Triple-addition assay protocols for detecting and characterizing modulators of seven-transmembrane receptors

The detection and characterization of seven-transmembrane-receptor modulators (orthosteric binding site agonists, antagonists, and more recently allosteric modulators) is an area of intense interest for both drug discovery and basic research. Traditionally, assays used to detect and characterize these different modes of modulation have been executed as separate, discrete protocols focused on a particular mode of action (e.g., agonism). In recent years, investigators have begun to combine aspects of these separate protocols to produce methods that detect multiple modes of modulation simultaneously. The power of such approaches is revealed not only in conservation of time and resources, but more importantly in a superior ability to discover and characterize novel modulators of the targets of interest. The protocols in this article describe a general procedure for developing, validating, and utilizing triple-addition assays to enable the simultaneous detection and characterization of multiple modes of seven-transmembrane-receptor modulation. Curr. Protoc. Chem. Biol. 3:119-140 © 2011 by John Wiley & Sons, Inc.