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Polyplexed Flow Cytometry Protein Interaction Assay: A Novel High-Throughput Screening Paradigm for RGS Protein Inhibitors

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan

Shodai Ota

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan

Richard R. Neubig

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, Center for Chemical Genomics, University of Michigan Life Sciences Institute, Ann Arbor, Michigan, RNeubig{at}umich.edu

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 Go, 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 Go 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. (Journal of Biomolecular Screening 2009: 610-619)

Key Words: G-protein • RGS • flow cytometry • FCPIA • high-throughput screening

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 610-619 (2009)
DOI: 10.1177/1087057109336590


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