Cell Free Assays

Drug discovery for GPCRs has involved either the use of cell free or cell-based HTS assays. Cell-free assays include ligand binding and GTPase assays. Ligand binding technologies have been one of the linchpins in the GPCR drug discovery process over the years, and most FDA approved GPCR drugs were discovered using this approach. However, ligand binding assays are not employed as much for drug discovery nowadays because they require the use of high-affinity, selective ligands that can be chemically radiolabeled, and for many receptors, such ligands are either not available or are cost prohibitive.

An alternative approach to ligand binding assays focuses on the interaction of GPCRs with G proteins and the fact that stimulation of GPCRs by an agonist results in an increase in GTPase catalytic activity of the G protein. Thus, one assay that has been employed for identifying GPCR agonists measures the ability of a compound to promote the binding of radiolabeled non-hydrolyzable GTP analogs, such as 35S-GTPyS, to G proteins which are coupled to a GPCR [18]. Such binding can be detected using a scintillation proximity assay (SPA) format and is easily adaptable for HTS. Furthermore, nonradioactive alternatives exist to measure GTPyS binding, such as the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) Eu-GTPyS. These non--sotopic ligands have also been adapted for HTS with the advantage of reduced cost due to a lack of radioactive waste. Importantly, novel technologies using GPCR-Ga fusion proteins eliminate the problem of whether the target cells expressing the recombinant GPCR under study have the appropriate endogenous G proteins that couple with the GPCR or whether the ratio of expression of GPCR to G protein is unnatural since by nature the stoichi-ometry of GPCR to G proteins in the fusion protein is 1:1 [19].

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