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MrgD, a member of the Mas-related gene family, is expressed exclusively in small diameter IB4+ neurons in the dorsal root ganglion. This unique expression pattern, the presence of a single copy of MrgD in rodents and humans, and the identification of a putative ligand, beta-alanine, make it an experimentally attractive therapeutic target for pain with limited likelihood of side effects. We have devised a high throughput calcium mobilization assay that enables identification of both agonists and antagonists from a single screen for MrgD. Screening of the Library of Pharmacologically Active Compounds (LOPAC) validated this assay approach, and we identified both agonists and antagonists active at micromolar concentrations in MrgD expressing but not in parental CHO-DUKX cell line. Further characterization was performed using a subset of these screening hits. Our results demonstrated that the dual agonist/antagonist assay format is feasible and likely can be extended to most GPCRs with known agonist.

Development of a FLIPR Assay for the Simultaneous Identification of MrgD Agonists and Antagonists from a Single Screen