Cell Based Assays

Although assays measuring binding of ligands to GPCRs have been used historically to identify GPCR drugs, most current technologies used for GPCR drug discovery are cell-based [20]. The primary readout of such assays is accumulation of second messenger levels in response to GPCR activation [21-24]. Assays measuring cellular levels of cAMP are dependent on the activity of adenylyl cyclase and detect GPCRs coupled to the G proteins, Gas and Gai. Many commercial assays are available to measure this cAMP response. These include radioimmunoassay, time - resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence resonance energy transfer (BRET)

assays, and those employing P-galactosidase enzyme fragment complementation (EFC) technology [3, 10, 11].

Activation of GPCRs coupled to Gq and Go is generally measured by detecting cellular levels of IP3, the end product of activation of phosphoinosit-ide phospholipase. IP3 levels can be detected using radioisotopes and biochemical approaches employing column chromatography. To adapt the assays for HTS, non -isotopic assays have been developed that measure IP3 levels such as PerkinElmer's ALPHAscreen (PerkinElmer, Waltham, MA) [3].

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