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A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity

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Phosphodiesterases as Drug Targets

Part of the book series: Handbook of Experimental Pharmacology ((HEP,volume 204))

Abstract

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors that possess “drug-like” characteristics, displaying activity in a growth stimulation assay over a 48-h period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme’s potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify whether different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.

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References

  • Alaamery MA, Hoffman CS (2008) Schizosaccharomyces pombe Hsp90/Git10 is required for glucose/cAMP signaling. Genetics 178:1927–1936

    Article  PubMed  CAS  Google Scholar 

  • Alaamery MA, Wyman AR, Ivey FD, Allain C, Demirbas D, Wang L, Ceyhan O, Hoffman CS (2010) New classes of PDE7 inhibitors identified by a fission yeast-based HTS. J Biomol Screen 15(4):359–367

    Article  PubMed  CAS  Google Scholar 

  • Bender AT, Beavo JA (2006) Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58:488–520

    Article  PubMed  CAS  Google Scholar 

  • Bischoff JR, Casso D, Beach D (1992) Human p53 inhibits growth in Schizosaccharomyces pombe. Mol Cell Biol 12:1405–1411

    PubMed  CAS  Google Scholar 

  • Bolger G, Michaeli T, Martins T, St John T, Steiner B, Rodgers L, Riggs M, Wigler M, Ferguson K (1993) A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs. Mol Cell Biol 13:6558–6571

    PubMed  CAS  Google Scholar 

  • Card GL, England BP, Suzuki Y, Fong D, Powell B, Lee B, Luu C, Tabrizizad M, Gillette S, Ibrahim PN, Artis DR, Bollag G, Milburn MV, Kim SH, Schlessinger J, Zhang KY (2004) Structural basis for the activity of drugs that inhibit phosphodiesterases. Structure 12:2233–2247

    Article  PubMed  CAS  Google Scholar 

  • Card GL, Blasdel L, England BP, Zhang C, Suzuki Y, Gillette S, Fong D, Ibrahim PN, Artis DR, Bollag G, Milburn MV, Kim SH, Schlessinger J, Zhang KY (2005) A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design. Nat Biotechnol 23:201–207

    Article  PubMed  CAS  Google Scholar 

  • Cherry JA, Thompson BE, Pho V (2001) Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse. Biochim Biophys Acta 1518:27–35

    PubMed  CAS  Google Scholar 

  • Colicelli J, Birchmeier C, Michaeli T, O’Neill K, Riggs M, Wigler M (1989) Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc Natl Acad Sci USA 86:3599–3603

    Article  PubMed  CAS  Google Scholar 

  • Colicelli J, Nicolette C, Birchmeier C, Rodgers L, Riggs M, Wigler M (1991) Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 88:2913–2917

    Article  PubMed  CAS  Google Scholar 

  • Conti M, Beavo J (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76:481–511

    Article  PubMed  CAS  Google Scholar 

  • D’Souza CA, Heitman J (2001) Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbiol Rev 25:349–364

    Article  PubMed  Google Scholar 

  • DeVoti J, Seydoux G, Beach D, McLeod M (1991) Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J 10:3759–3768

    PubMed  CAS  Google Scholar 

  • Engels P, Sullivan M, Muller T, Lubbert H (1995) Molecular cloning and functional expression in yeast of a human cAMP-specific phosphodiesterase subtype (PDE IV-C). FEBS Lett 358:305–310

    Article  PubMed  CAS  Google Scholar 

  • Fisher DA, Smith JF, Pillar JS, St Denis SH, Cheng JB (1998a) Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase. Biochem Biophys Res Commun 246:570–577

    Article  PubMed  CAS  Google Scholar 

  • Fisher DA, Smith JF, Pillar JS, St Denis SH, Cheng JB (1998b) Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase. J Biol Chem 273:15559–15564

    Article  PubMed  CAS  Google Scholar 

  • Friedmann Y, Shriki A, Bennett ER, Golos S, Diskin R, Marbach I, Bengal E, Engelberg D (2006) JX401, A p38alpha inhibitor containing a 4-benzylpiperidine motif, identified via a novel screening system in yeast. Mol Pharmacol 70:1395–1405

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Hoffman CS (2005a) Except in every detail: comparing and contrasting G protein signaling in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Eukaryot Cell 4:495–503

    Article  PubMed  CAS  Google Scholar 

  • Hoffman CS (2005b) Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe. Biochem Soc Trans 33:257–260

    Article  PubMed  CAS  Google Scholar 

  • Hoffman CS, Winston F (1990) Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe. Genetics 124:807–816

    PubMed  CAS  Google Scholar 

  • Hoffman CS, Winston F (1991) Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev 5:561–571

    Article  PubMed  CAS  Google Scholar 

  • Houslay MD (2010) Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci 35:91–100

    Article  PubMed  CAS  Google Scholar 

  • Houslay MD, Baillie GS, Maurice DH (2007) cAMP-Specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling. Circ Res 100:950–966

    Article  PubMed  CAS  Google Scholar 

  • Ivey FD, Wang L, Demirbas D, Allain C, Hoffman CS (2008) Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases. J Biomol Screen 13:62–71

    Article  PubMed  CAS  Google Scholar 

  • Jin M, Fujita M, Culley BM, Apolinario E, Yamamoto M, Maundrell K, Hoffman CS (1995) sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase. Genetics 140:457–467

    PubMed  CAS  Google Scholar 

  • Kao RS, Morreale E, Wang L, Ivey FD, Hoffman CS (2006) Schizosaccharomyces pombe Git1 is a C2-domain protein required for glucose activation of adenylate cyclase. Genetics 173:49–61

    Article  PubMed  CAS  Google Scholar 

  • Landry S, Hoffman CS (2001) The git5 Gβ and git11 Gγ form an atypical Gβγ dimer acting in the fission yeast glucose/cAMP pathway. Genetics 157:1159–1168

    PubMed  CAS  Google Scholar 

  • Landry S, Pettit MT, Apolinario E, Hoffman CS (2000) The fission yeast git5 gene encodes a Gβ subunit required for glucose-triggered adenylate cyclase activation. Genetics 154:1463–1471

    PubMed  CAS  Google Scholar 

  • Lee MG, Nurse P (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327:31–35

    Article  PubMed  CAS  Google Scholar 

  • Lengeler KB, Davidson RC, D’Souza C, Harashima T, Shen WC, Wang P, Pan X, Waugh M, Heitman J (2000) Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 64:746–785

    Article  PubMed  CAS  Google Scholar 

  • Lerner A, Epstein PM (2006) Cyclic nucleotide phosphodiesterases as targets for treatment of haematological malignancies. Biochem J 393:21–41

    Article  PubMed  CAS  Google Scholar 

  • Ma P, Wera S, Van Dijck P, Thevelein JM (1999) The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10:91–104

    PubMed  CAS  Google Scholar 

  • McAllister-Lucas LM, Sonnenburg WK, Kadlecek A, Seger D, Trong HL, Colbran JL, Thomas MK, Walsh KA, Francis SH, Corbin JD et al (1993) The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone. J Biol Chem 268:22863–22873

    PubMed  CAS  Google Scholar 

  • McHale MM, Cieslinski LB, Eng WK, Johnson RK, Torphy TJ, Livi GP (1991) Expression of human recombinant cAMP phosphodiesterase isozyme IV reverses growth arrest phenotypes in phosphodiesterase-deficient yeast. Mol Pharmacol 39:109–113

    PubMed  CAS  Google Scholar 

  • McPhee I, Pooley L, Lobban M, Bolger G, Houslay MD (1995) Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family. Biochem J 310(Pt 3):965–974

    PubMed  CAS  Google Scholar 

  • Michaeli T, Bloom TJ, Martins T, Loughney K, Ferguson K, Riggs M, Rodgers L, Beavo JA, Wigler M (1993) Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase-deficient Saccharomyces cerevisiae. J Biol Chem 268:12925–12932

    PubMed  CAS  Google Scholar 

  • Nikawa J, Cameron S, Toda T, Ferguson KM, Wigler M (1987a) Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. Genes Dev 1:931–937

    Article  PubMed  CAS  Google Scholar 

  • Nikawa J, Sass P, Wigler M (1987b) Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Mol Cell Biol 7:3629–3636

    PubMed  CAS  Google Scholar 

  • Nocero M, Isshiki T, Yamamoto M, Hoffman CS (1994) Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein α subunit encoded by gpa2 (git8). Genetics 138:39–45

    PubMed  CAS  Google Scholar 

  • Pillai R, Kytle K, Reyes A, Colicelli J (1993) Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase. Proc Natl Acad Sci USA 90:11970–11974

    Article  PubMed  CAS  Google Scholar 

  • Pillai R, Staub SF, Colicelli J (1994) Mutational mapping of kinetic and pharmacological properties of a human cardiac cAMP phosphodiesterase. J Biol Chem 269:30676–30681

    PubMed  CAS  Google Scholar 

  • Repaske DR, Swinnen JV, Jin SL, Van Wyk JJ, Conti M (1992) A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase. J Biol Chem 267:18683–18688

    PubMed  CAS  Google Scholar 

  • Sass P, Field J, Nikawa J, Toda T, Wigler M (1986) Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 83:9303–9307

    Article  PubMed  CAS  Google Scholar 

  • Schadick K, Fourcade HM, Boumenot P, Seitz JJ, Morrell JL, Chang L, Gould KL, Partridge JF, Allshire RC, Kitagawa K, Hieter P, Hoffman CS (2002) Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgtlp family, is required for glucose and cyclic AMP signaling, cell wall integrity, and septation. Eukaryot Cell 1:558–567

    Article  PubMed  CAS  Google Scholar 

  • Schudt C, Winder S, Eltze M, Kilian U, Beume R (1991) Zardaverine: a cyclic AMP specific PDE III/IV inhibitor. Agents Actions Suppl 34:379–402

    PubMed  CAS  Google Scholar 

  • Soderling SH, Beavo JA (2000) Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 12:174–179

    Article  PubMed  CAS  Google Scholar 

  • Stiefel J, Wang L, Kelly DA, Janoo RTK, Seitz J, Whitehall SK, Hoffman CS (2004) Suppressors of an adenylate cyclase deletion in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 3:610–619

    Article  PubMed  CAS  Google Scholar 

  • Thevelein JM, de Winde JH (1999) Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33:904–918

    Article  PubMed  CAS  Google Scholar 

  • Thevelein JM, Cauwenberg L, Colombo S, De Winde JH, Donation M, Dumortier F, Kraakman L, Lemaire K, Ma P, Nauwelaers D, Rolland F, Teunissen A, Van Dijck P, Versele M, Wera S, Winderickx J (2000) Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzyme Microb Technol 26:819–825

    Article  PubMed  CAS  Google Scholar 

  • Torphy TJ, Stadel JM, Burman M, Cieslinski LB, McLaughlin MM, White JR, Livi GP (1992) Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast. J Biol Chem 267:1798–1804

    PubMed  CAS  Google Scholar 

  • Wachtel H (1982) Characteristic behavioural alterations in rats induced by rolipram and other selective adenosine cyclic 3', 5'-monophosphate phosphodiesterase inhibitors. Psychopharmacol (Berl) 77:309–316

    Article  CAS  Google Scholar 

  • Wang L, Griffiths K, Zhang YH, Ivey FD, Hoffman CS (2005) Schizosaccharomyces pombe adenylate cyclase suppressor mutations suggest a role for cAMP phosphodiesterase regulation in feedback control of glucose/cAMP signaling. Genetics 171:1523–1533

    Article  PubMed  CAS  Google Scholar 

  • Welton RM, Hoffman CS (2000) Glucose monitoring in fission yeast via the gpa2 Gα, the git5 Gβ, and the git3 putative glucose receptor. Genetics 156:513–521

    PubMed  CAS  Google Scholar 

  • Zaks-Makhina E, Kim Y, Aizenman E, Levitan ES (2004) Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast. Mol Pharmacol 65:214–219

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Biology Department, Boston College, Higgins Hall 401B, Chestnut Hill, Boston, MA, 02467, USA

    Didem Demirbas, Ozge Ceyhan, Arlene R. Wyman & Charles S. Hoffman

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  1. Didem Demirbas
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  2. Ozge Ceyhan
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  3. Arlene R. Wyman
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  4. Charles S. Hoffman
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Corresponding author

Correspondence to Charles S. Hoffman .

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Editors and Affiliations

  1. School of Medicine, Dept. Molecular Physiology & Biophysics, Vanderbilt University, Light Hall 702, Nashville, 37232-0615, Tennessee, USA

    Sharron H. Francis

  2. HSW 1656, Center for Reproductive Sciences, Parnassus Avenue 513, San Francisco, 94143-0556, California, USA

    Marco Conti

  3. Wolfson Building, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

    Miles D. Houslay

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Demirbas, D., Ceyhan, O., Wyman, A.R., Hoffman, C.S. (2011). A Fission Yeast-Based Platform for Phosphodiesterase Inhibitor HTSs and Analyses of Phosphodiesterase Activity. In: Francis, S., Conti, M., Houslay, M. (eds) Phosphodiesterases as Drug Targets. Handbook of Experimental Pharmacology, vol 204. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17969-3_5

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