Structure–Activity Studies of Phosphopeptidomimetic Prodrugs Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3 (Stat3)

  • Pijus K. Mandal
  • Zhiyong Ren
  • Xiaomin Chen
  • Kumar Kaluarachchi
  • Warren S.-L. Liao
  • John S. McMurray


Signal transducer and activator of transcription 3 (Stat3) transmits signals from growth factors and interleukin-6 family cytokines by binding to their receptors via its Src homology 2 (SH2) domain. This results in phosphorylation of Tyr705, dimerization, translocation to the nucleus, and regulation of transcription of downstream genes. Stat3 is constitutively activated in several human cancers and is a target for anti-cancer drug design. We have shown previously phosphorylation of Tyr705 in intact cancer cells can be inhibited with prodrugs of phosphopeptide mimics targeting the SH2 domain. In a series of prodrugs consisting of bis-pivaloyloxymethyl esters of 4′-phosphonodifluoromethyl cinnamoyl-Haic-Gln-NHBn, appending methyl group to the β-position of the cinnamate increased potency ca. twofold, which paralleled the increase in affinity of the corresponding phosphopeptide models. However, dramatic increases in potency were observed when the C-terminal C(O)NHBn of Gln-NHBn was replaced with a simple methyl group. In this communication we continue to explore the effects of structural modifications of prodrugs on their ability to inhibit Tyr705 phosphorylation. A set of 4-substituted prolines incorporated into β-methyl-4-phosphocinnamoyl-leucinyl-Xaa-4-aminopentamide model peptides exhibited affinities of 88–317 nM by fluorescence polarization (Pro IC50 = 156 nM). In corresponding prodrugs, Pro inhibited constitutive Stat3 phosphorylation at 10 μM in MDA-MB-468 breast tumor cells. However, 4,4-difluoroproline and 4,4-dimethylproline resulted in complete inhibition at 0.5 μM. These results suggest that the prodrug with native proline undergoes metabolism that those with substituted prolines do not. In conclusion, changes in structure with minimal impact on intrinsic affinity can nevertheless have profound effects on the cellular potency of prodrug inhibitors of Stat3.


Signal transducer and activator of transcription 3 Stat3 Src homology domain 2 SH2 domain Peptidomimetic Phosphopeptide Prodrug 



















5-(Amino)-1,2,4,5,6,7-hexahydro-4-oxo-(2S,5S)-azepino[3,2,1-hi]indole-2-carboxylic acid






Janus kinase






β-Methyl pCinn or [2E] 3-(4-phosphoryloxyphenyl)-2-butenamide




Tyr705 phosphorylated Stat3


1H-Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

SH2 domain

Src homology 2 domain


Signal transducer and activator of transcription 3


Trifluoroacetic acid





We are grateful to the National Cancer Institute (CA096652) for support of this work. Partial support by a Grant from the Center for Targeted Therapy of The University of Texas M. D. Anderson Cancer Center and the Texas Institute of Drug and Diagnostic Development at The University of Texas at Austin is acknowledged. We also acknowledge the NCI Cancer Center Support Grant CA016672 for the support of both our NMR facility and the Translation Chemistry Core Facility which performed the mass spectrometry. Funding as an Odyssey Fellow (Z.R.) was supported by the Odyssey Program and the Cockrell Foundation Award for Scientific Achievement at UTMDACC.

Conflict of interest

The authors have no association with commercial entities associated with this work.

Supplementary material

10989_2012_9313_MOESM1_ESM.pdf (281 kb)
Supplementary material 1 (PDF 281 kb)


  1. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T (1994) Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77:63–71PubMedCrossRefGoogle Scholar
  2. Becker S, Groner B, Muller CW (1998) Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 394:145–151PubMedCrossRefGoogle Scholar
  3. Burke TR Jr, Smyth MS, Otaka A, Nomizu M, Roller PP, Wolf G, Case R, Shoelson SE (1994) Nonhydrolyzable phosphotyrosyl mimetics for the preparation of phosphatase-resistant SH2 domain inhibitors. Biochemistry 33:6490–6494PubMedCrossRefGoogle Scholar
  4. Chen J, Nikolovska-Coleska Z, Yang CY, Gomez C, Gao W, Krajewski K, Jiang S, Roller P, Wang S (2007) Design and synthesis of a new, conformationally constrained, macrocyclic small-molecule inhibitor of STAT3 via ‘click chemistry’. Bioorg Med Chem Lett 17:3939–3942PubMedCrossRefGoogle Scholar
  5. Chen J, Bai L, Bernard D, Nikolovska-Coleska Z, Gomez C, Zhang J, Yi H, Wang S (2010) Structure-based design of conformationally constrained, cell-permeable STAT3 inhibitors. ACS Med Chem Lett 1:85–89PubMedCrossRefGoogle Scholar
  6. Chiang YC, Lin YJ, Horng JC (2009) Stereoelectronic effects on the transition barrier of polyproline conformational interconversion. Protein Sci 18:1967–1977PubMedCrossRefGoogle Scholar
  7. Chiba J, Takayama G, Takashi T, Yokoyama M, Nakayama A, Baldwin JJ, McDonald E, Moriarty KJ, Sarko CR, Saionz KW, Swanson R, Hussain Z, Wong A, Machinaga N (2006) Synthesis, biological evaluation, and pharmacokinetic study of prolyl-1-piperazinylacetic acid and prolyl-4-piperidinylacetic acid derivatives as VLA-4 antagonists. Bioorg Med Chem 14:2725–2746PubMedCrossRefGoogle Scholar
  8. Coleman DR IV, Ren Z, Mandal PK, Cameron AG, Dyer GA, Muranjan S, Campbell M, Chen X, McMurray JS (2005) Investigation of the binding determinants of phosphopeptides targeted to the SRC homology 2 domain of the signal transducer and activator of transcription 3. Development of a high-affinity peptide inhibitor. J Med Chem 48:6661–6670PubMedCrossRefGoogle Scholar
  9. Coleman DR IV, Kaluarachchi K, Ren Z, Chen X, McMurray JS (2008) Solid phase synthesis of phosphopeptides incorporating 2,2-dimethyloxazolidine pseudoproline analogs: evidence for trans Leu-Pro peptide bonds in Stat3 inhibitors. Int J Pept Res Ther 14:1–9CrossRefGoogle Scholar
  10. Darnell JE Jr (2002) Transcription factors as targets for cancer therapy. Nat. Rev. Cancer 2:740–749PubMedCrossRefGoogle Scholar
  11. Demange L, Menez A, Dugave C (1998) Practical synthesis of Boc and Fmoc protected 4-fluoro and 4-difluoroprolines from trans-4-hydroxyproline. Tetrahedron Lett 39:1169–1172CrossRefGoogle Scholar
  12. Ezquerra J, Pedregal C, Rubio A, Vaquero JJ, Matia MP, Martin J, Diaz A, Navio JLG, Deeter JB (1994) Stereoselective double alkylation of ethyl N-boc-pyroglutamate. J Org Chem 59:4327–4331CrossRefGoogle Scholar
  13. Farquhar D, Khan S, Srivastva DN, Saunders PP (1994) Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2′-deoxy-5-fluorouridine 5′-monophosphate (FdUMP): a strategy to introduce nucleotides into cells. J Med Chem 37:3902–3909PubMedCrossRefGoogle Scholar
  14. Fletcher S, Turkson J, Gunning PT (2008) Molecular approaches towards the inhibition of the signal transducer and activator of transcription 3 (Stat3) protein. ChemMedChem 3:1159–1168PubMedCrossRefGoogle Scholar
  15. Fletcher S, Singh J, Zhang X, Yue P, Page BD, Sharmeen S, Shahani VM, Zhao W, Schimmer AD, Turkson J, Gunning PT (2009) Disruption of transcriptionally active Stat3 dimers with non-phosphorylated, salicylic acid-based small molecules: potent in vitro and tumor cell activities. ChemBioChem 10:1959–1964PubMedCrossRefGoogle Scholar
  16. Gunning PT, Katt WP, Glenn M, Siddiquee K, Kim JS, Jove R, Sebti SM, Turkson J, Hamilton AD (2007) Isoform selective inhibition of STAT1 or STAT3 homo-dimerization via peptidomimetic probes: structural recognition of STAT SH2 domains. Bioorg Med Chem Lett 17:1875–1878PubMedCrossRefGoogle Scholar
  17. Gunning PT, Glenn MP, Siddiquee KA, Katt WP, Masson E, Sebti SM, Turkson J, Hamilton AD (2008) Targeting protein–protein interactions: suppression of Stat3 dimerization with rationally designed small-molecule, nonpeptidic SH2 domain binders. ChemBioChem 9:2800–2803PubMedCrossRefGoogle Scholar
  18. Hecker SJ, Erion MD (2008) Prodrugs of phosphates and phosphonates. J Med Chem 51:2328–2345PubMedCrossRefGoogle Scholar
  19. Hedvat M, Huszar D, Herrmann A, Gozgit JM, Schroeder A, Sheehy A, Buettner R, Proia D, Kowolik CM, Xin H, Armstrong B, Bebernitz G, Weng S, Wang L, Ye M, McEachern K, Chen H, Morosini D, Bell K, Alimzhanov M, Ioannidis S, McCoon P, Cao ZA, Yu H, Jove R, Zinda M (2009) The JAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors. Cancer Cell 16:487–497PubMedCrossRefGoogle Scholar
  20. Kreis S, Munz GA, Haan S, Heinrich PC, Behrmann I (2007) Cell density dependent increase of constitutive signal transducers and activators of transcription 3 activity in melanoma cells is mediated by Janus kinases. Mol Cancer Res 5:1331–1341PubMedCrossRefGoogle Scholar
  21. Krise JP, Stella VJ (1996) Prodrugs of phosphates, phosphonates, and phosphinates. Adv Drug Deliv Rev 19:287–310CrossRefGoogle Scholar
  22. Levy DE, Darnell JE Jr (2002) Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662PubMedCrossRefGoogle Scholar
  23. Looyenga BD, Hutchings D, Cherni I, Kingsley C, Weiss GJ, Mackeigan JP (2012) STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma. PLoS One 7:e30820PubMedCrossRefGoogle Scholar
  24. Mandal PK, Heard PA, Ren Z, Chen X, McMurray JS (2007) Solid-phase synthesis of Stat3 inhibitors incorporating O-carbamoylserine and O-carbamoylthreonine as glutamine mimics. Bioorg Med Chem Lett 17:654–656PubMedCrossRefGoogle Scholar
  25. Mandal PK, Liao WS, McMurray JS (2009a) Synthesis of phosphatase-stable, cell-permeable peptidomimetic prodrugs that target the SH2 domain of Stat3. Org Lett 11:3394–3397PubMedCrossRefGoogle Scholar
  26. Mandal PK, Limbrick D, Coleman DR, Dyer GA, Ren Z, Birtwistle JS, Xiong C, Chen X, Briggs JM, McMurray JS (2009b) Conformationally constrained peptidomimetic inhibitors of signal transducer and activator of transcription 3: evaluation and molecular modeling. J Med Chem 52:2429–2442PubMedCrossRefGoogle Scholar
  27. Mandal PK, Ren Z, Chen X, Xiong C, McMurray JS (2009c) Structure-affinity relationships of glutamine mimics incorporated into phosphopeptides targeted to the SH2 domain of signal transducer and activator of transcription 3. J Med Chem 52:6126–6141PubMedCrossRefGoogle Scholar
  28. Mandal PK, Gao F, Lu Z, Ren Z, Ramesh R, Birtwistle JS, Kaluarachchi KK, Chen X, Bast RC, Liao WS, McMurray JS (2011) Potent and Selective phosphopeptide mimetic prodrugs targeted to the Src homology 2 (SH2) domain of signal transducer and activator of transcription 3. J Med Chem 54:3549–5463PubMedCrossRefGoogle Scholar
  29. McMurray JS (2008) Structural basis for the binding of high affinity phosphopeptides to Stat3. Biopolymers 90:69–79PubMedCrossRefGoogle Scholar
  30. Pearson DA, Blanchette M, Baker ML, Guindon CA (1989) Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis. Tetrahedron Lett 30:2739–2742CrossRefGoogle Scholar
  31. Ren Z, Cabell LA, Schaefer TS, McMurray JS (2003) Identification of a high-affinity phosphopeptide inhibitor of Stat3. Bioorg Med Chem Lett 13:633–636PubMedCrossRefGoogle Scholar
  32. Sagnard I, Sasaki NA, Chiaroni A, Riche C, Potier P (1995) Enantioselective synthesis of cyclopropane a-amino acids: synthesis of N-Boc-cis. (2S,3R,4S)-3,4-methanoproline and N-Boc-(2S,3R,4S)-3,4-methanoglutamic acid. Tetrahedron Lett 36:3149–3152CrossRefGoogle Scholar
  33. Schultz C (2003) Prodrugs of biologically active phosphate esters. Bioorg Med Chem 11:885–898PubMedCrossRefGoogle Scholar
  34. Shahani VM, Yue P, Fletcher S, Sharmeen S, Sukhai MA, Luu DP, Zhang X, Sun H, Zhao W, Schimmer AD, Turkson J, Gunning PT (2011a) Design, synthesis, and in vitro characterization of novel hybrid peptidomimetic inhibitors of STAT3 protein. Bioorg Med Chem 19:1823–1838PubMedCrossRefGoogle Scholar
  35. Shahani VM, Yue P, Haftchenary S, Zhao W, Lukkarila JL, Zhang X, Ball D, Nona C, Gunning PT, Turkson J (2011b) Identification of purine-scaffold small-molecule inhibitors of Stat3 activation by QSAR studies. ACS Med Chem Lett 2:79–84PubMedCrossRefGoogle Scholar
  36. Siddiquee KA, Gunning PT, Glenn M, Katt WP, Zhang S, Schrock C, Sebti SM, Jove R, Hamilton AD, Turkson J (2007) An oxazole-based small-molecule Stat3 inhibitor modulates Stat3 stability and processing and induces antitumor cell effects. ACS Chem Biol 2:787–798PubMedCrossRefGoogle Scholar
  37. Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE Jr, Yancopoulos GD (1995) Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267:1349–1353PubMedCrossRefGoogle Scholar
  38. Turkson J, Kim JS, Zhang S, Yuan J, Huang M, Glenn M, Haura E, Sebti S, Hamilton AD, Jove R (2004) Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity. Mol Cancer Ther 3:261–269PubMedGoogle Scholar
  39. Witkop B, Fujimoto Y, Irreverre F, Karle JM, Karle IL (1971) Synthesis and x-ray analysis of cis-3,4-methylene-l-proline, the new natural amino acid from horse chestnuts, and of its trans isomer. J Am Chem Soc 93:3471–3477PubMedCrossRefGoogle Scholar
  40. Yu H, Jove R (2004) The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 4:97–105PubMedCrossRefGoogle Scholar
  41. Zhang X, Yue P, Fletcher S, Zhao W, Gunning PT, Turkson J (2010) A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes. Biochem Pharmacol 79:1398–1409PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Pijus K. Mandal
    • 1
  • Zhiyong Ren
    • 2
    • 3
  • Xiaomin Chen
    • 2
    • 4
  • Kumar Kaluarachchi
    • 1
  • Warren S.-L. Liao
    • 1
  • John S. McMurray
    • 1
  1. 1.Department of Experimental TherapeuticsThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of Biochemistry and Molecular BiologyThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  3. 3.Department of PathologyUniversity of Alabama at BirminghamBirminghamUSA
  4. 4.Pfizer Global Research & DevelopmentGrotonUSA

Personalised recommendations