A Luminescent cAMP Assay for determining cellular and tissue cAMP and monitoring the Modulation of Gs and Gi Protein Coupled Receptors

Abstract

G Protein Coupled Receptors represent a large validated target for Drug discovery research. The Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase ƒÒ. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, fast, HTS formatted with high Z¡¦, and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.