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Duplexed Label-Free G Protein—Coupled Receptor Assays for High-Throughput Screening

This article describes duplexed label-free optical biosensor cellular assays for simultaneously assaying 2 endogenous receptors, the Gq-coupled histamine receptor (H 1) and the Gs-coupled β2-adrenergic receptor (β2AR), in A431 cells. The biosensor cellular assays consist of 2 sequential steps—an initial agonist screening using Sigma LOPAC (Library of Pharmaceutically Active Compounds) and a subsequent antagonist screening using a solution mixture containing the H1 agonist histamine and the β2AR agonist epinephrine. Results showed that costimulating A431 cells with histamine and epinephrine led to an optical response additive to individual responses. The agonist screening not only identified all full agonists for both the H1 and β2 receptors, but also detected pathway-biased ligands for the β2AR. Furthermore, the succeeding antagonist screening documented all known antagonists in the library for either the H1 or β2 receptors. This is the 1st demonstration of a single cellular assay that is capable of screening ligands against 2 GPCRs coupled to distinct G proteins, and highlights the power of pathway-unbiased and label-free biosensor cellular assays for GPCR screens. (Journal of Biomolecular Screening 2008:975-985)