A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening.

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gαs gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC®1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.

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

We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.

A yeast-based high-throughput screen for modulators of phosphodiesterase activity.

Cell-based high-throughput screens (HTSs) targeting heterologously expressed proteins in yeast identify compounds that often display relevant biological activity when tested in cell culture. We developed a fission yeast-based HTS to detect small-molecule inhibitors of mammalian cyclic nucleotide phosphodiesterases (PDEs). These screens are carried out in Schizosaccharomyces pombe using a PKA-repressed fbp1-ura4 reporter whose expression due to low PKA activity prevents cells from growing in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA). We describe here the steps required to construct strains for screening and to optimize conditions for successful screens.

Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators.

Features of the fission yeast Schizosaccharomyces pombe cAMP/PKA pathway make S. pombe particularly amenable for heterologous expression of cAMP pathway proteins such as GαS subunits and their cognate adenylyl cyclases, PKA catalytic and regulatory subunits, and cyclic nucleotide phosphodiesterases. We have constructed two PKA-repressed reporters for use in high-throughput screens to detect compounds that elevate or reduce PKA activity, thus facilitating the discovery of both inhibitors and activators of these target proteins. Here, we describe steps to construct screening strains and to optimize and conduct these screens.

Use of PKA-mediated phenotypes for genetic and small-molecule screens in Schizosaccharomyces pombe.

PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of genes involved in metabolism, cell growth and sexual development. In the present review, we discuss phenotypes associated with either high or low PKA activity in the context of how they can be used to carry out genetic or small-molecule screens that affect components of the PKA pathway. Although our recent research has focused on the study of heterologously expressed cyclic nucleotide PDEs (phosphodiesterases), these same methods can be used to target other S. pombe proteins or their functionally equivalent orthologues that act in the PKA pathway.