A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ~0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC50 value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.
References
[1]
Bender AT, Beavo JA (2006) Cyclic nucleotide phosphodiesterases: Molecular regulation to clinical use. Pharmacol Rev 58: 488–520.
[2]
Iacob RE, Pene-Dumitrescu T, Zhang J, Gray NS, Smithgall TE, et al. (2009) Conformational disturbance in abl kinase upon mutation and deregulation. Proc Natl Acad Sci U S A 106: 1386–1391.
[3]
Alaamery MA, Wyman AR, Ivey FD, Allain C, Demirbas D, et al. (2010) New classes of PDE7 inhibitors identified by a fission yeast-based HTS J Biomol Screen. 15: 359–367.
[4]
Kadoshima-Yamaoka K, Murakawaa M, Goto M, Tanaka Y, Inoue H, et al. (2009) ASB16165, a novel inhibitor for phosphodiesterase 7A (PDE7A), suppresses IL-12-induced IFN-gamma production by mouse activated T lymphocytes. Immunol Lett 122: 193–197.
[5]
Smith SJ, Cieslinski LB, Newton R, Donnelly LE, Fenwick PS, et al. (2004) Discovery of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-ni？trobenzene],a selective inhibitor of phosphodiesterase 7: In vitro studies in human monocytes, lung macrophages, and CD8+ T-lymphocytes. Mol Pharmacol 66: 1679–1689.
[6]
Soderling SH, Bayuga SJ, Beavo JA (1998) Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci U S A 95: 8991–8996.
[7]
Vasta V, Shimizu-Albergine M, Beavo JA (2006) Modulation of leydig cell function by cyclic nucleotide phosphodiesterase 8A. Proc Natl Acad Sci U S A 103: 19925–19930.
[8]
Vang AG, Ben-Sasson SZ, Dong H, Kream B, DeNinno MP, et al. (2010) PDE8 regulates rapid teff cell adhesion and proliferation independent of ICER. PLoS One 5: e12011.
[9]
Patrucco E, Albergine MS, Santana LF, Beavo JA (2010) Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes. J Mol Cell Cardiol 49: 330–333.
[10]
Tsai LC, Shimizu-Albergine M, Beavo JA (2011) The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland Mol Pharmacol. 79: 639–648.
[11]
Hayashi M, Matsushima K, Ohashi H, Tsunoda H, Murase S, et al. (1998) Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 250: 751–756.
[12]
Shimizu-Albergine M, Tsai LC, Patrucco E, Beavo JA (2012) cAMP-specific phosphodiesterases 8A and 8B, essential regulators of leydig cell steroidogenesis Mol Pharmacol. 81: 556–566.
[13]
Bol SM, Booiman T, Bunnik EM, Moerland PD, van Dort K, et al. (2011) Polymorphism in HIV-1 dependency factor PDE8A affects mRNA level and HIV-1 replication in primary macrophages. Virology 410: 32–42.
[14]
Horvath A, Giatzakis C, Tsang K, Greene E, Osorio P, et al. (2008) A cAMP-specific phosphodiesterase (PDE8B) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: A novel PDE8B isoform in human adrenal cortex. Eur J Hum Genet 16: 1245–1253.
[15]
Hoffman CS (2005) Glucose sensing via the protein kinase A pathway in schizosaccharomyces pombe. Biochem Soc Trans 33: 257–260.
[16]
Hoffman CS, Winston F (1990) Isolation and characterization of mutants constitutive for expression of the fbp1 gene of schizosaccharomyces pombe. Genetics 124: 807–816.
[17]
Demirbas D, Ceyhan O, Wyman AR, Hoffman CS (2011) A fission yeast-based platform for phosphodiesterase inhibitor HTSs and analyses of phosphodiesterase activity Handb Exp Pharmacol. (204): 135–149.
[18]
Demirbas D, Ceyhan O, Wyman AR, Ivey FD, Allain C, et al. (2011) Use of a schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases Cell Signal. 23: 594–601.
[19]
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.
[20]
Gutz H, Heslot H, Leupold U, Loprieno N. (1974) Schizosaccharomyces pombe. In: King RC, editor. Handbook of genetics. New York, NY: Plenum Press. 395–446.
[21]
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.
[22]
Ivey FD, Taglia FX, Yang F, Lander MM, Kelly DA, et al. (2010) Activated alleles of the schizosaccharomyces pombe gpa2+ galpha gene identify residues involved in GDP-GTP exchange Eukaryot Cell. 9: 626–633.
[23]
Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4: 67–73.
[24]
Ceyhan O, Birsoy K, Hoffman CS (2012) Identification of biologically active PDE11-selective inhibitors using a yeast-based high-throughput screen Chem Biol. 19: 155–163.
[25]
Wang H, Liu Y, Chen Y, Robinson H, Ke H (2005) Multiple elements jointly determine inhibitor selectivity of cyclic nucleotide phosphodiesterases 4 and 7. J Biol Chem 280: 30949–30955.
[26]
Morris G, Goodsell D, Halliday R, Huey R, Hart W, et al. (1998) Automated docking using a lamarckian genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry 19: 1639–1662.
[27]
Seiler KP, George GA, Happ MP, Bodycombe NE, Carrinski HA, et al. (2008) ChemBank: A small-molecule screening and cheminformatics resource database. Nucleic Acids Res 36: D351–D359.
[28]
Mellon SH, Vaisse C (1989) cAMP regulates P450scc gene expression by a cycloheximide-insensitive mechanism in cultured mouse leydig MA-10 cells. Proc Natl Acad Sci U S A 86: 7775–7779.
[29]
Dong H, Osmanova V, Epstein PM, Brocke S (2006) Phosphodiesterase 8 (PDE8) regulates chemotaxis of activated lymphocytes. Biochem Biophys Res Commun 345: 713–719.
[30]
Glavas NA, Ostenson C, Schaefer JB, Vasta V, Beavo JA (2001) T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3. Proc Natl Acad Sci U S A 98: 6319–6324.
[31]
Wang H, Yan Z, Yang S, Cai J, Robinson H, et al. (2008) Kinetic and structural studies of phosphodiesterase-8A and implication on the inhibitor selectivity. Biochemistry 47: 12760–12768.
[32]
DeNinno MP, Wright SW, Visser MS, Etienne JB, Moore DE, et al. (2011) 1,5-substituted nipecotic amides: Selective PDE8 inhibitors displaying diastereomer-dependent microsomal stability. Bioorg Med Chem Lett 21: 3095–3098.
[33]
Haider SG (2007) Leydig cell steroidogenesis: Unmasking the functional importance of mitochondria. Endocrinology 148: 2581–2582.
[34]
D'Andrea MR, Qiu Y, Haynes-Johnson D, Bhattacharjee S, Kraft P, et al. (2005) Expression of PDE11A in normal and malignant human tissues. J Histochem Cytochem. 53: 895–903.
[35]
Fawcett L, Baxendale R, Stacey P, McGrouther C, Harrow I, et al. (2000) Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A. Proc Natl Acad Sci U S A 97: 3702–3707.
