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.