Polyplexed flow cytometry protein interaction assay: a novel high-throughput screening paradigm for RGS protein inhibitors

J Biomol Screen. 2009 Jul;14(6):610-9. doi: 10.1177/1087057109336590. Epub 2009 Jun 16.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Dose-Response Relationship, Drug
  • Flow Cytometry / methods*
  • GTP-Binding Protein alpha Subunits / metabolism
  • Humans
  • Kinetics
  • Protein Binding / drug effects
  • RGS Proteins / antagonists & inhibitors*
  • Small Molecule Libraries / analysis*
  • Small Molecule Libraries / pharmacology*

Substances

  • GTP-Binding Protein alpha Subunits
  • RGS Proteins
  • Small Molecule Libraries