Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

Abstract

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3′,5′-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens. (Journal of Biomolecular Screening 2008:62-71)

Identification and characterization of a potent and biologically-active PDE4/7 inhibitor via fission yeast-based assays

“…5FOA growth assays to screen for and to characterize PDE inhibitors were carried out using strains that express the PKA-repressed fbp1-ura4 + reporter as previously described [13, 20, 22–25]. Dose response profiling of BC54 and rolipram was carried out at concentrations of 0.05 μM to 50 μM.…”

Anti-inflammatory effects of novel barbituric acid derivatives in T lymphocytes

“…HTS screens for PDE inhibitors that promote 5FOA-resistant (5FOA R ) growth of yeast strains due to PKA activation were performed at the Broad Institute's Chemical Biology Program screening facility as previously described [19,20]. In vitro PDE assays were conducted as described by Wang et al [21] in which the amount of cAMP remaining in the reaction is measured by scintillation counting following differential Ba(OH) 2 precipitation of the 3 H-AMP produced by PDE-mediated hydrolysis of 3 H-cAMP.…”

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

“…In addition to the known inhibitor controls, several specific inhibitors were identified, with the assay having a hit rate of 0·8% to 3·2%. In a secondary screen, a subset of these compounds demonstrated the ability to raise the level of cellular cAMP, indicating PDE inhibition (Ivey et al 2008). PDE7 has been similarly screened, this time with a library of nearly 50 000 compounds (Alaamery et al 2010).…”

“…This robust assay system, measuring growth by optical density in 384-well plates, has been utilized for HTS of compound libraries against several human PDEs. A small subset of compounds (3120, including known PDE inhibitors) were screened against human PDE2A, PDE4A and PDE4B and yeast Cgs2 (Ivey et al 2008). In addition to the known inhibitor controls, several specific inhibitors were identified, with the assay having a hit rate of 0·8% to 3·2%.…”