Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast.
Heterologous expression of cyclic nucleotide phosphodiesterases (PDEs) and adenylyl cyclases (ACs) in the fission yeast Schizosaccharomyces pombe can be used in combination with PKA-repressed reporters to either carry out high throughput screens for small molecule inhibitors of these target enzymes or to assess hit compounds and their analogs from such screens. Here, we describe two methods for testing panels of such compounds. The first uses a growth assay for which growth in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA) occurs in response to inhibiting PDE activity to activate PKA. The second uses mass spectrometry to directly measure the impact of compound treatment to study compounds that modulate either PDE or AC activity.
Domin M, Hoffman CS