhibition of eukaryotic translation initiation by the marine natural product pateamine A. Mol Cell 20:709–722
4.

Bordeleau ME, Cencic R, Lindqvist L, Oberer M, Northcote P, Wagner G, Pelletier J (2006) RNA-mediated sequestration of the RNA helicase eIF4A by pateamine A inhibits translation initiation. Chem Biol 13:1287–1295
5.

Dang Y, Low WK, Xu J, Gehring NH, Dietz HC, Romo D, Liu JO (2009) Inhibition of nonsense-mediated mRNA decay by the natural product pateamine A through eukaryotic initiation factor 4AIII. J Biol Chem 284:23613–23621
6.

Dang Y, Kedersha N, Low WK, Romo D, Gorospe M, Kaufman R, Anderson P, Liu JO (2006) Eukaryotic initiation factor 2α-independent pathway of stress granule induction by the natural product pateamine A. J Biol Chem 281:32870–32878
7.

Di Marco S, Cammas A, Lian XJ, Kovacs EN, Ma JF, Hall DT, Mazroui R, Richardson J, Pelletier J, Gallouzi IE (2012) The translation inhibitor pateamine A prevents cachexia-induced muscle wasting in mice. Nat Commun 3:896

2-Phenylethynesulfonamide* (PES, Pifithrin-μ)

Keywords: [Heat shock protein 70 (HSP70) inhibitor] [p53-dependent apoptosis inhibitor]

Structure

	PES/PFTμ
Molecular formula	C₈H₇NO₂S
Molecular weight	181.21
Solubility	DMSO, ++; H₂O, −; MeOH, ++

Discovery/Isolation

2-phenylethynesulfonamide (PES, phenylacetylenylsulfonamide, Pifithrin-μ (PFTμ)) was identified as an inhibitor of apoptosis induced in Saos-2 cells by lentiviral transduction of p53 [1].

Biological Studies

PES inhibited p53-dependent apoptosis by reducing the amount of mitochondria-bound p53 and the binding of p53 to both Bcl-xL and Bcl-2 proteins [1]. Leu et al. identified HPS70 as a PES binding protein using thiol-cleavable biotinylated PES [2]. PES interacted with the carboxy-terminal substrate binding domain, and inhibited the chaperone function of HSP70 by disrupting the association between HSP70 and several of its co-chaperones and substrate proteins. Furthermore, PES treatment induced dysfunction of the autophagy–lysosome system and the proteasome pathway [2, 3]. These results suggest that HSP70 is a crucial factor for the cancer survival pathway and that targeting HSP70 is a potential approach for cancer therapeutics [3, 4].

Prevention of lipopolysaccharide (LPS)–induced liver injury and reduction of proinflammatory factor induction by disruption of the sodium–hydrogen antiporter 1 (NHE1)–HSP70 interaction was also reported [5].

Biological Activity of 2-Phenylethynesulfonamide

Inhibition of p53-dependent cell death: 10 μM [1]

IC₅₀ in approximately 50 solid tumor cell lines: 4–10 μM [3]

References

1.

Strom E, Sathe S, Komarov PG, Chernova OB, Pavlovska I, Shyshynova I, Bosykh DA, Burdelya LG, Macklis RM, Skaliter R, Komarova EA, Gudkov AV (2006) Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol 2:474–479
2.

Leu JI, Pimkina J, Frank A, Murphy ME, George DL (2009) A small molecule inhibitor of inducible heat shock protein 70. Mol Cell 36:15–27
3.

Leu JI, Pimkina J, Pandey P, Murphy ME, George DL (2011) HSP70 inhibition by the small-molecule 2-phenylethynesulfonamide impairs protein clearance pathways in tumor cells. Mol Cancer Res 9:936–947
4.

Balaburski GM, Leu JI, Beeharry N, Hayik S, Andrake MD, Zhang G, Herlyn M, Villanueva J, Dunbrack RL, Jr., Yen T, George DL, Murphy ME (2013) A modified HSP70 inhibitor shows broad activity as an anticancer agent. Mol Cancer Res 11:219–229
5.

Huang C, Wang J, Chen Z, Wang Y, Zhang W (2013) 2-phenylethynesulfonamide Prevents Induction of Pro-inflammatory Factors and Attenuates LPS-induced Liver Injury by Targeting NHE1-Hsp70 Complex in Mice. PLoS One 8:e67582

Phoslactomycin A and F

Keywords: [Protein phosphatase type 2A (PP2A) inhibitor]

Structure

	Phoslactomycin A	Phoslactomycin F
Molecular formula	C₂₉H₄₆NO₁₀P	C₃₂H₅₂NO₁₀P
Molecular weight	599.65	641.73
Solubility	DMSO, ++; H₂O, +; MeOH, ++	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation/Chemical Studies

Phoslactomycins were originally isolated as antifungal antibiotics from soil Streptomyces bacteria species [1, 2]. Several related compounds, phosphazomycins [3, 4], phospholine [5, 6], and leustroducsins [7, 8], were reported as antifungal antibiotics, or as inducers of a colony-stimulating factor in bone marrow stromal cells. These compounds commonly contain not only an α,β-unsaturated δ-lactone, but also an amino group, a phosphate ester, and a cyclohexane ring, and have a different substituents bound to the cyclohexane ring.

Biological Studies

Phoslactomycin F reversibly induces actin filament depolymerization in murine fibroblast NIH/3T3 cells, but did not inhibit actin polymerization in vitro [9]. An in situ and in vitro phosphorylation assay revealed that phoslactomycin F stimulates the phosphorylation of intracellular vimentin by the inhibition of the protein phosphatase type-2A (PP2A). These results suggest that phoslactomycins are PP2A-specific inhibitors and that PP2A is involved in regulation of the organization of the actin cytoskeleton. Because phoslactomycins contain α,β-unsaturated δ-lactone, it has been thought that these compounds covalently bind to their target molecule. Teruya et al. synthesized biotinylated phoslactomycin A and investigated the binding proteins in mouse liver extract [10]. Three proteins to which biotinylated phoslactomycin A covalently binds were identified as PP2A family proteins: PP2Ac (a catalytic subunit of PP2A), PP4, and PP6. Furthermore, the binding site of phoslactomycins on PP2Ac was identified as Cys269 which is located at the entrance of the catalytic site. Interestingly, this cysteine residue is conserved in PP2A family proteins but not even in PP1, the most similar phosphatase. These results strongly suggest that Cys269 is a PP2A-specific gatekeeper residue, and that compounds targeting this residue are expected to be PP2A-specific inhibitors.

Biological Activity of Phoslactomycin F

Disassembly of actin cytoskeleton in NIH/3T3 cells: 10 μM [9]

IC₅₀ for PP2A: 4.7 μM [9]

References

1.

Fushimi S, Furihata K, Seto H (1989) Studies on new phosphate ester antifungal antibiotics phoslactomycins. II. Structure elucidation of phoslactomycins A to F. J Antibiot (Tokyo) 42:1026–1036
2.

Fushimi S, Nishikawa S, Shimazu A, Seto H (1989) Studies on new phosphate ester antifungal antibiotics phoslactomycins. I. Taxonomy, fermentation, purification and biological activities. J Antibiot (Tokyo) 42:1019–1025
3.

Tomiya T, Uramoto M, Isono K (1990) Isolation and structure of phosphazomycin C. J Antibiot (Tokyo) 43:118–121
4.

Uramoto M, Shen YC, Takizawa N, Kusakabe H, Isono K (1985) A new antifungal antibiotic, phosphazomycin A. J Antibiot (Tokyo) 38:665–668
5.

Ozasa T, Tanaka K, Sasamata M, Kaniwa H, Shimizu M, Matsumoto H, Iwanami M (1989) Novel antitumor antibiotic phospholine. 2. Structure determination. J Antibiot (Tokyo) 42:1339–1343
6.

Ozasa T, Suzuki K, Sasamata M, Tanaka K, Kobori M, Kadota S, Nagai K, Saito T, Watanabe S, Iwanami M (1989) Novel antitumor antibiotic phospholine. 1. Production, isolation and characterization. J Antibiot (Tokyo) 42:1331–1338
7.

Kohama T, Nakamura T, Kinoshita T, Kaneko I, Shiraishi A (1993) Novel microbial metabolites of the phoslactomycins family induce production of colony-stimulating factors by bone marrow stromal cells. II. Isolation, physico-chemical properties and structure determination. J Antibiot (Tokyo) 46:1512–1519
8.

Kohama T, Enokita R, Okazaki T, Miyaoka H, Torikata A, Inukai M, Kaneko I, Kagasaki T, Sakaida Y, Satoh A, Shiraishi A (1993) Novel microbial metabolites of the phoslactomycins family induce production of colony-stimulating factors by bone marrow stromal cells. I. Taxonomy, fermentation and biological properties. J Antibiot (Tokyo) 46:1503–1511
9.

Usui T, Marriott G, Inagaki M, Swarup G, Osada H (1999) Protein phosphatase 2A inhibitors, phoslactomycins. Effects on the cytoskeleton in NIH/3T3 cells. J Biochem 125:960–965
10.

Teruya T, Simizu S, Kanoh N, Osada H (2005) Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells. FEBS Lett 579:2463–2468

Piperlongumine

Keywords: [Antitumor] [Glutathione-S-transferase P1 (GSTP1) inhibitor]

Structure

	Piperlongumine
Molecular formula	C₁₇H₁₉NO₅
Molecular weight	317.34
Solubility	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation/Chemical Studies

Piperlongumine was originally isolated from the root of Piper longum Linn [1]. This compound was screened by a cell-based and quantitative proteomics approach and showed a cancer-cell-selective killing property [2]. SAR analysis suggested that the electrophilicity of the C2-C3 olefin of piperlogumine is critical for selective toxicity against cancer cells [3].

Biological Studies

Piperlongumine shows several biological activities including insecticidal activity [4], platelet aggregation inhibition [5], and antitumor activity [6]. Piperlongumine increases the level of reactive oxygen species and apoptotic cell death in cells with a cancer genotype, but have little effect on primary normal cells. Furthermore, a combination method of affinity enrichment with stable-isotope labeling and quantitative proteomics revealed that GSTP1 is the target molecule of piperlongumine, suggesting that piperlongumine induces apoptosis by interfering with redox and GSTP1 reactive oxygen species (ROS) homeostatic regulators. Further detailed analysis using synthetic analogs suggested that piperlongumine possesses two sites of reactivity, one with great and the other with lesser electrophilicity [3]. Great electrophilicity is required for covalent binding with glutathione, and less is probably required for cellular cross-linking with proteins, which is associated with toxicity. Therefore, both electrophilic and oxidative stress phenotypes can contribute to cancer-selective toxicity of piperlongumine.

Inhibitory activity of piperlongumine on atherosclerotic plaque formation and vascular smooth muscle cell proliferation has also been reported [7].

Biological Activity of Piperlongumine

IC₅₀ for cell proliferation of HL-60, HCT-8, SF295, and MDA-MB-435: >25 μg/ml [6]

Cellular ROS production in EJ, MDA-MB-231, U2OS, and MDA-MB-435: 10 μM [2]

EC₅₀ values for cellular ATP level in H1703 and HeLa cells: 2.8 and 7.1 μM, respectively [3]

References

1.

Chatterjee A, Dutta CP (1967) Alkaloids of Piper longum Linn–I: Structure and synthesis of piperlongumine and piperlonguminine. Tetrahedron 23:1769–1781
2.

Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, Tolliday NJ, Golub TR, Carr SA, Shamji AF, Stern AM, Mandinova A, Schreiber SL, Lee SW (2011) Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 475:231–234
3.

Adams DJ, Dai M, Pellegrino G, Wagner BK, Stern AM, Shamji AF, Schreiber SL (2012) Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs. Proc Natl Acad Sci U S A 109:15115–15120
4.

Bernard CB, Krishanmurty HG, Chauret D, Durst T, Philogene BJR, Sanchez-Vindas P, Hasbun C, Poveda L, San Roman L, Arnason JT (1995) Insecticidal defenses of Piperaceae from the neotropics. J Chem Ecol 21:801–814
5.

Iwashita M, Oka N, Ohkubo S, Saito M, Nakahata N (2007) Piperlongumine, a constituent of Piper longum L., inhibits rabbit platelet aggregation as a thromboxane A2 receptor antagonist. Eur J Pharmacol 570:38–42
6.

Bezerra DP, Pessoa C, Moraes MO, Alencar NM, Mesquita RO, Lima MW, Alves AP, Pessoa OD, Chaves JH, Silveira ER, Costa-Lotufo LV (2008) In vivo growth inhibition of sarcoma 180 by piperlonguminine, an alkaloid amide from the Piper species. J Appl Toxicol 28:599–607
7.

Son DJ, Kim SY, Han SS, Kim CW, Kumar S, Park BS, Lee SE, Yun YP, Jo H, Park YH (2012) Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling. Biochem Biophys Res Commun 427:349–354

Pironetin

Keywords: [Tubulin polymerization inhibitor]

Structure

	Pironetin
Molecular formula	C₁₉H₃₂O₄
Molecular weight	324.46
Solubility	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation

Pironetin was isolated as a plant growth regulator which induced shortening of the plant height of rice [1, 2], and as an immunosuppressor which inhibited the blastogenesis of both T and B cells.

Biological Studies

Pironetin showed antitumor activity against a murine tumor cell line, P388 leukemia, transplanted in mice [3], and induced apoptosis via disruption of the cellular microtubule network in situ [4]. The compound inhibited the polymerization of pure tubulin, showing that pironetin is a tubulin inhibitor which inhibits tubulin polymerization by binding to tubulin directly. Binding competition experiments with [³H]colchicine and [³H]vinblastine strongly suggested that pironetin binds on the vinblastine binding site on β-tubulin.

According to structure–activity relationship analyses, it was speculated that pironetin covalently binds to tubulin by Michael addition via its α,β-unsaturated δ-lactone [5]. The biotinylated pironetin (2), which inhibited tubulin assembly both in vitro and in situ, covalently bound with tubulin, and its binding was inhibited by natural pironetin [6]. The finding that inactive pironetin derivatives failed to bind to tubulin in situ suggested that covalent binding is important to inhibit tubulin polymerization. Partial proteolytic analyses of biotinylated pironetin–bound peptides, followed by systematic alanine scanning of both cysteine and lysine residues, strongly suggested that Lys352 of α-tubulin is the pironetin binding site. This is surprising, because the reactivity of the ε-amino group of lysine is quite low compared with that of the sulfhydryl group of cysteine. The in silico analysis of the pironetin–tubulin complex suggested that α helixes 8/10 and β sheet 8 form a cavity that fits pironetin well, and that pironetin binding with Lys352 and Asn258 disrupts the hydrogen network among Glu254, Lys352, and Asn258. Discrepancy with [³H]-vinblastine competition experiments were explained by the structure of vinblastine bound to the tubulin–colchicine:RB3-SLD complex [7, 8]. Gigant et al. revealed that vinblastine binds not only to β-tubulin but also on a hydrophobic groove on the α-tubulin surface that is located at an intermolecular contact in microtubules, and pironetin binding residue, Lys352, is part of the vinblastine binding site. These results strongly suggest that the binding sites of pironetin and vinblastine are overlapped, and that these drugs turn out each other.

Biological Activity of Pironetin

23% inhibition of growth of rice plants without any loss of crop yield 9 days before heading: 10 g/a [1]

Complete inhibition of cell proliferation of 3Y1 cells: 10–20 ng/ml [3]

Antiproliferative effects in several tumor cell lines: 5–25 ng/ml [3]

References

1.

Kobayashi S, Tsuchiya K, Harada T, Nishide M, Kurokawa T, Nakagawa T, Shimada N, Kobayashi K (1994) Pironetin, a novel plant growth regulator produced by Streptomyces sp. NK10958. I. Taxonomy, production, isolation and preliminary characterization. J Antibiot (Tokyo) 47:697–702
2.

Kobayashi S, Tsuchiya K, Kurokawa T, Nakagawa T, Shimada N, Iitaka Y (1994) Pironetin, a novel plant growth regulator produced by Streptomyces sp. NK10958. II. Structural elucidation. J Antibiot (Tokyo) 47:703–707
3.

Kondoh M, Usui T, Kobayashi S, Tsuchiya K, Nishikawa K, Nishikiori T, Mayumi T, Osada H (1998) Cell cycle arrest and antitumor activity of pironetin and its derivatives. Cancer Lett 126:29–32
4.

Kondoh M, Usui T, Nishikiori T, Mayumi T, Osada H (1999) Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin. Biochem J 340:411–416
5.

Watanabe H, Watanabe H, Usui T, Kondoh M, Osada H, Kitahara T (2000) Synthesis of pironetin and related analogs: studies on structure-activity relationships as tubulin assembly inhibitors. J Antibiot (Tokyo) 53:540–545
6.

Usui T, Watanabe H, Nakayama H, Tada Y, Kanoh N, Kondoh M, Asao T, Takio K, Watanabe H, Nishikawa K, Kitahara T, Osada H (2004) The anticancer natural product pironetin selectively targets Lys352 of α-tubulin. Chem Biol 11:799–806
7.

Gigant B, Cormier A, Dorleans A, Ravelli RBG, Knossow M (2009) Microtubule-destabilizing agents: structural and mechanistic insights from the interaction of colchicine and vinblastine with tubulin. Top Curr Chem 286:259–278
8.

Gigant B, Wang C, Ravelli RBG, Roussi F, Steinmetz MO, Curmi PA, Sobel A, Knossow M (2005) Structural basis for the regulation of tubulin by vinblastine. Nature 435:519–522

Pladienolide B

Keywords: [Antitumor] [Splicing inhibitor] [SF3b]

Structure

	Pladienolide B
Molecular formula	C₃₀H₄₈O₈
Molecular weight	536.70
Solubility	DMSO, +++; H₂O, ±; MeOH, +++

Discovery/Isolation/Chemical Studies

Pladienolide B is a 12-member macrolide purified from Streptomyces platensis Mer-11107, possessing inhibitory activity against vascular endothelial growth factor (VEGF) expression and cancer cell proliferation [1]. Absolute configurations at 10 chiral centers were determined on the basis of spectral data of pladienolide B and its chemical transformation products [2]. Total synthesis has done by Kanada et al. [3].

Biological Studies

Pladienolide B potently inhibited hypoxia-induced VEGF expression and proliferation of the U251 cancer cell line with strong antitumor activities in vitro and in vivo [4]. Using ³H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)–tagged “chemical probes,” the target protein of pladienolide was determined as a 140 kDa protein in splicing factor SF3b [5]. Pladienolide B impaired in vivo splicing in a dose-dependent manner. The pladienolide-resistant clones have a mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex and impaired the binding of pladienolide [6]. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

Biological Activity of Pladienolide B

IC₅₀ values for anti-VEGF-PLAP and antiproliferative activities in U251 cells: 1.8 and 3.5 nM, respectively [7]

Inhibition of splicing of DNAJB1 and RIOK3 in HeLa cells for 4 hours: 10 nM [5]

References

1.

Sakai T, Asai N, Okuda A, Kawamura N, Mizui Y (2004) Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. II. Physico-chemical properties and structure elucidation. J Antibiot (Tokyo) 57:180–187
2.

Asai N, Kotake Y, Niijima J, Fukuda Y, Uehara T, Sakai T (2007) Stereochemistry of pladienolide B. J Antibiot (Tokyo) 60:364–369
3.

Kanada RM, Itoh D, Nagai M, Niijima J, Asai N, Mizui Y, Abe S, Kotake Y (2007) Total synthesis of the potent antitumor macrolides pladienolide B and D. Angew Chem Int Ed Eng 46:4350–4355
4.

Mizui Y, Sakai T, Iwata M, Uenaka T, Okamoto K, Shimizu H, Yamori T, Yoshimatsu K, Asada M (2004) Pladienolides, new substances from culture of Streptomyces platensis Mer-11107 III. In vitro and in vivo antitumor activities . J Antibiot (Tokyo) 57:188–196
5.

Kotake Y, Sagane K, Owa T, Mimori-Kiyosue Y, Shimizu H, Uesugi M, Ishihama Y, Iwata M, Mizui Y (2007) Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol 3:570–575
6.

Yokoi A, Kotake Y, Takahashi K, Kadowaki T, Matsumoto Y, Minoshima Y, Sugi NH, Sagane K, Hamaguchi M, Iwata M, Mizui Y (2011) Biological validation that SF3b is a target of the antitumor macrolide pladienolide. FEBS J 278:4870–4880
7.

Sakai T, Sameshima T, Matsufuji M, Kawamura N, Dobashi K, Mizui Y (2004) Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. I. Taxonomy, fermentation, isolation and screening. J Antibiot (Tokyo) 57:173–179

Prodigiosin 25-C

Keywords: [Antitumor] [Immunosuppressant]

Structure

	Prodigiosin 25-C
Molecular formula	C₂₅H₃₅N₃O
Molecular weight	393.57
Solubility	DMSO, +++; H₂O, −; MeOH, ++

Discovery/Isolation

Prodigiosins were originally isolated as pigments from Serratia marcescens and Streptomyces sp. Prodigiosin 25-C was reisolated as an immunomodulating substance showing inhibition of cytotoxic T-cell induction in a mixed lymphocyte reaction [1].

Biological Studies

Prodigiosin 25-C completely inhibited induction of cytotoxic T cells in vitro and in vivo [2], and moderately prolonged survival of MHC-mismatched skin grafts [3]. This compound uncoupled vacuolar H⁺-ATPase (V-ATPase), inhibited vacuolar acidification, and affected glycoprotein processing [4]. Further analyses suggested that prodigiosins promote H⁺/Cl⁻ symport (or OH⁻/Cl⁻ exchange, in its equivalence) across vesicular membranes [5]. Because prodigiosin facilitates chloride/bicarbonate exchange (antiport) across liposomal membranes [6], the disturbance of ions endows these immunosuppressive activities.

Suppressive activity on osteoclast bone resorption [7], and antitumor activity by mTOR inhibition [8] and by up-regulating p73 in p53-deficient cells [9] have been reported.

Biological Activity of Prodigiosins: Prodigiosin 25-C

IC₅₀ for proton pump activity in rat liver lysosomes: 30 nM [4]

Inhibition of ATPase activity in rat liver lysosomes: >1 μM [4]

Prodigiosin

IC₅₀ for intralysosomal pH through inhibition of lysosomal acidification driven by V-ATPase: 30–120 pmol/mg of protein [10]

Activation of both autophagic and apoptotic mechanisms in SK-MEL-28 and SK-MEL-5 cells: 4.51 ± 0.47 μM and 1.02 ± 0.15 μM, respectively [8]

Induction of p53 target proteins and PARP cleavage in various cancer cells: 1 μM [9]

References

1.

Nakamura A, Nagai K, Ando K, Tamura G (1986) Selective suppression by prodigiosin of the mitogenic response of murine splenocytes. J Antibiot (Tokyo) 39:1155–1159
2.

Nakamura A, Magae J, Tsuji RF, Yamasaki M, Nagai K (1989) Suppression of cytotoxic T cell induction in vivo by prodigiosin 25-C. Transplantation 47:1013–1016
3.

Tsuji RF, Magae J, Yamashita M, Nagai K, Yamasaki M (1992) Immunomodulating properties of prodigiosin 25-C, an antibiotic which preferentially suppresses induction of cytotoxic T cells. J Antibiot (Tokyo) 45:1295–1302
4.

Kataoka T, Muroi M, Ohkuma S, Waritani T, Magae J, Takatsuki A, Kondo S, Yamasaki M, Nagai K (1995) Prodigiosin 25-C uncouples vacuolar type H(+)-ATPase, inhibits vacuolar acidification and affects glycoprotein processing. FEBS Lett 359:53–59
5.

Sato T, Konno H, Tanaka Y, Kataoka T, Nagai K, Wasserman HH, Ohkuma S (1998) Prodigiosins as a new group of H⁺/Cl^- symporters that uncouple proton translocators. J Biol Chem 273:21455–21462
6.

Davis JT, Gale PA, Okunola OA, Prados P, Iglesias-Sanchez JC, Torroba T, Quesada R (2009) Using small molecules to facilitate exchange of bicarbonate and chloride anions across liposomal membranes. Nat Chem 1:138–144
7.

Woo JT, Ohba Y, Tagami K, Sumitani K, Kataoka T, Nagai K (1997) Prodigiosin 25-C and metacycloprodigiosin suppress the bone resorption by osteoclasts. Biosci Biotechnol Biochem 61:400–402
8.

Espona-Fiedler M, Soto-Cerrato V, Hosseini A, Lizcano JM, Guallar V, Quesada R, Gao T, Perez-Tomas R (2012) Identification of dual mTORC1 and mTORC2 inhibitors in melanoma cells: prodigiosin vs. obatoclax. Biochem Pharmacol 83:489–496
9.

Hong B, Prabhu VV, Zhang S, van den Heuvel AP, Dicker DT, Kopelovich L, El-Deiry WS (2014) Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53. Cancer Res 74:1153–1165
10.

Ohkuma S, Sato T, Okamoto M, Matsuya H, Arai K, Kataoka T, Nagai K, Wasserman HH (1998) Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H⁺/Cl^- symport. Biochem J 334:731–741

Pseudolaric Acid B

Keywords: [Antifungal] [Tubulin inhibitor]

Structure

	Pseudolaric acid B
Molecular formula	C₂₃H₂₈O₈
Molecular weight	432.47
Solubility	DMSO, ++; H₂O, ±; MeOH, ++

Discovery/Isolation/Chemical Study

Pseudolaric acid B is a diterpene acid isolated from Tu-Jin-Pi, Pseudolarix kaempferi Golden (Pinaceae), which has been used in traditional Chinese medicine for skin diseases caused by fungi. The chemical structure and total synthesis have been reported [1, 2].

Biological Studies

Several biological activities of pseudolaric acid B including cytotoxicity against cancer cells [3], antifungal activities [4], antiangiogenesis activity [5], apoptosis induction [6], and anti-inflammatory activity [7] have been reported.

Wong et al. investigated the molecular target of pseudolaric acid B in detail [8]. They found that pseudolaric acid B induced cell cycle arrest at G2/M transition, leading to apoptosis. Furthermore, this compound dose-dependently inhibited not only cellular microtubule networks but also tubulin polymerization in vitro. The binding site of pseudolaric acid B is thought to be the distinct binding site on tubulin compared with those of colchicine and vinblastine [9].

Biological Activity of Pseudolaric Acid B

IC₅₀ for VEGF-stimulated migration of HUVECs: 2.92 μM [5]

IC₅₀ for FBS-stimulated tube formation of HUVECs: 2.60 μM [5]

IC₅₀ for cytotoxicities against several cancer cell lines: ~3 μM [8]

IC₅₀ for in vitro microtubule polymerization: 10.9 ± 1.8 μM [8]

Depolymerization of micortubule network in HMEC cells: 1 μM [9]

References

1.

Li Z-L, Pan D-J, Chang-Qi H, Qin-Li W, Song-Song Y, Guang-Yi X (1982) Studies on the novel diterpenic constituents of Tu-Jin-Pi. I. Strucutre of pseudolaric acid A and pseudolaric acid B. Acta Chim Sin 05
2.

Trost BM, Waser J, Meyer A (2008) Total synthesis of (−)-pseudolaric acid B. J Am Chem Soc 130:16424–16434
3.

Pan DJ, Li ZL, Hu CQ, Chen K, Chang JJ, Lee KH (1990) The cytotoxic principles of Pseudolarix kaempferi: pseudolaric acid-A and -B and related derivatives. Planta Med 56:383–385
4.

Li E, Clark AM, Hufford CD (1995) Antifungal evaluation of pseudolaric acid B, a major constituent of Pseudolarix kaempferi. J Nat Prod 58:57–67
5.

Li MH, Miao ZH, Tan WF, Yue JM, Zhang C, Lin LP, Zhang XW, Ding J (2004) Pseudolaric acid B inhibits angiogenesis and reduces hypoxia-inducible factor 1α by promoting proteasome-mediated degradation. Clin Cancer Res 10:8266–8274
6.

Gong X, Wang M, Wu Z, Tashiro S, Onodera S, Ikejima T (2004) Pseudolaric acid B induces apoptosis via activation of c-Jun N-terminal kinase and caspase-3 in HeLa cells. Exp Mol Med 36:551–556
7.

Li T, Wong VK, Yi XQ, Wong YF, Zhou H, Liu L (2009) Pseudolaric acid B suppresses T lymphocyte activation through inhibition of NF-κB signaling pathway and p38 phosphorylation. J Cell Biochem 108:87–95
8.

Wong VK, Chiu P, Chung SS, Chow LM, Zhao YZ, Yang BB, Ko BC (2005) Pseudolaric acid B, a novel microtubule-destabilizing agent that circumvents multidrug resistance phenotype and exhibits antitumor activity in vivo. Clin Cancer Res 11:6002–6011
9.

Tong YG, Zhang XW, Geng MY, Yue JM, Xin XL, Tian F, Shen X, Tong LJ, Li MH, Zhang C, Li WH, Lin LP, Ding J (2006) Pseudolarix acid B, a new tubulin-binding agent, inhibits angiogenesis by interacting with a novel binding site on tubulin. Mol Pharmacol 69:1226–1233

Pyrrolizilactone

Keywords: [Proteasome inhibitor]

Structure

	Pyrrolizilactone
Molecular formula	C₂₄H₃₃NO₅
Molecular weight	415.53
Solubility	DMSO, ++; H₂O, −; MeOH, +++

Discovery/Isolation/Chemical Studies

Pyrrolizilactone was isolated from uncharacterized fungus in the course of screening program to find structurally unique metabolites from microorganisms on the basis of spectral data collected through LC/MS analysis [1].

Biological Studies

A molecular target of pyrrolizilactone was identified as a proteasome inhibitor by using two phenotypic profiling systems, cell morphology–based profiling analysis and proteome-based profiling analysis. Pyrrolizilactone is a novel type of proteasome inhibitor inhibiting the trypsin-like activity of the proteasome [2].

Biological Activity of Pyrrolizilactone

IC₅₀ values for HeLa and HL60: 9.5 and 2.5 μM, respectively [2]

IC₅₀ values for trypsin-, chymotrypsin-, and caspase-like protease activity of proteasome: 1.6, 29, and 84 μM, respectively [2]

Inhibition of cell cycle progression of HeLa in G2/M phase: 10 μM [2]

References

1.

Nogawa T, Kawatani M, Uramoto M, Okano A, Aono H, Futamura Y, Koshino H, Takahashi S, Osada H (2013) Pyrrolizilactone, a new pyrrolizidinone metabolite produced by a fungus. J Antibiot (Tokyo) 66:621–623
2.

Futamura Y, Kawatani M, Muroi M, Aono H, Nogawa T, Osada H (2013) Identification of a molecular target of a novel fungal metabolite, pyrrolizilactone, by phenotypic profiling systems. ChemBioChem 14:2456–2463

QS11*

Keywords: [Wnt/β-catenin signaling activator] [GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1) inhibitor]

Structure

	QS11
Molecular formula	C₃₆H₃₃N₅O₂
Molecular weight	567.68
Solubility	DMSO, +++; H₂O, −; MeOH, +++

Discovery/Isolation

QS11 is a purine derivative identified as a Wnt-3a synergist by a cell-based assay of Wnt/β-catenin signal transduction [1].

Biological Studies

QS11 specifically activated the reporter activity of Wnt/β-catenin signal transduction in the presence, but not in the absence, of Wnt-3a [1]. GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1) was identified as the QS11-target protein by an affinity-probe approach. Therefore, it is thought that QS11 activates ARF activity by inhibition of ARFGAP1, and modulates the localization of β-catenin in the cells. Further analysis suggested that the activation of ARF is necessary for both the production of phosphatidylinositol-4,5-bisphosphate and LRP6 phosphorylation, which is a critical initiation step for the transmission of Wnt/β-catenin signaling [2].

QS11 has also been used for investigation of other ARF-mediated processes including intercellular trafficking [3], interleukin signaling pathway [4], and endothelin-1-induced lipolysis [5].

Biological Activity of QS11

EC₅₀ for activation of the Wnt signal reporter in the presence of Wnt-3a: 0.5 μM [1]

K_d for ARFGAP1: 620 nM [1]

80% inhibition of MDA-MB-231 migration: 2.5 μM [1]

References

1.

Zhang Q, Major MB, Takanashi S, Camp ND, Nishiya N, Peters EC, Ginsberg MH, Jian X, Randazzo PA, Schultz PG, Moon RT, Ding S (2007) Small-molecule synergist of the Wnt/β-catenin signaling pathway. Proc Natl Acad Sci U S A 104:7444–7448
2.

Kim W, Kim SY, Kim T, Kim M, Bae DJ, Choi HI, Kim IS, Jho E (2013) ADP-ribosylation factors 1 and 6 regulate Wnt/β-catenin signaling via control of LRP6 phosphorylation. Oncogene 32:3390–3396
3.

Kanamarlapudi V, Thompson A, Kelly E, Lopez Bernal A (2012) ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling. J Biol Chem 287:20443–20455
4.

Zhu W, London NR, Gibson CC, Davis CT, Tong Z, Sorensen LK, Shi DS, Guo J, Smith MC, Grossmann AH, Thomas KR, Li DY (2012) Interleukin receptor activates a MYD88-ARNO-ARF6 cascade to disrupt vascular stability. Nature 492:252–255
5.

Davies JC, Bain SC, Kanamarlapudi V (2014) ADP-ribosylation factor 6 regulates endothelin-1-induced lipolysis in adipocytes. Biochem Pharmacol 90:406–413

Radicicol

Keywords: [HSP90 inhibitor] [ATP citrate lyase inhibitor (ACL)]

Structure

	Radicicol
Molecular formula	C₁₈H₁₇O₆Cl
Molecular weight	364.78
Solubility	DMSO, ++; H₂O, +; MeOH, +++

Discovery/Isolation/Chemical Studies

Radicicol was isolated as an antifungal substance from Monosporuim bonorden [1]. The compound was assigned the molecular formula C₁₇H₁₆O₇ on the basis of preliminary analytical data, but McCapra et al. revised the formula to C₁₈H₁₇O₆Cl [2].

Biological Studies

Radicicol was reisolated as the compound that induces the reversal of transformed phenotypes of v-src-transformed fibroblasts [3]. This compound showed various effects on mammalian cells, such as antiangiogenesis activity [4], suppression of transformation by RAS and MOS [5], differentiation of HL60 cells [6], and selective depletion of Raf-1 kinase [7].

The first report of the molecular target of radicicol was made by Sharma et al. [8]. They synthesized a biotinylated radicicol, and identified HSP90 as the molecular target of radicicol. Radicicol binds on the N-terminal ATP/ADP binding domain of HSP90 as a nucleotide mimic and inhibits inherent ATPase activity [9, 10]. Radicicol destabilizes Hsp90 client proteins including telomerase, mutated p53, Bcr-Abl, Raf-1, Akt, HER2/Neu, mutated B-Raf, mutated EGF receptor, and HIF-1α, and shows promising antitumor activity in various preclinical tumor models [11].

The second report was made by Ki et al. [12]. They also synthesized two functional biotinylated derivatives, and found that one derivative binds HSP90, but the other derivative binds another protein, ATP citrate lyase (ACL), a potential target for hypolipidaemic intervention. Because ACL was inhibited noncompetitively in vitro, these results imply that a different part in radicicol is required for each specific binding and that unmodified radicicol can bind both the proteins. It is likely that the introduction of the bulky biotin probe prevents these biotinylated compounds from access to one of these target molecules. It was also reported that radiciol binds on an ATP binding motif belonging to the GHKL superfamily, for example in archeal topoisomerase VI; however, radicicol inhibits the ATPase activity of yeast HSP90 at least 1000-fold more effectively than is seen for archaeal TopoVI [13].

Biological Activity of Radicicol

Morphological reversion and inhibition of src kinase of src-transformed 3Y1 cells: 0.1–1 μg/ml [3]

ID₅₀ for inhibition embryonic angiogenesis: 200 ng/egg [4]

IC₅₀ for Raf-1 depletion in KNRK5.2 cells: 3.1 μM [7]

K_d for HSP90: 19 nM [9]

K_i values for citrate and ATP against ACL: 13 and 7 μM, respectively [12]

References

1.

Delmotte P, Delmotte-Plaque J (1953) A new antifungal substance of fungal origin. Nature 171:344
2.

McCapra F, Scott AI (1964) The constitution of monorden, an antibiotic with tranquilising action. Tetrahedron Lett 15:869–875
3.

Kwon HJ, Yoshida M, Abe K, Horinouchi S, Beppu T (1992) Radicicol, an agent inducing the reversal of transformed phenotypes of src-transformed fibroblasts. Biosci Biotechnol Biochem 56:538–539
4.

Oikawa T, Ito H, Ashino H, Toi M, Tominaga T, Morita I, Murota S (1993) Radicicol, a microbial cell differentiation modulator, inhibits in vivo angiogenesis. Eur J Pharmacol 241:221–227
5.

Zhao JF, Nakano H, Sharma S (1995) Suppression of RAS and MOS transformation by radicicol. Oncogene 11:161–173
6.

Shimada Y, Ogawa T, Sato A, Kaneko I, Tsujita Y (1995) Induction of differentiation of HL-60 cells by the anti-fungal antibiotic, radicicol. J Antibiot (Tokyo) 48:824–830
7.

Soga S, Kozawa T, Narumi H, Akinaga S, Irie K, Matsumoto K, Sharma SV, Nakano H, Mizukami T, Hara M (1998) Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway. J Biol Chem 273:822–828
8.

Sharma SV, Agatsuma T, Nakano H (1998) Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene 16:2639–2645
9.

Roe SM, Prodromou C, O″Brien R, Ladbury JE, Piper PW, Pearl LH (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem 42:260–266
10.

Schulte TW, Akinaga S, Soga S, Sullivan W, Stensgard B, Toft D, Neckers LM (1998) Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. Cell Stress Chaperones 3:100–108
11.

Neckers L (2006) Using natural product inhibitors to validate Hsp90 as a molecular target in cancer. Curr Top Med Chem 6:1163–1171
12.

Ki SW, Ishigami K, Kitahara T, Kasahara K, Yoshida M, Horinouchi S (2000) Radicicol binds and inhibits mammalian ATP citrate lyase. J Biol Chem 275:39231–39236
13.

Gadelle D, Bocs C, Graille M, Forterre P (2005) Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol. Nucleic Acids Res 33:2310–2317

Rapamycin

Keywords: [Antifungal] [Immunosuppressant] [mTOR inhibitor]

Structure

	Rapamycin
Molecular formula	C₅₁H₇₉NO₁₃
Molecular weight	914.19
Solubility	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation

Rapamycin was isolated as an antifungal antibiotic produced by Streptomyces hygroscopicus [1, 2].

Biological Studies

Rapamycin inhibited the immune response in rats and the immunosuppressant activity of rapamycin appears to be related to inhibition of the lymphatic system [3]. Interestingly, FK506, which is structurally related to rapamycin, showed immunosuppressive properties by a different mechanism. Remarkably, these two drugs inhibit each other’s actions, raising the possibility that both act by means of a common immunophilin, FKBP [4]. Heitman et al. found that Saccharomyces cerevisiae treated with rapamycin was irreversibly arrested in the G1 phase and isolated TOR1 and TOR2 genes, which participate in rapamycin toxicity [5]. Brown et al. and Sabatini et al. identified a mammalian protein targeted by the G1-arresting rapamycin–receptor complex, FRAP, that is highly related to TOR1/2 [6, 7]. The rapamycin/FKBP complex selectively inhibits the p70^S6k activation cascade; however, the kinase activity of FRAP alone is not sufficient for control of p70^S6k [8–10]. This inhibition cascade results in the decrease of cap-dependent, but not cap-independent, translation, which is related to dephosphorylation and consequent activation of 4E–BP1/PHAS-I, a protein identified as a repressor of the cap binding protein, eIF-4E [11, 12].

Now it is known that mTOR is an atypical serine/threonine kinase that nucleates two distinct multiprotein complexes commonly known as mTORC1 that is generally associated with cell growth, and mTORC2 that promotes cell proliferation and survival [13]. Rapamycin selectively inhibits mTORC1, but not mTORC2.

Biological Activity of Rapamycin

K_d for rapamycin to FKBP: 0.2 nM [5]

Inhibition of IL-2-induced S-phase entry of T cells: 0.05–0.2 nM [8]

References

1.

Sehgal SN, Baker H, Vezina C (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J Antibiot (Tokyo) 28:727–732
2.

Vezina C, Kudelski A, Sehgal SN (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot (Tokyo) 28:721–726
3.

Martel RR, Klicius J, Galet S (1977) Inhibition of the immune response by rapamycin, a new antifungal antibiotic. Can J Physiol Pharmacol 55:48–51
4.

Bierer BE, Mattila PS, Standaert RF, Herzenberg LA, Burakoff SJ, Crabtree G, Schreiber SL (1990) Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc Natl Acad Sci U S A 87:9231–9235
5.

Heitman J, Movva NR, Hall MN (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905–909
6.

Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, Schreiber SL (1994) A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369:756–758
7.

Sabatini DM, Erdjument-Bromage H, Lui M, Tempst P, Snyder SH (1994) RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78:35–43
8.

Brown EJ, Beal PA, Keith CT, Chen J, Shin TB, Schreiber SL (1995) Control of p70 S6 kinase by kinase activity of FRAP in vivo. Nature 377:441–446
9.

Chung J, Kuo CJ, Crabtree GR, Blenis J (1992) Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69:1227–1236
10.

Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR (1992) Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 358 (6381):70–73
11.

Beretta L, Gingras AC, Svitkin YV, Hall MN, Sonenberg N (1996) Rapamycin blocks the phosphorylation of 4E–BP1 and inhibits cap-dependent initiation of translation. EMBO J 15:658–664
12.

Brunn GJ, Hudson CC, Sekulic A, Williams JM, Hosoi H, Houghton PJ, Lawrence JC, Jr., Abraham RT (1997) Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science 277:99–101
13.

Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149:274–293

Rebeccamycin

Keywords: [Topoisomerase I inhibitor] [Chk1 inhibitor]

Structure

	Rebeccamycin
Molecular formula	C₂₇H₂₁Cl₂N₃O₇
Molecular weight	570.38
Solubility	DMSO, ++; H₂O, ? ; MeOH, ?

Discovery/Isolation

Rebeccamycin is an indolocarbazole which was isolated from the mycelium of an actinomycete, strain C-38,383 as a novel antitumor agents [1].

Biological Studies

Rebeccamycin showed antitumor activity against P388 leukemia, L1210 leukemia, and B16 melanoma implanted in mice [1]. However rebeccamycin is structurally related to staurosporine, this compound has no inhibitory activity on protein kinases [2]. Instead, rebeccamycin induced topoisomerase I–mediated DNA cleavage [2]. Structure–activity relationship analyses using rebeccamycin analogs suggested that the stereochemistry of the sugar residue is the most important factor for interaction with DNA [3, 4].

Biological Activity of Rebeccamycin

DNA break in A549 cells: 0.15–5.0 μg/mL [1]

IC₅₀ for protein kinase A and protein kinase C in vitro: >100 μM [2]

Topoisomerase I–mediated DNA cleavage: 5 μM [3]

References

1.

Bush JA, Long BH, Catino JJ, Bradner WT, Tomita K. (1987) Production and biological activity of rebeccamycin, a novel antitumor agent. J Antibiot (Tokyo) 40:668–678
2.

Sancelme M, Fabre S, Prudhomme M. (1994) Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors. J Antibiot (Tokyo) 47:792–798
3.

Yamashita Y, Fujii N, Murakata C, Ashizawa T, Okabe M, Nakano H. (1992) Induction of mammalian DNA topoisomerase I mediated DNA cleavage by antitumor indolocarbazole derivatives. Biochemistry 31:12069–12075
4.

Bailly C, Qu X, Graves DE, Prudhomme M, Chaires JB. (1999) Calories from carbohydrates: energetic contribution of the carbohydrate moiety of rebeccamycin to DNA binding and the effect of its orientation on topoisomerase I inhibition. Chem Biol 6:277–286

Reveromycin A

Keywords: [Antiosteoporotic] [Detransforming agent] [Protein translation inhibitor]

Structure

	Reveromycin A
Molecular formula	C₃₆H₅₂O₁₁
Molecular weight	660.80
Solubility	DMSO, ++; H₂O, +; MeOH, ++

Discovery/Isolation

Reveromycin A was isolated as an inhibitor of mitogenic activity induced by EGF in a mouse epidermal keratinocyte [1, 2].

Biological Studies

Reveromycin A showed inhibitory activity against EGF-stimulated mitogen response in Balb/MK cells and exhibited morphological reversion of src^ts-NRK cells, antiproliferative activity against human tumor cell lines, and antifungal activity. In vitro studies revealed that reveromycin A is a selective inhibitor of protein synthesis in eukaryotic cells [3], and showed little antitumor effect against three murine tumors tested, but showed a strong antitumor effect against a human ovarian carcinoma BG-1 [4].

The molecular target of reveromycin A was determined by yeast genetics [5]. A Saccharomyces cerevisiae dominant mutant specifically resistant to reveromycin A was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNA^Ile synthetase (IleRS). Reveromycin A inhibited the activity of IleRS specifically and the mutation IleRS(N660D), near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for reveromycin A resistance. These results indicated that IleRS is a target of reveromycin A in vivo.

Because reveromycin A has three carboxyl groups in its structure, this compound accumulates in an acidic microenvironment, a prominent characteristic of mature bone-resorbing osteoclasts. In fact, reveromycin A induced apoptosis specifically in mature bone-resorbing osteoclasts, but not in osteoclast progenitors, nonfunctional osteoclasts, or osteoblasts [6]. Furthermore, reveromycin A inhibited the osteolytic bone metastasis of human small lung cancer cells in vivo [7]. These findings suggest that reveromycin A may inhibit osteolytic bone metastasis through suppression of osteoclast activity by directly inducing apoptosis and indirectly inhibiting tumor cell–derived parathyroid hormone–related peptide production.

Biological Activity of Reveromycin A

IC₅₀ for inhibition of protein synthesis in src^ts-NRK cells: 1 μM [3]

IC₅₀ for inhibition of protein synthesis in rabbit reticulocyte lysate: 40 nM [3]

Inhibition of cell proliferation induced by TGF-α in BG-1 cells: 30–300 nM [4]

Growth inhibition of Saccharomyces cerevisiae on YPD plate adjusted at pH 4.5: 1 μg/ml [8]

References

1.

Osada H, Koshino H, Isono K, Takahashi H, Kawanishi G (1991) Reveromycin A, a new antibiotic which inhibits the mitogenic activity of epidermal growth factor. J Antibiot (Tokyo) 44:259–261
2.

Takahashi H, Osada H, Koshino H, Kudo T, Amano S, Shimizu S, Yoshihama M, Isono K (1992) Reveromycins, new inhibitors of eukaryotic cell growth. I. Producing organism, fermentation, isolation and physico-chemical properties. J Antibiot (Tokyo) 45:1409–1413
3.

Takahashi H, Osada H, Koshino H, Sasaki M, Onose R, Nakakoshi M, Yoshihama M, Isono K (1992) Reveromycins, new inhibitors of eukaryotic cell growth. II Biological activities. J Antibiot (Tokyo) 45:1414–1419
4.

Takahashi H, Yamashita Y, Takaoka H, Nakamura J, Yoshihama M, Osada H (1997) Inhibitory action of reveromycin A on TGF-α-dependent growth of ovarian carcinoma BG-1 in vitro and in vivo. Oncol Res 9:7–11
5.

Miyamoto Y, Machida K, Mizunuma M, Emoto Y, Sato N, Miyahara K, Hirata D, Usui T, Takahashi H, Osada H, Miyakawa T (2002) Identification of Saccharomyces cerevisiae isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor Reveromycin A. J Biol Chem 277:28810–28814
6.

Woo JT, Kawatani M, Kato M, Shinki T, Yonezawa T, Kanoh N, Nakagawa H, Takami M, Lee KH, Stern PH, Nagai K, Osada H (2006) Reveromycin A, an agent for osteoporosis, inhibits bone resorption by inducing apoptosis specifically in osteoclasts. Proc Natl Acad Sci U S A 103:4729–4734
7.

Muguruma H, Yano S, Kakiuchi S, Uehara H, Kawatani M, Osada H, Sone S (2005) Reveromycin A inhibits osteolytic bone metastasis of small-cell lung cancer cells, SBC-5, through an antiosteoclastic activity. Clin Cancer Res 11:8822–8828
8.

Cui Z, Hirata D, Tsuchiya E, Osada H, Miyakawa T (1996) The multidrug resistance-associated protein (MRP) subfamily (Yrs1/Yor1) of Saccharomyces cerevisiae is important for the tolerance to a broad range of organic anions. J Biol Chem 271:14712–14716

R-Roscovitine*

Keywords: [Cyclin-dependent kinase (CDK) inhibitor]

Structure

	R-Roscovitine
Molecular formula	C₁₉H₂₆N₆O
Molecular weight	354.46
Solubility	DMSO, +++; H₂O, +; MeOH, +++

Discovery

R-roscovitine (named after Roscoff—a French town, where the lab that discovered the compound was located; seliciclib, CYC202) is the second CDK inhibitor to enter clinical trials. Roscovitine belongs to the family of purines and acts through competion with ATP for binding at the ATP binding site of CDKs. Consistent with these structural motifs, R-roscovitine inhibits the activity of CDK2 and CDK1, 5, 7, 8, and 9. R-roscovitine exposure to human diffuse large B-cell lymphoma (DLBCL) cells results in G1- and G2/M-phase arrest and induction of apoptosis independently of any underlying chromosomal translocations [1, 2].

Biological Studies

Roscovitine is a selective inhibitor of cyclin-dependent kinase (CDK), which has a key role in cell cycle regulation. Since CDKs are also known to be involved in the disregulation of cell growth and tumorgenesis, roscovitine is used for cancer therapy. In a study of the effects on the cell cycle in Lovo colorectal carcinoma cells, the major effect was the induction of cell death at all phases of the cell cycle—not merely in specific phases. Roscovitine has also been found to inhibit transition from prophase to metaphase at a range of micromolar concentrations. It has also been shown that it inhibits progesterone-induced oocyte maturation of Xenopus oocytes [3].

Biological Activity of R-Roscovitine

IC₅₀ values for cdc2 (cdc2/cyclin B), cdk2 (cdk2/cyclin A and cdk2/cyclin E), and cdk5 (cdk5/p35): 0.65 μM, 0.7 μM, 0.7 μM, and 0.16 μM respectively [3]

IC₅₀ values for Erk1 and Erk2: 34 μM and 14 μM respectively [3]

Average IC₅₀ for CDK2/cyclin E kinase activity in a panel of 19 human tumor cell lines: 15.2 μM [4]

References

1.

Lacrima K, Rinaldi A, Vignati S, Martin V, Tibiletti MG, Gaidano G, Catapano CV, Bertoni F (2007) Cyclin-dependent kinase inhibitor seliciclib shows in vitro activity in diffuse large B-cell lymphomas. Leuk Lymphoma 48:158–167
2.

Cicenas J, Kalyan K, Sorokinas A, Stankunas E, Levy J, Meskinyte I, Stankevicius V, Kaupinis A, Valius M (2015) Roscovitine in cancer and other diseases. Ann Transl Med 3:135
3.

Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP (1997) Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 243:527–536
4.

McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer PM, MacKenzie M, Melville J, Stewart K, Wang S, Zhelev N, Zheleva D, Lane DP (2002) In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 102: 463–468

Rottlerin

Keywords: [Autophagy inducer] [Kinase inhibitor]

Structure

	Rottlerin
Molecular formula	C₃₀H₂₈O₈
Molecular weight	516.55
Solubility	DMSO, +++; H₂O, −; MeOH, +

Discovery/Isolation

The first description of rottlerin dates from 1855. In this paper, Anderson isolated a crystalline compound named rottlerin from the Indian coloring and anthelmintic drug “kamala,” which is obtained from the fruit glands of Mallotus philippensis (Muell) [1].

Biological Studies

Rottlerin has been reported as a protein kinase C (PKC) δ inhibitor and many researchers have used rottlerin as a specific inhibitor of PKCδ [2]. However, a lot of reports strongly suggest that rottlerin is not a PKCδ-specific inhibitor [3]. Rottlerin inhibits mitochondrial function independently of PKCδ, and induces apoptosis [4,5].

Recently, rottlerin was screened as a modulator of autophagy [6]. Rottlerin inhibited mTORC1 signaling and this inhibition was dependent on the presence of TSC2, a negative regulator of mTORC1.

Inhibitory activity of rottlerin on amyloid polymerization was also reported [7].

Biological Activity of Rottlerin

Rapid reduction of cellular ATP in parotid acinar cells: 10 μM [4]

Inhibition of amyloid polymerization: 30 μM [7]

Autophagy induction in MCF-7 cells: 3 μM [6]

References

1.

Anderson (1855) The Edinburgh new philosophical journal 1:300
2.

Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, Marks F (1994) Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199:93–98
3.

Soltoff SP (2007) Rottlerin: an inappropriate and ineffective inhibitor of PKCδ. Trends Pharmacol Sci 28:453–458
4.

Soltoff SP (2001) Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks protein kinase Cδ tyrosine phosphorylation. J Biol Chem 276:37986–37992
5.

Tillman DM, Izeradjene K, Szucs KS, Douglas L, Houghton JA (2003) Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C. Cancer Res 63:5118–5125
6.

Balgi AD, Fonseca BD, Donohue E, Tsang TC, Lajoie P, Proud CG, Nabi IR, Roberge M (2009) Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling. PLoS One 4:e7124
7.

Feng BY, Toyama BH, Wille H, Colby DW, Collins SR, May BC, Prusiner SB, Weissman J, Shoichet BK (2008) Small-molecule aggregates inhibit amyloid polymerization. Nat Chem Biol 4:197–199

S1201*

Keywords: [Lysine-specific demethylase I inhibitor]

Structure

	S1201
Molecular formula	C₁₆H₁₆FNO
Molecular weight	257.30
Solubility	DMSO, +++; H₂O, +; MeOH, +

Discovery

S1201 was synthesized as a compound to inhibit lysine-specific demethylase-1 (LSD1), based on the structures of LSD1 and monoamine oxidase B (MAO-B), in complex with an antidepressant tranylcypromine derivative [1].

Biological Studies

LSD1, a unique nuclear protein that utilizes FAD as a cofactor, regulates eukaryotic gene expression through chromatin structure alteration by demethylates histone H3 and DNA methyltransferase 1 (Dnmt1). S1201 strongly and specifically inhibited LSD1 [1]. The S1201-bound LSD1 structure revealed the enhanced stability of the S1201-FAD adducts by their interactions with the surrounding LSD1 residues. S1201 treatments resulted in the activation of mitochondrial respiration through the induction of a number of regulators of energy expenditure and mitochondrial methabolism in adipocytes [2]. Therefore, LSD1 is thought to regulate cellular energy balance through coupling with cellular FAD biosynthesis.

Biological Activity of S1201

k_inact/K_i for demethylation activity of LSD1: 4560 M⁻¹ s⁻¹ [1]

References

1.

Mimasu S, Umezawa N, Sato S, Higuchi T, Umehara T, Yokoyama S (2010) Structurally designed trans-2-phenylcyclopropylamine derivatives potently inhibit histone demethylase LSD1/KDM1. Biochemistry 49:6494–6503
2.

Hino S, Sakamoto A, Nagaoka K, Anan K, Wang Y, Mimasu S, Umehara T, Yokoyama S, Kosai K, Nakao M (2012) FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure. Nat Commun 3:758

SC1* (Pluripotin)

Keywords: [Self-renewal] [Dual inhibitor of RasGAP and ERK1]

Structure

	SC1 (pluripotin)
Molecular formula	C₂₇H₂₅F₃N₈O₂
Molecular weight	550.54
Solubility	DMSO, +++; H₂O, ±; MeOH, +

Discovery/Isolation

SC1/pluripotin was discovered by a cell-based screen of chemical libraries to identify small molecules that control the self-renewal of ES cells [1].

Biological Studies

SC1 propagated murine ES cells (mES) in an undifferentiated, pluripotent state for >10 passages under chemically defined conditions in the absence of feeder cells, serum, and LIF. SC1 inhibited differentiation of mES induced by either 20% FBS or 1 μM retinoic acid. After removal of SC1, SC1-expanded mES cells (>10 passages) were differentiated into neural/neuronal, cardiac muscle, and endodermal cells and could generate healthy chimeric mice in vivo.

Using SC1-affinity beads, ERK1 and RasGAP were identified as the SC1 binding proteins. Biochemical and cellular experiments suggest that SC1 acts through two targets: by inhibiting RasGAP, SC1 blocks RasGAPs differentiation-inducing activity and activates self-renewal pathways, while at the same time blocking ERK-dependent differentiation.

SC1 could be used for the efficient establishment of ES cells from immunodeficient mice on refractory backgrounds [2] and a tractable cancer stem cell model [3].

Biological Activity of SC1 (Pluripotin)

Self-renewal of mES for >10 passages in an undifferentiated/pluripotent state: 1 μM [1]

K_d values for ERK1 and RasGAP: 98 and 212 nM, respectively [1]

References

1.

Chen S, Do JT, Zhang Q, Yao S, Yan F, Peters EC, Scholer HR, Schultz PG, Ding S (2006) Self-renewal of embryonic stem cells by a small molecule. Proc Natl Acad Sci U S A 103:17266–17271
2.

Yang W, Wei W, Shi C, Zhu J, Ying W, Shen Y, Ye X, Fang L, Duo S, Che J, Shen H, Ding S, Deng H (2009) Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains. Stem Cells 27:383–389
3.

Mertins SD, Scudiero DA, Hollingshead MG, Divelbiss RD, Jr., Alley MC, Monks A, Covell DG, Hite KM, Salomon DS, Niederhuber JE (2013) A small molecule (pluripotin) as a tool for studying cancer stem cell biology: proof of concept. PLoS One 8:e57099

Sirtinol*

Keywords: [NAD-dependent lysine deacetylase inhibitor]

Structure

	Sirtinol
Molecular formula	C₂₆H₂₂N₂O₂
Molecular weight	394.47
Solubility	DMSO, +++; H₂O, ? ; MeOH, ?

Discovery/Isolation

Sirtinol is an NAD-dependent lysine deacetylase inhibitor with a structurally related α-substituted β-naphthol analog, which was identified by high-throughput cell-based phenotypic screening using yeast [1].

Biological Studies

Sirtinol inhibits Sir2p-mediated transcriptional silencing at three loci in yeast [2]. A chemical–genetic screen for Arabidopsis mutants resistant to sirtinol identified SIR1 as an upstream regulator in the auxin signaling pathway [2]. Sirtinol activates auxin-signal transduction and inhibits root elongation in Arabidopsis [1, 2]. In mammals, sirtinol has been shown to induce senescence-like growth arrest by impairing activation of MAPK pathways in human breast and lung cancer cells [3] and to induce apoptosis and autophagic cell death in human breast cancer cells [4]. Anti-inflammatory activity of sirtinol in human dermal microvascular endothelial cells was also reported [5].

Biological Activity of Sirtinol

In vitro IC₅₀ for yeast Sir2p and human SIRT2: 68 μM and 38 μM [1]

IC₅₀ for proliferation of human breast cancer MCF-7 cells: 48.6 μM (24 hours) and 43.5 μM (48 hours) [4]

References

1.

Grozinger CM, Chao ED, Blackwell HE, Moazed D, Schreiber SL (2001) Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 276:38837–38843
2.

Zhao Y, Dai X, Blackwell HE, Schreiber SL, Chory J (2003) SIR1, an upstream component in auxin signaling identified by chemical genetics. Science 301:1107–1110
3.

Ota H, Tokunaga E, Chang K, Hikasa M, Iijima K, Eto M, Kozaki K, Akishita M, Ouchi Y, Kaneki M (2006) Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells. Oncogene 25:176–85
4.

Wang J, Kim TH, Ahn MY, Lee J, Jung JH, Choi WS, Lee BM, Yoon KS, Yoon S, Kim HS (2011) Sirtinol, a class III HDAC inhibitor, induces apoptotic and autophagic cell death in MCF-7 human breast cancer cells. Int J Oncol 41:1101–1109
5.

Orecchia A, Scarponi C, Di Felice F, Cesarini E, Avitabile S, Mai A, Mauro ML, Sirri V, Zambruno G, Albanesi C, Camilloni G, Failla CM (2011) Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells. PLoS One 6:e24307

Spliceostatin A*

Keywords: [Antitumor] [Splicing inhibitor] [SF3b]

Structure

	Spliceostatin A
Molecular formula	C₂₈H₄₃NO₈
Molecular weight	521.64
Solubility	DMSO, +++; H₂O, ±; MeOH, +

Discovery/Isolation/Chemical Study

Spliceostatin A is a methylated derivative of FR901464, a natural product isolated from the culture broth of a bacterium of Pseudomonas sp. No.2663 as an antitumor compound [1–4].

Biological Studies

FR901464 inhibited cell cycle progression at G1 and G2 phases by producing a C-terminally truncated p27, named p27* [5]. Spliceostatin A inhibited in vitro splicing and promoted pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Detailed investigation of molecular mechanism revealed that spliceostatin A prevents interaction of an SF3b 155 kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5′ of the branch point [6], and abolishes 3′ end pausing of polymerases [7]. Furthermore, this inhibition induces leakage of unspliced transcripts to the nucleoplasm [7] and to the cytoplasm [5]. These results clearly indicate that spliceostatin A targets SF3b and inhibits splicing of pre-mRNA, and that the inhibition of pre-mRNA splicing at least during early steps involving SF3b allows unspliced mRNA leakage and translation.

Biological Activity of Spliceostatin A

Inhibition of in vitro splicing: >60 nM [5]

Inhibition of in vivo splicing and pre-mRNA translation: 100 ng/ml [5]

Inhibition of in vivo tumor angiogenesis in CAM assay: 2 nM [8]

References

1.

Motoyoshi H, Horigome M, Ishigami K, Yoshida T, Horinouchi S, Yoshida M, Watanabe H, Kitahara T (2004) Structure-activity relationship for FR901464: a versatile method for the conversion and preparation of biologically active biotinylated probes. Biosci Biotechnol Biochem 68:2178–2182
2.

Nakajima H, Hori Y, Terano H, Okuhara M, Manda T, Matsumoto S, Shimomura K (1996) New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action. J Antibiot (Tokyo) 49:1204–1211
3.

Nakajima H, Sato B, Fujita T, Takase S, Terano H, Okuhara M (1996) New antitumor substances, FR901463, FR901464 and FR901465. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities. J Antibiot (Tokyo) 49:1196–1203
4.

Nakajima H, Takase S, Terano H, Tanaka H (1997) New antitumor substances, FR901463, FR901464 and FR901465. III. Structures of FR901463, FR901464 and FR901465. J Antibiot (Tokyo) 50:96–99
5.

Kaida D, Motoyoshi H, Tashiro E, Nojima T, Hagiwara M, Ishigami K, Watanabe H, Kitahara T, Yoshida T, Nakajima H, Tani T, Horinouchi S, Yoshida M (2007) Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat Chem Biol 3:576–583
6.

Corrionero A, Minana B, Valcarcel J (2011) Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A. Genes Dev 25:445–459
7.

Martins SB, Rino J, Carvalho T, Carvalho C, Yoshida M, Klose JM, de Almeida SF, Carmo-Fonseca M (2011) Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes. Nat Struct Mol Biol 18:1115–1123
8.

Furumai R, Uchida K, Komi Y, Yoneyama M, Ishigami K, Watanabe H, Kojima S, Yoshida M (2010) Spliceostatin A blocks angiogenesis by inhibiting global gene expression including VEGF. Cancer Sci 101:2483–2489

Staurosporine

Keywords: [Cell cycle inhibitor] [Kinase inhibitor]

Structure

	Staurosporine
Molecular formula	C₂₈H₂₆N₄O₃
Molecular weight	466.53
Solubility	DMSO, ++; H₂O, −; MeOH, +

Discovery/Isolation

Staurosporine is the first natural indolocarbazole compound [1]. Thereafter, a lot of indolocarbazoles have been isolated from a variety of organisms [2] or synthesized by chemical synthesis and by engineered biosynthetic pathways.

Biological Studies

This compound was originally discovered as an antifungal antibiotic [1]. However, it was shown that staurosporine possesses various biological activities, including induction of differentiation [2], apoptosis induction [3], cell cycle inhibition [4], and so on. It is important to use this compound as a potent but nonspecific protein kinase inhibitor [5–7], and 100 kinases were identified as staurosporine binding kinases in HepG2 cells [8].

Recently, there has been application of analog-sensitive (AS) kinase technology of staurosporine to investigation of phospho-signaling pathways in diverse organisms and physiological processes [9].

There are good reviews about the characteristics, biological activities, and usage of staurosporine [10].

Biological Activity of Staurosporine

IC₅₀ for protein kinase C from rat brain: 2.7 nM [5]

Reversible cell cycle arrest of rat 3Y1 fibroblasts at the early G1 phase: 1–10 ng/ml [4]

Reversible cell cycle arrest of rat 3Y1 fibroblasts at G2: 100 ng/ml [4]

Apoptosis induction in MOLT-4 cells: 10–200 nM [3]

References

1.

Omura S, Iwai Y, Hirano A, Nakagawa A, Awaya J, Tsuchya H, Takahashi Y, Masuma R (1977) A new alkaloid AM-2282 of Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. J Antibiot (Tokyo) 30:275–282
2.

Morioka H, Ishihara M, Shibai H, Suzuki T (1985) Staurosporine-induced differentiation in a human neuroblastoma cell line, NB-1. Agric Biol Chem 49:1959–1963
3.

Falcieri E, Martelli AM, Bareggi R, Cataldi A, Cocco L (1993) The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation. Biochem Biophys Res Commun 193:19–25
4.

Abe K, Yoshida M, Usui T, Horinouchi S, Beppu T (1991) Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases. Exp Cell Res 192:122–127
5.

Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca²⁺ dependent protein kinase. Biochem Biophys Res Commun 135:397–402
6.

Nakano H, Kobayashi E, Takahashi I, Tamaoki T, Kuzuu Y, Iba H (1987) Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60. J Antibiot (Tokyo) 40:706–708
7.

Fujita-Yamaguchi Y, Kathuria S (1988) Characterization of receptor tyrosine-specific protein kinases by the use of inhibitors. Staurosporine is a 100-times more potent inhibitor of insulin receptor than IGF-I receptor. Biochem Biophys Res Commun 157:955–962
8.

Fischer JJ, Graebner OY, Dalhoff C, Michaelis S, Schrey AK, Ungewiss J, Andrich K, Jeske D, Kroll F, Glinski M, Sefkow M, Dreger M, Koester H (2010) Comprehensive identification of staurosporine-binding kinases in the hepatocyte cell line HepG2 using Capture Compound Mass Spectrometry (CCMS). J Proteome Res 9:806–817
9.

Lopez MS, Choy JW, Peters U, Sos ML, Morgan DO, Shokat KM (2013) Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology. J Am Chem Soc 135:18153–18159
10.

Nakano H, Omura S (2009) Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine. J Antibiot (Tokyo) 62:17–26

TC11*

Keywords: [Antitumor] [Nucleophosmin 1 inhibitor]

Structure

	TC11
Molecular formula	C₂₀H₂₂N₂O₂
Molecular weight	322.41
Solubility	DMSO, +++; H₂O, −; MeOH, ±

Discovery/Isolation

TC11 is a phthalimide derivative identified as antitumor compound against several multiple myeloma cell line [1].

Biological Studies

TC11 is a potent inhibitor of tumor cell proliferation and showed antitumor activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice [1]. The target protein was identified to be nucleophosmin 1 by mRNA display selection on a microfluidic chip. TC11 treatment and knockdown of nucleophosmin 1 inhibited centrosomal clustering, and induced multipolar cells. These results suggest that nucleophosmin 1 is a novel antitumor target protein. Further analyses revealed that TC11 also significantly inhibited osteoclasts and induced apoptosis of myeloma [2].

Biological Activity of TC11

IC₅₀ for several multiple myeloma cell lines: 4–8 μM [1]

K_d values for oligomeric and monomeric fractions of nucleophosmin 1: 1.3 × 10⁻⁴ and 6.6 × 10⁻⁸ M, respectively [1]

Formation of multipolar spindle in HeLa cells: 5 μM [1]

References

1.

Shiheido H, Terada F, Tabata N, Hayakawa I, Matsumura N, Takashima H, Ogawa Y, Du W, Yamada T, Shoji M, Sugai T, Doi N, Iijima S, Hattori Y, Yanagawa H (2012) A phthalimide derivative that inhibits centrosomal clustering is effective on multiple myeloma. PLoS One 7:e38878
2.

Matsushita M, Ozaki Y, Hasegawa Y, Terada F, Tabata N, Shiheido H, Yanagawa H, Oikawa T, Matsuo K, Du W, Yamada T, Hozumi M, Ichikawa D, Hattori Y. (2015) A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts. PLoS One 10:e0116135

Terpendole E

Keywords: [Kinesin spindle protein (KSP) inhibitor]

Structure

	Terpendole E	11-Ketopaspaline
Molecular formula	C₂₈H₃₉NO₃	C₂₈H₃₇NO₃
Molecular weight	437.62	435.61
Solubility	DMSO, +++; H₂O, ±; MeOH, +	DMSO, +++; H₂O, ±; MeOH, +

Discovery/Isolation/Chemical Studies

Terpendoles were isolated as acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors from Albophoma yamanashiensis [1–3]. 11-Ketopaspaline was isolated from the strain whose biosynthetic pathway of terpendole E was genetically modified [4].

Biological Studies

Terpendole E is a minor derivative and possesses only weak ACAT inhibitory activity [3]. However, this compound was rediscovered as an inhibititor of cell cycle progression in M phase from the Ascomycota fungus Tolypocladium album (syn. Chaunopycnis alba) [5]. Terpendole E accumulated M-phase cells containing monoastar spindle by inhibiting the bipolar spindle formation. Detailed in vitro analyses revealed that terpendole E is a natural Eg5/KSP-specific inhibitor. Terpendole E and its derivative 11-ketopaspaline inhibit the ATPase activity not only of wild-type but also S-trityl-L-cysteine- or GSK1-resistant Eg5, suggesting that these compounds have a different binding site and/or inhibitory mechanism from S-trityl-L-cysteine or GSK1 [4].

Biological Activity of Terpendole E

IC₅₀ for in vitro ACAT activity: 228 μM [3]

Inhibition of cell cycle progression in M phase in 3Y1, A549, and HeLa cells: 50 μM [5]

IC₅₀ for microtubule-stimulated ATPase activity of recombinant Eg5: 23 μM [5]

References

1.

Huang XH, Tomoda H, Nishida H, Masuma R, Omura S (1995) Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis. I. Production, isolation and biological properties. J Antibiot (Tokyo) 48: 1–4
2.

Huang XH, Nishida H, Tomoda H, Tabata N, Shiomi K, Yang DJ, Takayanagi H, Omura S (1995) Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis. II. Structure elucidation of terpendoles A, B, C and D. J Antibiot (Tokyo) 48: 5–11
3.

Tomoda H, Tabata N, Yang DJ, Takayanagi H, Omura S (1995) Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis. III. Production, isolation and structure elucidation of new components. J Antibiot (Tokyo) 48: 793–804
4.

Tarui Y, Chinen T, Nagumo Y, Motoyama T, Hayashi T, Hirota H, Muroi M, Ishii Y, Kondo H, Osada H, Usui T (2014) Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5. ChemBioChem 15: 934–938
5.

Nakazawa J, Yajima J, Usui T, Ueki M, Takatsuki A, Imoto M, Toyoshima YY, Osada H (2003) A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5. Chem Biol 10: 131–137

Terpestacin

Keywords: [Antiagiogenesis] [HIV-1 inhibitor] [Mitochondria complex III inhibitor]

Structure

	Terpestacin
Molecular formula	C₂₅H₃₈O₄
Molecular weight	402.57
Solubility	DMSO, ++; H₂O, ±; MeOH, +++

Discovery/Isolation/Chemical Study

Terpestacin was isolated from Arthrinium sp. FA1744 (ATCC 74132) as an inhibitor of syncytium formation of HIV-infected T4 cells [1]. Total synthesis of natural terpestacin [2] and enantioselective synthesis [3] have been reported.

Biological Studies

Terpestacin potently inhibited the syncytium formation of BSC-1 cells infected with recombinant vaccinia virus expressing HIV-1 gp120 and gp41 and HeLa-T4 cells expressing CD4 antigen [1]. This compound also showed antiangiogenic activity [4]. A 13.4 kDa subunit of mitochondrial complex III (ubiquinol–cytochrome c reductase binding protein, UQCRB) was identified as a molecular target protein of terpestacin by phage-display affinity selection using biotinylated terpestacin [5]. The UQCRB binding of terpestacin resulted in the inhibition of hypoxia-induced ROS generation, followed by blocking of HIF-1α activation and tumor angiogenesis in vivo. Further analyses revealed that UQCRB plays a crucial role in the oxygen-sensing mechanism that regulates responses to hypoxia including VEGF expression [5, 6].

Biological Activity of Terpestacin

ID₅₀ for syncytium formation: 0.46 μg/ml [1]

Inhibition of BAECs tube formation stimulated by 30 ng/ml bFGF: 10 μg/ml [4]

Instability of HIF-1α under hypoxia conditions: 50 μM [5]

References

1.

Oka M, Iimura S, Tenmyo O, Sawada Y, Sugawara M, Ohkusa N, Yamamoto H, Kawano K, Hu SL, Fukagawa Y, et al. (1993) Terpestacin, a new syncytium formation inhibitor from Arthrinium sp. J Antibiot (Tokyo) 46:367–373
2.

Tatsuta K, Masuda N (1998) The first total synthesis of natural (+)-terpestacin, syncytium formation inhibitor. J Antibiot (Tokyo) 51:602–606
3.

Myers AG, Siu M, Ren F (2002) Enantioselective synthesis of (−)-terpestacin and (−)-fusaproliferin: clarification of optical rotational measurements and absolute configurational assignments establishes a homochiral structural series. J Am Chem Soc 124:4230–4232
4.

Jung HJ, Lee HB, Kim CJ, Rho JR, Shin J, Kwon HJ (2003) Anti-angiogenic activity of terpestacin, a bicyclo sesterterpene from Embellisia chlamydospora. J Antibiot (Tokyo) 56:492–496
5.

Jung HJ, Shim JS, Lee J, Song YM, Park KC, Choi SH, Kim ND, Yoon JH, Mungai PT, Schumacker PT, Kwon HJ (2010) Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing. J Biol Chem 285:11584–11595
6.

Cho YS, Jung HJ, Seok SH, Payumo AY, Chen JK, Kwon HJ (2013) Functional inhibition of UQCRB suppresses angiogenesis in zebrafish. Biochem Biophys Res Commun 433:396–400

Thalidomide, Lenalidomide

Keywords: [Cereblon inhibitor] [E3 ubiquitin ligase inhibitor]

Structure

	Thalidomide	Lenalidomide
Molecular formula	C₁₃H₁₀N₂O₄	C₁₃H₁₃N₃O₃
Molecular weight	258.23	259.26
Solubility	DMSO, +++; H₂O, −; MeOH, +	DMSO, +++; H₂O, −; MeOH, +

Discovery

Thalidomide is a synthetic drug marketed in 1957 by Chemie Grunenthal as a sedative, hypnotic, nausea, and morning sickness drug for pregnant women [1], but the use of thalidomide was prohibited in many countries because of its teratogenic properties [2].

Biological Studies

Thalidomide caused many infants to be born with phocomelia and amelia because of its teratogenic effects. However, the discovery of benefical effects in erythema nodosum leprosum remarked the potential of thalidomide as a medicine again [3]. Furthermore, its antiangiogenesis activity has suggested that thalidomide is a promising medicine for cancer therapy [4]. Today, thalidomide and its derivatives, for example lenalidomide [5], have been used as a treatment for a complication of leprosy and multiple myeloma.

Using high-performance affinity beads, cereblon (CRBN), a component of E3 ubiquitin ligase complex, was identified as the binding protein of thalidomide [6]. This E3 complex has been shown to be important for limb outgrowth and expression of FGF8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity.

Thalidomide and its derivatives promote the degradation of Ikaros proteins, transcription factors IKZF1 and IKZF3, in a CRBN-dependent fashion by altering the specificity of the E3 ubiquitin ligase complex [7, 8]. The crystal structure of the human CRBN-DDB1-thalidomide derivative complex was reported [9]. Based on the complex structure, it has revealed that the key drug binding residues in the thalidomide binding domain are critical for antiproliferative effects. These studies reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

Biological Activity of Thalidomide and Lenalidomide: Thalidomide

Thalidomide-induced teratogenicity in zebrafish: 400 μM [6]

Lenalidomide

Down-regulation of Ikaros proteins by lenalidomide: 2 μM [8]

References

1.

Franks ME, Macpherson GR, Figg WD. (2004) Thalidomide. Lancet 363:1802–1811
2.

Speirs AL (1962) Thalidomide and congenital abnormalities. Lancet 279:303–305
3.

Sheskin L. Thalidomide in the treatment of lepra reaction. (1965) Clin Pharmacol Ther 6303–306
4.

D’Amato RJ, Loughnan MS, Flynn E, Folkman J.(1994) Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A 91:4082–4085
5.

Tsenova L, Mangaliso B, Muller G, Chen Y, Freedman VH, Stirling D, Kaplan G. (2002) Use of IMiD3, a thalidomide analog, as an adjunct to therapy for experimental tuberculous meningitis. Antimicrob Agents Chemother 46:1887–1895
6.

Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, Handa H (2010) Identification of a primary target of thalidomide teratogenicity. Science 327:1345–1350
7.

Licht JD, Shortt J, Johnstone R (2014) From anecdote to targeted therapy: the curious case of thalidomide in multiple myeloma. Cancer Cell 25:9–11
8.

Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, Wong KK, Bradner JE, Kaelin WG, Jr. (2014) The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343:305–309
9.

Chamberlain PP, Lopez-Girona A, Miller K, Carmel G, Pagarigan B, Chie-Leon B, Rychak E, Corral LG, Ren YJ, Wang M, Riley M, Delker SL, Ito T, Ando H, Mori T, Hirano Y, Handa H, Hakoshima T, Daniel TO, Cathers BE (2014) Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat Struct Mol Biol 21:803–809

Theonellamide F

Keywords: [Antifungal] [3β-hydroxysterol]

Structure

	Theonellamide F
Molecular formula	C₆₉H₈₆Br₂N₁₆O₂₂
Molecular weight	1651.35
Solubility	DMSO, +++; H₂O, ++; MeOH, +++

Discovery/Isolation/Chemical Study

Theonellamide F was isolated as an antifungal dodecapeptide from a marine sponge, genus Theonella. Its bicyclic structure, which contains an unprecedented histidinoalanine, was unequivocally determined [1].

Biological Studies

Theonellamide F induced the formation of many abnormally large vacuoles around the nucleus in the cells [2]. This effect was more drastic in an amino acid–deficient medium, suggesting that theonellamide F affects cellular autophagy by inhibiting the degradation of the organelles and turnover of proteins. Acidification by vacuolar ATPase (V-ATPase) was required to maintain the induced vacuoles [3].

Using affinity bead covalently bound theonellamide A, 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase were identified as the binding proteins [4]. However, the in vitro inhibitory activity could not account for the effects of theollenamide F. A chemical–genomic profile of theonellamide F used a collection of fission yeast strains in which each open reading frame is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-β-D-glucan synthesis. Furthermore analysis revealed that theonellamides specifically bind to 3β-hydroxysterols, including ergosterol, and cause membrane damage. These results suggest that theonellamides are a new class of sterol binding molecules that induce membrane damage and activate Rho1-mediated 1,3-β-D-glucan synthesis [5].

Biological Activity of Theonellamide F

Vacuolation in exponentially growing 3Y1 cells: 6 μM [2]

Elevation of 1,3-β-D-glucan synthesis in Schizosaccharomyces pombe: 5 μg/ml [5]

References

1.

Matsunaga S, Fusetani N, Hashimoto K, Walchli M (1989) Theonellamide F. A novel antifungal bicyclic peptide from a marine sponge Theonella sp. J Am Chem Soc 111:2582–2588
2.

Wada S, Matsunaga S, Fusetani N, Watabe S (1999) Theonellamide F, a bicyclic peptide marine toxin, induces formation of vacuoles in 3Y1 rat embryonic fibroblast. Mar Biotechnol (NY) 1:337–341
3.

Wada S, Kantha S, Yamashita T, Matsunaga S, Fusetani N, Watabe S (2002) Accumulation of H⁺ in vacuoles induced by a marine peptide toxin, theonellamide F, in rat embryonic 3Y1 fibroblasts. Mar Biotechnol (NY) 4:571–582
4.

Wada S, Matsunaga S, Fusetani N, Watabe S (2000) Interaction of cytotoxic bicyclic peptides, theonellamides A and F, with glutamate dehydrogenase and 17β-hydroxysteroid dehydrogenase IV. Mar Biotechnol (NY) 2:285–292
5.

Nishimura S, Arita Y, Honda M, Iwamoto K, Matsuyama A, Shirai A, Kawasaki H, Kakeya H, Kobayashi T, Matsunaga S, Yoshida M (2010) Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling. Nat Chem Biol 6:519–526

Trapoxin B

Keywords: [Cell cycle inhibitor] [Histone deacetylase (HDAC) inhibitor]

Structure

	Trapoxin B
Molecular formula	C₃₃H₄₀N₄O₆
Molecular weight	588.71
Solubility	DMSO, +++; H₂O, ±; MeOH, ++

Discovery/Isolation

Trapoxin B, a cyclotetrapeptide, was isolated from the culture broth of Helicoma ambiens RF-1023 as antitumor compound [1].

Biological Studies

Trapoxin B exhibits detransformation activities against v-sis oncogene-transformed NIH/3T3 cells as antitumor agents [1]. Kijima et al. found that trapoxin B accumulated highly acetylated core histones in a variety of mammalian cell lines [2]. In vitro experiments using partially purified mouse histone deacetylase (HDAC) showed that trapoxin B irreversibly inhibited deacetylation of acetylated histone molecules probably by covalent binding to histone deacetylase via the epoxide. This covalent binding ability made it possible to isolate histone deacetylase HDAC1 by using a synthesized trapoxin affinity matrix [3]. Isolated histone deacetylase protein is very similar to the yeast transcriptional regulator Rpd3p; these results support a role for histone deacetylase as a key regulator of eukaryotic transcription, and expression of several proteins has been shown, including transcription factor [4], cell cycle inhibitor [5, 6], and so on.

It was also revealed that HDAC proteins form complexes with protein phosphatases [7] or hypoxia-inducible factor 1α [8], suggesting that HDACs regulate not only protein expression but also phosphorylation/stabilization of protein complexes.

Another HDAC inhibitor, trichostatin A, inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin B cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. Using this differential sensitivity to HDAC6, it was revealed that the acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules [9].

Biological Activity of Trapoxin B

ID₅₀ for growth of sis-transformed NIH3T3 cells: 200 ng/ml [1]

Histone hyperacetylation in various cell lines: 100 nM [2]

MIC values for FM3A and TR303, trichostatin-resistant mutant of FM3A: 2 and 50 ng/ml, respectively [2]

References

1.

Itazaki H, Nagashima K, Sugita K, Yoshida H, Kawamura Y, Yasuda Y, Matsumoto K, Ishii K, Uotani N, Nakai H, et al. (1990) Isolation and structural elucidation of new cyclotetrapeptides, trapoxins A and B, having detransformation activities as antitumor agents. J Antibiot (Tokyo) 43:1524–1532
2.

Kijima M, Yoshida M, Sugita K, Horinouchi S, Beppu T (1993) Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase. J Biol Chem 268:22429–22435
3.

Taunton J, Hassig CA, Schreiber SL (1996) A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272:408–411
4.

Hassig CA, Fleischer TC, Billin AN, Schreiber SL, Ayer DE (1997) Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89:341–347
5.

Kim YB, Ki SW, Yoshida M, Horinouchi S (2000) Mechanism of cell cycle arrest caused by histone deacetylase inhibitors in human carcinoma cells. J Antibiot (Tokyo) 53:1191–1200
6.

Sambucetti LC, Fischer DD, Zabludoff S, Kwon PO, Chamberlin H, Trogani N, Xu H, Cohen D (1999) Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 274:34940–34947
7.

Brush MH, Guardiola A, Connor JH, Yao TP, Shenolikar S (2004) Deacetylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases. J Biol Chem 279:7685–7691
8.

Qian DZ, Kachhap SK, Collis SJ, Verheul HM, Carducci MA, Atadja P, Pili R (2006) Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1α. Cancer Res 66:8814–8821
9.

Matsuyama A, Shimazu T, Sumida Y, Saito A, Yoshimatsu Y, Seigneurin-Berny D, Osada H, Komatsu Y, Nishino N, Khochbin S, Horinouchi S, Yoshida M (2002) In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 21:6820–6831

Trichostatin A

Keywords: [Cell cycle inhibitor] [Histone deacetylase (HDAC) inhibitor]

Structure

	Trichostatin A
Molecular formula	C₁₇H₂₂N₂O₃
Molecular weight	302.37
Solubility	DMSO, ++; H₂O, −; MeOH, ++

Discovery/Isolation

Trichostatin A was isolated from the metabolites of Streptomyces hygroscopicus as active compounds against trichophytons and some fungi [1], and reisolated as a potent inducer of differentiation in murine erythroleukemia cells [2].

Biological Studies

Trichostatin A blocked cell proliferation by inhibiting cell cycle progression specifically at the G1 and G2 phases [3]. Interestingly, when trichostatin A–arrested cells at the G2 phase were released from the inhibition, cells with 4C DNA entered a new S phase without passage through the M phase, resulting in the formation of proliferative tetraploid cells. Trichostatin A induces not only the morphological reversion of sis- or ras-transformed NIH/3T3, T24, and HeLa [4, 5], but also apoptosis [6, 7].

The molecular target of trichostatin A was biochemically determined as histone deacetylase (HDAC) [8]. Now it has been revealed that trichostatin A is a potent and specific inhibitor of the class I and II HDACs, and induces histone hyperacetylation. Because transcriptionally active chromatin is associated with hyperacetylated histones in general, the treatment of trichostatin A drastically changes the gene expression profiles [9, 10], and in vitro effects of trichostatin A on cell proliferation and differentiation can be attributed to the inhibition of HDACs [8].

Trichostatin A is a good bioprobe for investigating the functions of acetylation of histone on gene expression [9, 11], the relationships of histone acetylation and methylation [12, 13], and so on. Furthermore, it is known that HDAC deacetylates not only acetylated histones, but also the other proteins including p53 [14, 15], NF-κB [16], sp3 [17], Bcl-6 [18], and α-tubulin [19]. The functions of these proteins are also posttranslationally regulated by reversible acetylation; the regulatory mechanisms has been investigated using trichostatin A.

Biological Activity of Trichostatin A

Differentiation in murine erythroleukemia cells: 15 nM [2]

K_i for partially purified histone deacetylase from FM3A cells, noncompetitive: 3.4 nM [8]

Induction of normal and flat phenotype in sis-transformed NIH3T3 cells: 1 ng/ml [4]

References

1.

Tsuji N, Kobayashi M, Nagashima K, Wakisaka Y, Koizumi K (1976) A new antifungal antibiotic, trichostatin. J Antibiot (Tokyo) 29:1–6
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Yoshida M, Nomura S, Beppu T (1987) Effects of trichostatins on differentiation of murine erythroleukemia cells. Cancer Res 47:3688–3691
3.

Yoshida M, Beppu T (1988) Reversible arrest of proliferation of rat 3Y1 fibroblasts in both the G1 and G2 phases by trichostatin A. Exp Cell Res 177:122–131
4.

Sugita K, Koizumi K, Yoshida H (1992) Morphological reversion of sis-transformed NIH3T3 cells by trichostatin A. Cancer Res 52:168–172
5.

Hoshikawa Y, Kwon HJ, Yoshida M, Horinouchi S, Beppu T (1994) Trichostatin A induces morphological changes and gelsolin expression by inhibiting histone deacetylase in human carcinoma cell lines. Exp Cell Res 214:189–197
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Lee E, Furukubo T, Miyabe T, Yamauchi A, Kariya K (1996) Involvement of histone hyperacetylation in triggering DNA fragmentation of rat thymocytes undergoing apoptosis. FEBS Lett 395:183–187
7.

McBain JA, Eastman A, Nobel CS, Mueller GC (1997) Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors. Biochem Pharmacol 53:1357–1368
8.

Yoshida M, Kijima M, Akita M, Beppu T (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem 265:17174–17179
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Bernstein BE, Tong JK, Schreiber SL (2000) Genomewide studies of histone deacetylase function in yeast. Proc Natl Acad Sci U S A 97:13708–13713
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Schlake T, Klehr-Wirth D, Yoshida M, Beppu T, Bode J (1994) Gene expression within a chromatin domain: the role of core histone hyperacetylation. Biochemistry 33:4197–4206
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Sheridan PL, Mayall TP, Verdin E, Jones KA (1997) Histone acetyltransferases regulate HIV-1 enhancer activity in vitro. Genes Dev 11:3327–3340
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Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet 21:103–107
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Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386–389
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Roy S, Tenniswood M (2007) Site-specific acetylation of p53 directs selective transcription complex assembly. J Biol Chem 282:4765–4771
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Zhao Y, Lu S, Wu L, Chai G, Wang H, Chen Y, Sun J, Yu Y, Zhou W, Zheng Q, Wu M, Otterson GA, Zhu WG (2006) Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1). Mol Cell Biol 26:2782–2790
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Chen L, Fischle W, Verdin E, Greene WC (2001) Duration of nuclear NF-κB action regulated by reversible acetylation. Science 293:1653–1657
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Ammanamanchi S, Freeman JW, Brattain MG (2003) Acetylated Sp3 is a transcriptional activator. J Biol Chem 278:35775–35780
18.

Bereshchenko OR, Gu W, Dalla-Favera R (2002) Acetylation inactivates the transcriptional repressor BCL6. Nat Genet 32:606–613
19.

Matsuyama A, Shimazu T, Sumida Y, Saito A, Yoshimatsu Y, Seigneurin-Berny D, Osada H, Komatsu Y, Nishino N, Khochbin S, Horinouchi S, Yoshida M (2002) In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 21:6820–6831

Tunicamycin

Keywords: [N-linked glycosylation inhibitor] [Secretion inhibitor]

Structure

	Tunicamycin
	n = 8	n = 9	n = 10	n = 11
Molecular formula	C₃₇H₆₀N₄O₁₆	C₃₈H₆₂N₄O₁₆	C₃₉H₆₄N₄O₁₆	C₄₀H₆₆N₄O₁₆
Molecular weight	816.90	830.93	844.95	858.99
Solubility	DMSO, +++; H₂O, ±; MeOH, +

Discovery/Isolation/Chemical Studies

Tunicamycin was isolated as an antiviral antibiotic produced by Streptomyces lysosuperificus, nov. sp. [1]. Analytical studies on fatty acids which were obtained by hydrolysis of tunicamycin suggested that tunicamycin is a mixture of at least 10 homologous antibiotics [2].

Biological Studies

Tunicamycin is active against not only Gram-positive bacteria, yeast, and fungi but also animal and plant viruses [1]. Interestingly, this antiviral activity was partially reversed with some amino sugars and their derivatives [3], and the tunicamycin inhibition point was identified as the transfer of N-acetylglucosamine from UDP-GlcNAc to a lipid intermediate [4]. An in vitro glycolipid synthesis system revealed that tunicamycin inhibits UDP-N-acetylglucosamine:dolichol phosphate GlcNAc-1-P transferase (GPT) that catalyzes the first step in protein N-glycosylation [5, 6].

Because tunicamycin blocks the transfer of the common pentasaccharide core of N-glycan on asparagin residues of newly synthesized proteins, tunicamycin has been used as a bioprobe for investigating the numerous aspects of N-glycan function, including protein folding [7] and trafficking [8]. Furthermore, tunicamycin has been used as the reagent to induce the unfolded protein response (UPR)/ER stress [9, 10] and cellular response against UPR [11–13].

Biological Activity of Tunicamycin

MIC for Newcastle disease virus production by the agar-diffusion plaque inhibition method: 0.5 mg/ml [1]

Apoptosis induction in HL-60 cells: 0.05 μg/ml [14]

Inhibition of VEGF glycosylation in RPE-J cells: 10 μg/ml [8]

Induction of GRP78 mRNA in HeLa cells: 10 μg/ml [10]

Induction of autophagy in yeast: 2 μg/ml [12]

References

1.

Takatsuki A, Arima K, Tamura G (1971) Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. J Antibiot (Tokyo) 24:215–223
2.

Takatsuki A, Kawamura K, Okina M, Kodama Y, Ito T, Tamura G (1977) The structure of tunicamycin. Agric Biol Chem 41:2307–2309
3.

Takatsuki A, Tamura G (1971) Tunicamycin, a new antibiotic. 3. Reversal of the antiviral activity of tunicamycin by aminosugars and their derivatives. J Antibiot (Tokyo) 24:232–238
4.

Takatsuki A, Kohno K, Tamura G (1975) Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Arg BiolChem 39:2089–2091
5.

Datema R, Schwarz RT (1978) Formation of 2-deoxyglucose-containing lipid-linked oligosaccharides. Interference with glycosylation of glycoproteins. Eur J Biochem 90:505–516
6.

Xu L, Appell M, Kennedy S, Momany FA, Price NP (2004) Conformational analysis of chirally deuterated tunicamycin as an active site probe of UDP-N-acetylhexosamine: polyprenol-P N-acetylhexosamine-1-P translocases. Biochemistry 43:13248–13255
7.

McCormick LM, Urade R, Arakaki Y, Schwartz AL, Bu G (2005) Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein. Biochemistry 44:5794–5803
8.

Marmorstein AD, Csaky KG, Baffi J, Lam L, Rahaal F, Rodriguez-Boulan E (2000) Saturation of, and competition for entry into, the apical secretory pathway. Proc Natl Acad Sci U S A 97:3248–3253
9.

Brewer JW, Cleveland JL, Hendershot LM (1997) A pathway distinct from the mammalian unfolded protein response regulates expression of endoplasmic reticulum chaperones in non-stressed cells. EMBO J 16:7207–7216
10.

Shinjo S, Mizotani Y, Tashiro E, Imoto M (2013) Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds. Biosci Biotechnol Biochem 77:729–735
11.

Brewer JW, Hendershot LM, Sherr CJ, Diehl JA (1999) Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proc Natl Acad Sci U S A 96:8505–8510
12.

Yorimitsu T, Nair U, Yang Z, Klionsky DJ (2006) Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281:30299–30304
13.

Kim H, Tu HC, Ren D, Takeuchi O, Jeffers JR, Zambetti GP, Hsieh JJ, Cheng EH (2009) Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell 36:487–499
14.

Perez-Sala D, Mollinedo F (1995) Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells. J Cell Physiol 163:523–531

TWS119*

Keywords: [Glycogen synthase kinase-3β (GSK-3β) inhibitor] [Neurogenesis inducer]

Structure

	TWS119
Molecular formula	C₁₈H₁₄N₄O₂
Molecular weight	318.33
Solubility	DMSO, +++; H₂O, −; MeOH, −

Discovery/Isolation/Chemical Studies

TWS119 is a 4,6-disubstituted pyrrolopyrimidine identified as a neurogenesis inducer of ES cells by high-throughput phenotypic cell-based screening of kinase-directed combinatorial libraries [1].

Biological Studies

Affinity-based and biochemical methods revealed that the target molecule of TWS119 is glycogen synthase kinase-3β (GSK-3β) [1]. GSK-3β stmulates the degradation of β-catenin in the Wnt signaling pathway by phosphorylation. TWS119 treatment resulted in increases in the amount and nuclear translocation of β-catenin, resulting in the activation of β-catenin-induced TCF/LEF reporter activity in a dose-dependent manner.

GSK-3β is a multifunctional Ser/Thr kinase involved in pattern formation during embryonic development, cell fate determination, transcriptional control, metabolism, oncogenesis, and neurological diseases. Therefore, TWS119 has been used for the investigation of biological events involving GSK-3β kinase [2–4].

Biological Activity of TWS119

Neuronal differentiation of P19 EC cells and primary mouse ES cells: 0.4–1 μM [1]

K_d for GSK-3β: 126 nM [1]

IC₅₀ for kinase activity of GSK-3β: 30 nM [1]

References

1.

Ding S, Wu TY, Brinker A, Peters EC, Hur W, Gray NS, Schultz PG (2003) Synthetic small molecules that control stem cell fate. Proc Natl Acad Sci U S A 100:7632–7637
2.

Morioka N, Abe H, Araki R, Matsumoto N, Zhang FF, Nakamura Y, Hisaoka-Nakashima K, Nakata Y (2014) A β1/2 adrenergic receptor-sensitive intracellular signaling pathway modulates CCL2 production in cultured spinal astrocytes. J Cell Physiol 229:323–332
3.

Muralidharan S, Hanley PJ, Liu E, Chakraborty R, Bollard C, Shpall E, Rooney C, Savoldo B, Rodgers J, Dotti G (2011) Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes. J Immunol 187:5221–5232
4.

Zeng FY, Dong H, Cui J, Liu L, Chen T (2010) Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells. Biochem Biophys Res Commun 391:1049–1055

UCN-01

Keywords: [G2 checkpoint inhibitor] [Chk1 inhibitor] [α1-acid glycoprotein binder]

Structure

	UCN-01
Molecular formula	C₂₈H₂₆N₄O₄
Molecular weight	482.53
Solubility	DMSO, +++; H₂O, −; MeOH, +

Discovery/Isolation/Chemical Studies

UCN-01 was found in a culture broth of Streptomyces sp., which produces staurosporine. The structure of UCN-01 differs from staurosporine in that the C-7 carbon bears a hydroxyl group [1].

Biological Studies

UCN-01 was originally found as a protein kinase C–specific inhibitor compared with staurosporine [1] and inhibits cPKC [2]. UCN-01 shows strong cytotoxic effects on the growth of mammalian cells and shows antitumor activity in vivo against three human tumor xenografts [3]. UCN-01 retards but does not prevent cell cycle progression through phase, but cells are clearly blocked from exit of G1 and entry into S phase [4]. In clinically, UCN-01 sensitizes cells to ionizing radiation and chemotherapeutic agents. The important determinants of the sensitivity of cancer cells to UCN-01 are apoptosis induction and cell cycle arrest in G1 [5]. Synergic effects with histone deacetylase inhibitors were also reported [6].

UCN-01 abrogates G2 arrest following DNA damage through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C [7]. Because DNA damage induces G2 checkpoint activation by preventing the activation of Cdc25C and arrests cell cycle progression in G2 phase to provide time for repair, it has been thought that UCN-01 inhibits an upstream kinase of Cdc25C. Indeed, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro and caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells, strongly suggesting the Chk1 kinase and the Cdc25C pathway as potential targets of G2 checkpoint abrogation by UCN-01 [8]. UCN-01 also potently inhibits p38MAPK/MK2, a protein kinase involved in the cell cycle checkpoint pathway, suggesting that UCN-01 simultaneously disrupts two critical checkpoint pathways [9]. Furthermore, high concentrations of UCN-01 caused rapid premature mitosis by inhibiting C-TAK1, a constitutive kinase phosphorylating Cdc25C in S phase [10]. These results suggest that UCN-01 shows multiple phenotypes in a dose-dependent manner. The crystal structure of the Chk1 kinase domain in the complex revealed that UCN-01 binds the ATP binding pocket of Chk1. The potency and selectivity of UCN-01 to Chk1 is accounted for by high structural complementarity of both hydrophobic and hydrogen-bonding interactions [11]. UCN-01 also potently inhibits 3-phosphoinositide-dependent protein kinase-1 (PDK1). The crystal structure of UCN-01 in complex with the kinase domain of PDK1 has been reported [12].

UCN-01 shows unusual pharmacokinetics with extremely low distribution volumes, low systemic clearance, and long elimination half-lives in humans. Human α1-acid glycoprotein, but not human serum albumin, completely inhibited the initial uptake of UCN-01 into isolated rat hepatocytes. Therefore, the high-affinity binding of UCN-01 to α1-acid glycoprotein at least partly results in high plasma concentrations in humans [13, 14]. The binding site was thought to be around Trp160 by photoaffinity labeling and site-directed mutation analysis [15].

Biological Activity of UCN-01

IC₅₀ values for protein kinase C, protein kinase A, and pp60^src kinase: 4.1, 42, and 45 nM, respectively [1]

IC₅₀ values for cdk2, 4, and 6: 42, 32, and 58 nM, respectively [16]

IC₅₀ for Chk1 autophosphorylation and Cdc25C-serine 216 phosphorylation: ~25 nM [8]

References

1.

Takahashi I, Kobayashi E, Asano K, Yoshida M, Nakano H. (1987) UCN-01, a selective inhibitor of protein kinase C from Streptomyces. J Antibiot (Tokyo) 40:1782–1784
2.

Mizuno K, Noda K, Ueda Y, Hanaki H, Saido TC, Ikuta T, Kuroki T, Tamaoki T, Hirai S, Osada S, Ohno S (1995) UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily. FEBS Lett 359:259–261
3.

Akinaga S, Gomi K, Morimoto M, Tamaoki T, Okabe M. (1991) Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res 51:4888–4892
4.

Seynaeve CM, Stetler-Stevenson M, Sebers S, Kaur G, Sausville EA, Worland PJ. (1993) Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 53:2081–2086
5.

Sugiyama K, Akiyama T, Shimizu M, Tamaoki T, Courage C, Gescher A, Akinaga S. (1999) Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine). Cancer Res 59:4406–4412
6.

Lee JH, Choy ML, Ngo L, Venta-Perez G, Marks PA. (2011) Role of checkpoint kinase 1 (Chk1) in the mechanisms of resistance to histone deacetylase inhibitors. Proc Natl Acad Sci U S A 108:19629–19634
7.

Shao RG, Cao CX, Zhang H, Kohn KW, Wold MS, Pommier Y. (1999) Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J. 18:1397–1406. EMBO J 1999 Mar 1;18(5):1397–406
8.

Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O’Connor PM, Piwnica-Worms H. (2000) The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 275:5600–5605
9.

Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. (2007) p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11:175–189
10.

Kohn EA, Ruth ND, Brown MK, Livingstone M, Eastman A. (2002) Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation. J Biol Chem 277:26553–26564
11.

Zhao B, Bower MJ, McDevitt PJ, Zhao H, Davis ST, Johanson KO, Green SM, Concha NO, Zhou BB. (2002) Structural basis for Chk1 inhibition by UCN-01. J Biol Chem 277:46609–46615
12.

Komander D, Kular GS, Bain J, Elliott M, Alessi DR, Van Aalten DM. (2003) Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3-phosphoinositide-dependent protein kinase-1) inhibition. Biochem J 375:255–262
13.

Fuse E, Tanii H, Kurata N, Kobayashi H, Shimada Y, Tamura T, Sasaki Y, Tanigawara Y, Lush RD, Headlee D, Figg WD, Arbuck SG, Senderowicz AM, Sausville EA, Akinaga S, Kuwabara T, Kobayashi S. (1998) Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human α1-acid glycoprotein. Cancer Res 58:3248–3253
14.

Fuse E, Tanii H, Takai K, Asanome K, Kurata N, Kobayashi H, Kuwabara T, Kobayashi S, Sugiyama Y. (1999) Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to α1-acid glycoprotein in humans. Cancer Res 59:1054–1060
15.

Katsuki M, Chuang VT, Nishi K, Kawahara K, Nakayama H, Yamaotsu N, Hirono S, Otagiri M. (2005) Use of photoaffinity labeling and site-directed mutagenesis for identification of the key residue responsible for extraordinarily high affinity binding of UCN-01 in human α1-acid glycoprotein. J Biol Chem 280:1384–1391
16.

Kawakami K, Futami H, Takahara J, Yamaguchi K. (1996) UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 219:778–783

UNC0638*

Keywords: [Protein lysine methyltransferases G9a and GLP inhibitor]

Structure

	UNC0638
Molecular formula	C₃₀H₄₇N₅O₂
Molecular weight	509.73
Solubility	DMSO, +++; H₂O, −; MeOH, ±

Discovery/Isolation/Chemical Studies

UNC0638 is a synthetic compound from the structure–activity relationships study of UNC0321, a potent and selective G9a and GLP inhibitor [1] which was synthesized based on the X-ray structures of complexes of GLP-BIX01294 and G9a-UNC0224 [2]. UNC0638 has balanced in vitro potency and physicochemical properties aiding cell penetration.

Biological Studies

UNC0638 potently and selectively inhibits G9a and GLP activities in a substrate-competitive manner [2]. The crystal structure of G9a-UNC0638-SAH (S-adenosyl-L-homocysteine) revealed that UCN0638 occupies the substrate binding groove without interaction with the SAM (S-adenosyl-L-methionine) binding pocket. Because UCN0638 possesses the cell penetration potency and is stable under cellular assay conditions, this compound potently inhibits cellular G9a and GLP without cytotoxicity and reactivated the silenced gene expression in mES cell by decreasing H3K9me2 (Lys9-dimethylated histone H3) levels.

It is thought that histone modification by protein methyltransfereases is associated with transcriptional repression during development and this reinforces lineage choice by affecting high-order chromatin structure. UNC0638-treated hematopoietic stem and progenitor cells retained stem cell–like phenotypes and function during in vitro expansion, suggesting that this compound is useful for stem cell therapy [3].

Chromatin modification is also seen in human cancers and is thought to be an attractive drug target for cancer therapy. The report that methyltransferase activity is connected with the activity of the leukomogenic transcription factor HoxA9 suggests a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription [4].

Biological Activity of UNC0638

IC₅₀ values for G9a and GLP: <15 and 19 ± 1 nM, respectively [2]

Stem cell–like phenotypes maintenance in hematopoietic stem and progenitor cells: 1 μM [3]

Growth inhibition and induction of myeloid differentiation of AML cells: 1 μM [4]

References

1.

Liu F, Chen X, Allali-Hassani A, Quinn AM, Wigle TJ, Wasney GA, Dong A, Senisterra G, Chau I, Siarheyeva A, Norris JL, Kireev DB, Jadhav A, Herold JM, Janzen WP, Arrowsmith CH, Frye SV, Brown PJ, Simeonov A, Vedadi M, Jin J (2010) Protein lysine methyltransferase G9a inhibitors: design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines. J Med Chem 53:5844–5857
2.

Vedadi M, Barsyte-Lovejoy D, Liu F, Rival-Gervier S, Allali-Hassani A, Labrie V, Wigle TJ, Dimaggio PA, Wasney GA, Siarheyeva A, Dong A, Tempel W, Wang SC, Chen X, Chau I, Mangano TJ, Huang XP, Simpson CD, Pattenden SG, Norris JL, Kireev DB, Tripathy A, Edwards A, Roth BL, Janzen WP, Garcia BA, Petronis A, Ellis J, Brown PJ, Frye SV, Arrowsmith CH, Jin J (2011) A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat Chem Biol 7:566–574
3.

Chen X, Skutt-Kakaria K, Davison J, Ou YL, Choi E, Malik P, Loeb K, Wood B, Georges G, Torok-Storb B, Paddison PJ (2012) G9a/GLP-dependent histone H3K9me2 patterning during human hematopoietic stem cell lineage commitment. Genes Dev 26:2499–2511
4.

Lehnertz B, Pabst C, Su L, Miller M, Liu F, Yi L, Zhang R, Krosl J, Yung E, Kirschner J, Rosten P, Underhill TM, Jin J, Hebert J, Sauvageau G, Humphries RK, Rossi FM (2014) The methyltransferase G9a regulates HoxA9-dependent transcription in AML. Genes Dev 28:317–327

UTKO1*

Keywords: [Migration inhibitor] [14-3-3ζ] [Protein-Protein interaction inhibitor]

Structure

	UTKO1
Molecular formula	C₂₃H₃₄O₄
Molecular weight	374.52
Solubility	DMSO, +++; H₂O, ±; MeOH, +

Discovery/Isolation

UTKO1 is a synthetic analog of moverastins which were isolated from Aspergillus sp. F7720 as inhibitors of cancer cell migration [1, 2].

Biological Studies

UTKO1 was found to have the most potent inhibitory activity against the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay [1]. Interestingly, although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase (FTase), UTKO1 did not inhibit FTase.

During the investigation of EGF-stimulating migration, Kobayashi et al. found that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1 inhibits the second EGF-induced wave of Rac1 activation but not the first wave [3]. They focused on 14-3-3ζ, one of the UTKO1 binding proteins which were identified by affinity purification using a biotinylated UTKO1 probe, because it has been previously reported to relate to the formation of lamellipodia. Further investigation revealed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event.

Biological Activity of UTKO1

IC₅₀ values for cell migration and cell viability against EC17 cells: 1.98 and 46 μM, respectively [1]

IC₅₀ for second EGF-induced wave of lamellipodia formation in A431 cells: 0.78 μM [3]

IC₅₀ for cell migration induced by EGF against A431 cells: 0.67 μM [3]

References

1.

Sawada M, Kubo S, Matsumura K, Takemoto Y, Kobayashi H, Tashiro E, Kitahara T, Watanabe H, Imoto M (2011) Synthesis and anti-migrative evaluation of moverastin derivatives. Bioorg Med Chem Lett 21:1385–1389
2.

Takemoto Y, Watanabe H, Uchida K, Matsumura K, Nakae K, Tashiro E, Shindo K, Kitahara T, Imoto M (2005) Chemistry and biology of moverastins, inhibitors of cancer cell migration, produced by Aspergillus. Chem Biol 12:1337–1347
3.

Kobayashi H, Ogura Y, Sawada M, Nakayama R, Takano K, Minato Y, Takemoto Y, Tashiro E, Watanabe H, Imoto M (2011) Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration. J Biol Chem 286:39259–39268

Withaferin A

Keywords: [Antiangiogenesis] [Anti-inflammation] [Antitumor]

Structure

	Withaferin A
Molecular formula	C₂₈H₃₈O₆
Molecular weight	470.61
Solubility	DMSO, ++; H₂O, ±; MeOH, ++

Discovery/Isolation/Chemical Studies

Withaferin A was isolated from Withania somnifera Dun (Solanaceae) in 1962 [1], and its structure was reported in 1965 [2].

Biological Studies

There are many reports about biological activities of withaferin A: antitumor [3], antiangiogenesis [4], anti-inflammation, and so on. Several proteins including annexin II [5], protein kinase C [6], tumor proteasome [7], IKKβ [8], vimentin [9], HSP90 [10], glial fibrillary acidic protein (GFAP) [11], and β-tubulin [12] were suggested as target molecules of withaferin A. Because withaferin A possesses the α,β-unsaturated ketone in its structure, it has been thought that some of these proteins are inhibited by covalent binding of withaferin A. The binding sites of annexin II (Cys133), GFAP (Cys294), β-tubulin (Cys303), and IKKβ (Cys179) have also been reported [12, 11, 5, 13].

Biological Activity of Withaferin A

Actin filament disruption in WI-38 and HepG2 cells: 4 μM [5]

IC₅₀ for protein kinase C activity of Leishmania: ~5 μM [6]

IC₅₀ for chymotrypsin-like activity of a purified rabbit 20S proteasome: 4.5 μM [7]

IC₅₀ for pancreatic cancer cells: 1.24–2.93 μM [10]

References

1.

Yarden A, Lavie D (1962) Withaferin – a new constituent of Withania somnifera. B Res Counc Israel A 11:35
2.

Lavie D, Glotter E, Shvo Y (1965) Constituents of Withania somnifera Dun .4. Structure of withaferin A. J Chem Soc (Dec):7517–7531
3.

Shohat B, Gitter S, Abraham A, Lavie D (1967) Antitumor activity of withaferin A (NSC-101088). Cancer Chemother Rep 51:271–276
4.

Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, Zhang L, Hanson AD, Conner BP, Rougas J, Pribluda VS (2004) Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 7:115–122
5.

Falsey RR, Marron MT, Gunaherath GM, Shirahatti N, Mahadevan D, Gunatilaka AA, Whitesell L (2006) Actin microfilament aggregation induced by withaferin A is mediated by annexin II. Nat Chem Biol 2:33–38
6.

Sen N, Banerjee B, Das BB, Ganguly A, Sen T, Pramanik S, Mukhopadhyay S, Majumder HK (2007) Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex. Cell Death Differ 14:358–367
7.

Yang H, Shi G, Dou QP (2007) The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from “Indian winter cherry”. Mol Pharmacol 71:426–437
8.

Kaileh M, Vanden Berghe W, Heyerick A, Horion J, Piette J, Libert C, De Keukeleire D, Essawi T, Haegeman G (2007) Withaferin A strongly elicits IκB kinase β hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282:4253–4264
9.

Bargagna-Mohan P, Hamza A, Kim YE, Khuan Abby Ho Y, Mor-Vaknin N, Wendschlag N, Liu J, Evans RM, Markovitz DM, Zhan CG, Kim KB, Mohan R (2007) The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin. Chem Biol 14:623–634
10.

Yu Y, Hamza A, Zhang T, Gu M, Zou P, Newman B, Li Y, Gunatilaka AA, Zhan CG, Sun D (2010) Withaferin A targets heat shock protein 90 in pancreatic cancer cells. Biochem Pharmacol 79:542–551
11.

Bargagna-Mohan P, Paranthan RR, Hamza A, Dimova N, Trucchi B, Srinivasan C, Elliott GI, Zhan CG, Lau DL, Zhu H, Kasahara K, Inagaki M, Cambi F, Mohan R (2010) Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis. J Biol Chem 285:7657–7669
12.

Antony ML, Lee J, Hahm ER, Kim SH, Marcus AI, Kumari V, Ji X, Yang Z, Vowell CL, Wipf P, Uechi GT, Yates NA, Romero G, Sarkar SN, Singh SV (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of β-tubulin. J Biol Chem 289:1852–1865
13.

Heyninck K, Lahtela-Kakkonen M, Van der Veken P, Haegeman G, Vanden Berghe W (2014) Withaferin A inhibits NF-κB activation by targeting cysteine 179 in IKKβ. Biochem Pharmacol 91:501–509

Wortmannin

Keywords: [Apoptosis inducer] [PI3 kinase inhibitor]

Structure

	Wortmannin
Molecular formula	C₂₃H₂₄O₈
Molecular weight	428.43
Solubility	DMSO, +++; H₂O, ±; MeOH, ±

Discovery/Isolation

Wortmannin is an antifungal antibiotic isolated from a culture of Penicillium wortmanni Klocker [1].

Biological Studies

Wortmannin showed a potent anti-inflammatory activity [2], and inhibited the signal transduction initiated by the bacterial stimulus which leads to the activation of the respiratory burst oxidase [3]. Okada et al. found that wortmannin is a potent and selective inhibitor of phosphatidylinositol 3-kinase (PI3 kinase, PI3K) [4]. Wortmannin inhibits PI3K irreversibly by covalent modification of Lys802, a residue involved in the phosphate transfer reaction [5, 6].

Wortmannin has been used as a bioprobe for investigating the function of PI3P and PI3P signaling pathway including Akt [7, 8] and ATM [9], downstream of PI3K. Wortmannin is also used for cell surviving signals [10–12], the autophagy mechanism [13], and endosome regulation [14].

Biological Activity of Wortmannin

Growth inhibition of Botrytis allii, B. cincerea, B. fabae, Cladosporium herbarum and Rhizopus stolonifer: 0.4–3.2 μg/ml [1]

IC₅₀ for noncompetitive inhibition of all three of the PI3-kinase activities found in the cytosol fraction of guinea pig neutrophils: 5 nM [4]

Inhibition of the phosphorylation of Akt in HEK293 and HeLa cells: 100 nM [12]

IC₅₀ for antiproteolytic effect on autophagic sequestration: 30 nM [13]

Inhibition of early endosome fusion in vitro prepared wortmannin cell: 100 nM [14]

References

1.

Brian PW, Curtis PJ, Hemming HG, Norris GLF (1957) Wortmannin, an antibiotic produced by Penicillium wortmanni. Trans Br Mycol Soc 40:365–368
2.

Wiesinger D, Gubler HU, Haefliger W, Hauser D (1974) Antiinflammatory activity of the new mould metabolite 11-desacetoxy-wortmannin and of some of its derivatives. Experientia 30:135–136
3.

Baggiolini M, Dewald B, Schnyder J, Ruch W, Cooper PH, Payne TG (1987) Inhibition of the phagocytosis-induced respiratory burst by the fungal metabolite wortmannin and some analogues. Exp Cell Res 169:408–418
4.

Okada T, Sakuma L, Fukui Y, Hazeki O, Ui M (1994) Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J Biol Chem 269:3563–3567
5.

Norman BH, Shih C, Toth JE, Ray JE, Dodge JA, Johnson DW, Rutherford PG, Schultz RM, Worzalla JF, Vlahos CJ (1996) Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs. J Med Chem 39:1106–1111
6.

Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield MD, Panayotou G (1996) Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 16:1722–1733
7.

Burgering BM, Coffer PJ (1995) Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602
8.

Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN (1995) The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736
9.

Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281:1674–1677
10.

Yao R, Cooper GM (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267:2003–2006
11.

Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME (1997) Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275:661–665
12.

Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB (1999) NF-κB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85
13.

Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelárová H, Meijer AJ. (1997) The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243:240–246
14.

Simonsen A, Lippe R, Christoforidis S, Gaullier JM, Brech A, Callaghan J, Toh BH, Murphy C, Zerial M, Stenmark H (1998) EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394:494–498

Xanthohumol

Keywords: [Antiangiogesis] [Antitumor] [Autophagy] [Valosin-containing protein (VCP) inhibitor]

Structure

	Xanthohumol
Molecular formula	C₂₁H₂₂O₅
Molecular weight	354.40
Solubility	DMSO, ++; H₂O, ±; MeOH, +

Discovery/Isolation/Chemical Study

Xanthohumol is a natural chalcone isolated from hops [1, 2]. The structure was determined by Verzele et al. [3].

Biological Studies

There are several reports about biological activities of xanthohumol as a bone resorption inhibitor [4], diacylglycerol acyltransferase inhibitor [5], farnesoid X receptor agonist [6], angiogenesis inhibitor [7], antitumor compound [8], and so on. It has been reported that xanthohumol represses NF-κB and Akt pathways and induces apoptosis [7, 8].

Sasazawa et al. rediscovered xanthohumol as an autophagy modulator, and identified valosin-containing protein (VCP) as a xanthohumol binding protein by using xanthohumol-immobilized beads [9]. Xanthohumol bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. VCP knockdown also induced autophagy, and xanthohumol increased the protein levels of CHOP and GRP78, suggesting that xanthohumol bound to and inactivated VCP. These results may explain how xanthohumol exhibits various biological activities that have been reported previously.

Biological Activity of Xanthohumol

IC₅₀ for diacylglycerol acyltransferase: 50.3 μM [5]

Inhibition of endothelial cell chemotaxis and invasion: 5–10 μM [7]

Growth inhibition of prostate cancer cells, LNCaP, C4-2, PC-3 and DU145: 20–40 μM [8]

Autophagy induction in HeLa cells: 10–30 μM [9]

References

1.

Power FB, Tutin F, Rogerson H (1913) CXXXV.-The constituents of hops. J Chem Soc Trans 103:1267–1292
2.

Stevens JF, Page JE (2004) Xanthohumol and related prenylflavonoids from hops and beer: to your good health! Phytochemistry 65:1317–1330
3.

Verzele M, Stockx J, Fontijn F, Anteunis M (1957) Xanthohumol, a new natural chalcone. Bull Soc Chim Belg 66:452–475
4.

Tobe H, Muraki Y, Kitamura K, Komiyama O, Sato Y, Sugioka T, Maruyama HB, Matsuda E, Nagai M (1997) Bone resorption inhibitors from hop extract. Biosci Biotechnol Biochem 61:158–159
5.

Tabata N, Ito M, Tomoda H, Omura S (1997) Xanthohumols, diacylglycerol acyltransferase inhibitors, from Humulus lupulus. Phytochemistry 46:683–687
6.

Nozawa H (2005) Xanthohumol, the chalcone from beer hops (Humulus lupulus L.), is the ligand for farnesoid X receptor and ameliorates lipid and glucose metabolism in KK-A(y) mice. Biochem Biophys Res Commun 336:754–761
7.

Albini A, Dell”Eva R, Vene R, Ferrari N, Buhler DR, Noonan DM, Fassina G (2006) Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets. FASEB J 20:527–529
8.

Deeb D, Gao X, Jiang H, Arbab AS, Dulchavsky SA, Gautam SC (2010) Growth inhibitory and apoptosis-inducing effects of xanthohumol, a prenylated chalone present in hops, in human prostate cancer cells. Anticancer Res 30:3333–3339
9.

Sasazawa Y, Kanagaki S, Tashiro E, Nogawa T, Muroi M, Kondoh Y, Osada H, Imoto M (2012) Xanthohumol impairs autophagosome maturation through direct inhibition of valosin-containing protein. ACS Chem Biol 7:892–900

XAV939*

Keywords: [Antitumor] [PARP/tankyrase inhibitior] [Wnt signaling]

Structure

	XAV939
Molecular formula	C₁₄H₁₁F₃N₂OS
Molecular weight	312.31
Solubility	DMSO, ++; H₂O, −; MeOH, −

Discovery/Isolation

XAV939 was identifed as a selective inhibitor of β-catenin-mediated transcription by a chemical–genetic screen [1].

Biological Studies

Using a quantitative chemical proteomic approach, it has been revealed that XAV939 stimulated the degradation of β-catenin through the stabilization of axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2 [1]. The cocrystal structure of the PARP domain of human tankyrase-2 revealed that XAV939 is anchored in the nicotinamide crevice of the NAD⁺ binding site [2].

Because Wnt/β-catenin signaling regulates several biological mechanisms including embryonic development, cell proliferation, cell migration, and so on, several research groups have used XAV939 for investigations. Wang et al. reported that XAV939 robustly induced cardiomyogenesis in mouse ES cells [3], and Fancy et al. found the essential function of Axin2 in remyelination after hypoxic and demyelinating injury using XAV939 [4].

Poly-ADP-ribosylation is involved not only in the Wnt/β-catenin signaling pathway, but also in mitotic spindle formation, sister telomere resolution, and other functions. Li et al. investigated the function of poly-ADP-ribosylation in interphase, and found that poly-ADP-ribosylation is required for efficient replication of herpes simplex virus, and that the tankyrase inhibition by VAX939 decreased viral titers [5]. The other function of tankyrase, the assembly of 26S proteasome through the inhibition of tankyrase-mediated ADP-ribosylation of PI31, a proteaseome regulator, has also been confirmed using XAV939 [6].

Biological Activity of XAV939

IC₅₀ values for TNKS1, TNKS2, and PARP1: 11, 4, and 2194 nM, respectively [1]

K_d values for TNKS1, TNKS2, and PARP1: 14 ± 8, 8 ± 3, and 620 ± 130 nM, respectively [2]

IC₅₀ for growth of MDA-MB-231 cells in low-serum conditions: 1.5 μM [7]

References

1.

Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, Michaud GA, Charlat O, Wiellette E, Zhang Y, Wiessner S, Hild M, Shi X, Wilson CJ, Mickanin C, Myer V, Fazal A, Tomlinson R, Serluca F, Shao W, Cheng H, Shultz M, Rau C, Schirle M, Schlegl J, Ghidelli S, Fawell S, Lu C, Curtis D, Kirschner MW, Lengauer C, Finan PM, Tallarico JA, Bouwmeester T, Porter JA, Bauer A, Cong F (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461:614–620
2.

Karlberg T, Markova N, Johansson I, Hammarstrom M, Schutz P, Weigelt J, Schuler H (2010) Structural basis for the interaction between tankyrase-2 and a potent Wnt-signaling inhibitor. J Med Chem 53:5352–5355
3.

Wang H, Hao J, Hong CC (2011) Cardiac induction of embryonic stem cells by a small molecule inhibitor of Wnt/β-catenin signaling. ACS Chem Biol 6:192–197
4.

Fancy SP, Harrington EP, Yuen TJ, Silbereis JC, Zhao C, Baranzini SE, Bruce CC, Otero JJ, Huang EJ, Nusse R, Franklin RJ, Rowitch DH (2011) Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination. Nat Neurosci 14:1009–1016
5.

Li Z, Yamauchi Y, Kamakura M, Murayama T, Goshima F, Kimura H, Nishiyama Y (2012) Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1. J Virol 86:492–503
6.

Cho-Park PF, Steller H (2013) Proteasome regulation by ADP-ribosylation. Cell 153:614–627
7.

Bao R, Christova T, Song S, Angers S, Yan X, Attisano L (2012) Inhibition of tankyrases induces Axin stabilization and blocks Wnt signalling in breast cancer cells. PLoS One 7:e48670

Xestospongin B and C

Keywords: [Vasodilative activity] [IP3 receptor agonist]

Structure

	Xestospongin B	Xestospongin C
Molecular formula	C₂₉H₅₂N₂O₃	C₂₈H₅₀N₂O₂
Molecular weight	476.75	446.72
Solubility	DMSO, +; H₂O, ±; MeOH, +	DMSO, +; H₂O, ±; MeOH, +

Discovery/Isolation

Xestospongin B was isolated from Australian marine sponge Xestospongia exugua as a novel vasodilative compound which induces relaxation of blood vessel in vivo [1].

Biological Studies

By screening of marine sponges for inhibitor of 1,4,5-trisphosphaste receptor (IP₃R), it was found that Xestospongia species contained potent blockers of IP₃-mediated Ca²⁺ release including xestospongin A, C, D, araguspongine B, and demethylxestospongin B [2]. Xestospongin C, a most potent IP₃R inhibitor among these five compounds, shows a noncompetitive inhibition mechanism with respect to the IP₃ binding site. Interestingly, xestospongin B is a competitive inhibitor of IP₃-mediated Ca²⁺ release [3]. Both compounds are membrane-permeable blockers of IP₃-mediated Ca²⁺ release in intact cells, and selectively inhibited bradykinin- and carbamylocholine-induced Ca²⁺ efflux from ER.

Xestospongin B induced autophagy in human cells by disrupting a molecular complex of IP₃R and Beclin 1 [4]. ER-targeted Bcl-2 increased the interaction of IP₃R and Beclin 1, resulting in the suppression of xestospongin B-induced autophagy [4, 5]. These results suggest that IP₃R is a new regulator of the Beclin 1 complex involving the autophagy cascade.

Biological Activity of Xestospongin B and C

IC₅₀ for IP₃-mediated Ca²⁺ release of xestospongin C in rabbit cerebellar microsomes: 358 nM [2]

K_i for xestospongin B on IP₃ binding for cerevellar membrane: 31 μM [3]

Inhibition of xestospongin B on bradykinin-induced Ca²⁺ signaling in NG108-15 cells: 27 μM [3]

Autophagy induction in HeLa cells: 2 μM [4]

References

1.

Nakagawa M, Endo M, Tanaka N, Lee GP (1984) Structures of xestospongin-A, xestospongin-B, xestospongin-C and xestospongin-D, novel vasodilative compounds from marine sponge, Xestospongia exigua. Tetrahedron Lett 25:3227–3230
2.

Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, Pessah IN (1997) Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19:723–733
3.

Jaimovich E, Mattei C, Liberona JL, Cardenas C, Estrada M, Barbier J, Debitus C, Laurent D, Molgo J (2005) Xestospongin B, a competitive inhibitor of IP3-mediated Ca²⁺ signalling in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells. FEBS Lett 579:2051–2057
4.

Vicencio JM, Ortiz C, Criollo A, Jones AW, Kepp O, Galluzzi L, Joza N, Vitale I, Morselli E, Tailler M, Castedo M, Maiuri MC, Molgo J, Szabadkai G, Lavandero S, Kroemer G (2009) The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1. Cell Death Differ 16:1006–1017
5.

Criollo A, Vicencio JM, Tasdemir E, Maiuri MC, Lavandero S, Kroemer G (2007) The inositol trisphosphate receptor in the control of autophagy. Autophagy 3:350–353

ZM447439*

Keywords: [Aurora kinase inhibitor]

Structure

	ZM447439
Molecular formula	C₂₉H₃₁N₅O₄
Molecular weight	513.60
Solubility	DMSO, +++; H₂O, +; MeOH, +++

Discovery/Isolation

ZM447439 is one of the first-generation Aurora kinase inhibitors which was developed by AstraZeneca [1]. ZM-447439 inhibits Aurora kinase A (IC₅₀ 1000 nM, K_i 100 nM), Aurora kinase B (IC₅₀ 50 nM, K_i 0.36 nM) and Aurora kinase C. The compound specifically interacts at the ATP binding site and at the adjacent cleft, found only in Aurora kinases.

Biological Studies

ZM447439 has been shown to induce growth arrest, inhibit histone H3 phosphorylation, and effectively activate apoptosis. In the presence of ZM447439, several properties during mitotic progression such as chromosome alignment, segregation, and cytokinesis during mitosis are perturbed. It is also shown that ZM447439 can override the process of the spindle assembly checkpoint and accelerate meiosis I in mouse oocytes. Based on the use of this inhibitor, Aurora B was found to be important for chromosomal alignment through kinetochore–microtubule attachments in metaphase, activation of the spindle assembly checkpoint, and cytokinesis [2].

Biological Activity of ZM447439

IC₅₀ values for Aurora kinase A and Aurora kinase B: 1000 nM and 50 nM, respectively [1]

K_i values for Aurora kinase A and Aurora kinase B: 100 nM and 0.36 nM, respectively [1]

References

1.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 161:267–280
2.

Gadea BB, Ruderman JV (2005) Aurora kinase inhibitor ZM447439 blocks chromosome-induced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts. Mol Biol Cell 16:1305–1318

ZSTK474*

Keywords: [Antiangiogeneisis] [Antitumor] [PI3K inhibitior]

Structure

	ZSTK474
Molecular formula	C₁₉H₂₁F₂N₇O₂
Molecular weight	417.41
Solubility	DMSO, ++; H₂O, −; MeOH, −

Discovery/Isolation

ZSTK474 was isolated as a strong antiproliferative compound from a chemical library consisting of 1500 compounds of s-triazine derivatives [1].

Biological Studies

COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines suggested phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474 [1]. An in vitro PI3K kinase assay and molecular modeling revealed that ZSTK474 competitively inhibited class I PI3K kinase by binding to the ATP binding pocket [1, 2]. Oral administration of ZSTK474 resulted in strong antitumor activity against human cancer xenografts without toxic effects in critical organs by cell cycle arrest at G0/G1 phase [1, 3]. ZSTK474 also has potent antiangiogenic activity by inhibition of HIF-1α expression and VEGF secretion from cancer cells [4].

Biological Activity of ZSTK474

Inhibition of cell cycle progression of OVCAR3 in G1 phase: >0.5 μM [1]

K_i for PI3Kδ: 1.8 nM [2]

IC₅₀ for inhibiting growth of HUVECs: 146 nM [4]

References

1.

Yaguchi S, Fukui Y, Koshimizu I, Yoshimi H, Matsuno T, Gouda H, Hirono S, Yamazaki K, Yamori T (2006) Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor. J Natl Cancer Inst 98:545–556
2.

Kong D, Yamori T (2007) ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms. Cancer Sci 98:1638–1642
3.

Dan S, Yoshimi H, Okamura M, Mukai Y, Yamori T (2009) Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest. Biochem Biophys Res Commun 379:104–109
4.

Kong D, Okamura M, Yoshimi H, Yamori T (2009) Antiangiogenic effect of ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor. Eur J Cancer 45:857–865

Pyrabactin B, Pyrabactin

Pyrabactin B was identified as germination inhibitor that acts as agonists of the abscisic acid signal transduction pathway in seeds of Arabidopsis thaliana. Park et al. pursed a chemical genetic strategy using pyrabactin, and isolated pyrabactin-resistant mutants named Pyr1, which binds and inhibits Mg²⁺- and Mn²⁺-dependent serine/threonine phosphatases type 2C (PP2C) in abscisic acid-dependent manner.

References

1.

Yang Zhao, Tszfung F Chow, Rachel S Puckrin, Simon E Alfred, Albert K Korir, Cynthia K Larive & Sean R Cutler (2007) Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated. Nat Chem Biol 3:716–721
2.

Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TF, Alfred SE, Bonetta D, Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK, Schroeder JI, Volkman BF, Cutler SR (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324:1068–1071

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Authors and Affiliations

Hiroyuki Koshino
- 1
Takeo Usui
- 2
Email author

1.Molecular Structure Characterization UnitRIKEN CSRSWakoJapan
2.Faculty of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

Bioprobes at a Glance

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