Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases

Abstract

The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1-ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5' AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1-ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2-GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increase the amount of exogenous cGMP required to activate PKA in order to repress fbp1-ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC 50 of 232 nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMPor cGMP-metabolising PDEs.

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

“…Cells were cultured at 30°C in YES-rich (yeast extract medium with supplements), EMM-defined, or 5FOA media as described previously [17–19]. Insertions of the murine PDE4A1 or PDE4B3 , rat PDE4A5 , human PDE4A1 , PDE4B2 , PDE4D3 , PDE7A1 or PDE7B1 gene into the S. pombe cgs2 + (PDE) locus as well as fbp1-ura4 + and fbp1-GFP reporters have been previously described [18, 20, 21]. …”

“…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.…”

“…A pool of alleles of the human PDE4B2 gene (Genbank accession number L20971) were generated by PCR amplification of PDE4B2 inserted into the S. pombe cgs2 locus in plasmid pKG3-PDE4B2 [20]. PCR was carried out using the FailSafe ™ PCR kit with 2X buffers B, C, J and L (Epicentre Biotechnologies), as these conditions produce both transition and transversion mutations (unpublished data).…”

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

“…Cells were grown at 30°C in YEA-rich, EMM-defined, or 5FOA media as previously described [16], [20], [21]. Insertions of the murine PDE8A1 gene or the human PDE4A1, PDE7A1 or PDE7B1 gene into the S. pombe cgs2 (PDE) locus have been previously described (the cgs2 PDE gene was chosen as the site of integration so as to express the mammalian PDEs at a physiologically-relevant level in our strains) [18]. A plasmid expressing the catalytic domain of murine PDE8A was constructed by PCR-amplifying and cloning a 1325 bp region of the PDE8A gene (codons 443 to 824 of PDE8A1) into EcoRI -linearized pNMT1 (Invitrogen, Carlsbad, CA) vector via gap repair transformation [22].…”

“…Cells were inoculated at an initial cell density of 0.5–2×10 5 cells/mL. Previously described optimized pre-growth conditions for each strain were used to repress the fbp1-ura4 + reporter prior to inoculation into 5FOA medium, as cells that are already expressing the Ura4 enzyme would be 5FOA-sensitive even upon exposure to a PDE inhibitor [18]. Following a 48-h incubation at 30°C, plates were vortexed and optical densities (600 nm) were measured.…”