Development and validation of a robust and sensitive assay for the discovery of selective inhibitors for serine/threonine protein phosphatases PP1α (PPP1C) and PP5 (PPP5C).
Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z'=0.93; representative PP1Cα assay data: Z'=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5.
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama , Mobile, Alabama.
Mark R
MR
Swingle
Richard E
RE
Honkanen
D002193:Cantharidin / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D017830:Octoxynol; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D013379:Substrate Specificity; D013501:Surface-Active Agents
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009687:Nuclear Proteins; D054852:Small Molecule Libraries; D013501:Surface-Active Agents; D017830:Octoxynol; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C089641:protein phosphatase 5; D002193:Cantharidin; D004121:Dimethyl Sulfoxide