title: A yeast-based high-throughput screen for modulators of phosphodiesterase activity.
abstract: 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.
authors.affiliation: Biology Department, Boston College, Higgins Hall 401b, 140 Commonwealth Avenue, Chestnut Hill, MA, 02467, USA.
authors.forename: Ana Santos
authors.initials: AS
authors.lastname: de Medeiros
authors.forename: Charles S
authors.initials: CS
authors.lastname: Hoffman
mesh_terms: D003470:Culture Media / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D010726:Phosphodiesterase Inhibitors; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases