A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition

PLoS One. 2013 Aug 14;8(8):e71279. doi: 10.1371/journal.pone.0071279. eCollection 2013.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cyclic Nucleotide Phosphodiesterases, Type 4 / chemistry
  • Cyclic Nucleotide Phosphodiesterases, Type 4 / metabolism*
  • Drug Evaluation, Preclinical / methods*
  • High-Throughput Screening Assays
  • Humans
  • Leydig Cells / drug effects*
  • Leydig Cells / metabolism*
  • Male
  • Mice
  • Molecular Docking Simulation
  • Phosphodiesterase 4 Inhibitors / chemistry
  • Phosphodiesterase 4 Inhibitors / metabolism
  • Phosphodiesterase 4 Inhibitors / pharmacology*
  • Protein Conformation
  • Schizosaccharomyces / genetics*
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / metabolism
  • Small Molecule Libraries / pharmacology
  • Steroids / biosynthesis*

Substances

  • Phosphodiesterase 4 Inhibitors
  • Small Molecule Libraries
  • Steroids
  • Cyclic Nucleotide Phosphodiesterases, Type 4