High throughput screening assays to identify modulators of GPCR function have evolved from filter binding to measuring GPCR internalization using high-content screening platforms. The decision to utilize a specific technology depends on the expected properties of a compound and the characteristics of the technology (sensitivity, quenching, cost, throughput, miniaturization, required instrumentation). For example, identification of ligands for orphan receptors will likely require functional cell-based assays (gene reporter systems, melanophores, Ca2+, etc.) rather than ligand binding assays. Also, the receptor can sometimes define the choice of technology; screening for antagonists of Gq-coupled receptors will preferably be performed with a Ca2+ assay in the FLIPR whereas a Gi-coupled receptor will be assayed by cAMP or Ca2+ using promiscuous or chimeric G proteins. If a compound needs to function as an allosteric modulator on a specific GPCR, ligand binding assays will not entirely reveal such compounds and signal transduction assay would be a better choice.

Tremendous progress has been made in understanding the signal transduction and regulation of GPCRs (transactivation, homodimerization, heterodimerization, novel effectors). Although GPCR assay tools have improved in terms of sensitivity, throughput and ease of use, most current screening technologies rely on classic GPCR mechanisms such as receptor binding, GTP binding, and cAMP and Ca2+ signaling. Some high content screening technologies take advantage of newer paradigms such as desensitization or internalization of GPCRs (Transfluor and CypHer dyes). These high-content screening methodologies are becoming integral parts of the functional analysis required for the lead discovery and optimization processes.

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