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.
KeywordsCyclic nucleotide phosphodiesterase Fission yeast Schizosaccharomyces pombe fbp1 High-throughput screen Inhibitors
This work was supported by NIH grant GM079662, the Peter Rieser Lectureship Fund, and a grant from Boston College to C.S.H.