The role of these GPCRs in human disease is being explored, although more complex animal models will be needed because there are no obviously homologous genes in the mouse, so it is not possible to create knockouts. Meanwhile, chemical libraries are being screened for leads against these new GPCRs for both target validation and drug development.
Meanwhile, DiscoveRx (www.discoverx.com) provides a range of assay solutions for GPCRs and other important drug targets, all based upon its enzyme fragment complementation (EFC) assay technology. EFC is a new way of measuring protein-protein interactions. An enzyme acceptor fragment is fused to one protein, an enzyme donor fragment to another protein. If the two proteins bind, they make up a-galactosidase, which hydrolyzes a substrate that produces a chemiluminescent or fluorescent signal.
The company has now applied EFC to its new b-arrestin assays for GPCR binding, which are lead products in the PathHunter™ cell-based assay portfolio. Arrestins are key players in the desensitization of most GPCRs after ligand binding. Keith Olson, Ph.D., vp of product and market development at DiscoveRx, explains how GPCR stimulation triggers G-protein dissociation and GPCR kinase phosphorylation. The latter, which only phosphorylates active GPCRs, in turn triggers the recruitment of arrestin from the cytoplasm to the membrane. Here it binds to the phosphorylated C-terminus of GPCR, causing its internalization for either recycling or degradation. “This is a generic system applicable to virtually all GPCRs,” he says.
The DiscoveRx cell-based assay involves the fusion of the enzyme acceptor to b-arrestin and fusion of the donor (known as the ProLink) to the GPCR. When the arrestin binds to the GPCR, complementation between the enzyme acceptor and the ProLink occurs, leading to generation of a readout signal. Dr. Olson explains that this new format offers a number of advantages: generic implementation for any GPCR, reduced interference in comparison to second messenger and reporter gene assays, and greatly improved ease of use compared to imaging approaches.
“The assay is applicable to primary screening, secondary screening, selectivity profiling, and deorphanization of novel GPCRs,” he says. Currently the b-arrestin assay is applicable in around 70 GPCR-driven cell lines, and this will be extended to cover more than 200 GPCR targets over the next few months.