Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. The pathophysiological cause of the disease is a decrease in the activity of the bone-forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the relaxin family peptide receptor 2 (RXFP2), the G protein-coupled receptor (GPCR) for insulin-like 3 peptide (INSL3), as an attractive target expressed in osteoblast cells to increase bone formation. The goal of this dissertation is to discover and characterize small molecule agonists of RXFP2 that are stable and can be delivered orally to promote bone growth. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a cAMP high-throughput screen of the NCATS small molecule library. An extensive structure-activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of these compounds for human and mouse RXFP2 was shown in counter-screens against the related relaxin receptor RXFP1 and other GPCRs. Using a series of RXFP2/RXFP1 chimeric receptors, in silico modeling and RXFP2 point mutants, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. We also showed that the candidate compounds promoted mineralization in primary human osteoblasts and had low cytotoxicity in various cell types. The compound with the highest activity in vitro was selected for pharmacokinetics profiling in mice, showing oral bioavailability and bone exposure. Moreover, an efficacy study in wild-type female mice treated orally with the lead compound showed a significant increase of the vertebral trabecular number and thickness compared to vehicle treated controls. Overall, our study has successfully identified and characterized the first-in-class small molecule series of RXFP2 agonists, which may lead to the development of a new class of orally bioavailable drugs for the treatment of diseases associated with bone loss
