Assessing GPCR activation using protein complementation: a novel technique for HTS

GPCRs (G-protein-coupled receptors) are critical targets in drug discovery. Although most HTS (high-throughput screening) assays are routinely used to identify functional agonism or antagonism, they are suboptimal as methods to screen for modulators of other, novel, aspects of GPCR function. Indeed, it is now evident that GPCRs are highly complex proteins that interact with RAMPs (receptor-activity-modifying partners), β-arrestins, G-proteins, as well as functioning in potential homo- or hetero-meric complexes. Consequently, novel HTS technologies are now required that would facilitate interrogation of GPCRs in terms of their cellular protein–protein interactions. One approach is oligomerization-assisted complementation of monomeric protein fragments and detection of fragment reassembly. Notably, the use of enzymes has advantages in this regard, since complementation results in catalytically competent protein. The assay signal generated in this fashion results in assays of high sensitivity, thereby enabling protocols to be developed in HTS systems that require extremely low fluid volumes. The use of complementing proteins that generate a luminescent signal also provides assays that are markedly free from artefactual interferences.