Polyplexed flow cytometry protein interaction assay: a novel high-throughput screening paradigm for RGS protein inhibitors.
Intracellular signaling cascades are a series of regulated protein-protein interactions that may provide a number of targets for potential drug discovery. Here, the authors examine the interaction of regulators of G-protein signaling (RGS) proteins with the G-protein Galphao, using a flow cytometry protein interaction assay (FCPIA). FCPIA accurately measures nanomolar binding constants of this protein-protein interaction and has been used in high-throughput screening. This report focuses on 5 RGS proteins (4, 6, 7, 8, and 16). To increase the content of screens, the authors assessed high-throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Galphao in isolation, and then in a multiplex format with 5 RGS proteins present. To use this methodology as a higher-content multiplex protein-protein interaction screen, they established Z-factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, they also compressed a set of 8000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in their mixtures using the polyplexed FCPIA method.
Roman
David L
DL
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Ota
Shodai
S
Neubig
Richard R
RR
D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects; D020710:RGS Proteins / Q000037:antagonists & inhibitors*; D054852:Small Molecule Libraries / Q000032:analysis* / Q000494:pharmacology*
D044385:GTP-Binding Protein alpha Subunits; D020710:RGS Proteins; D054852:Small Molecule Libraries