Normal bone development is a complex interplay of bone formation by osteoblasts and bone resorption byosteoclasts. Age-associated, environmental, and endogenous factors, as well as genetic abnormalities canalter that homeostasis, leading to osteoporosis and other diseases associated with bone loss. New therapeutictargets and especially osteoblast anabolic drugs are needed to treat such disorders. Insulin-like3 (INSL3)peptide has recently been shown to have an important role in bone metabolism. It signals through its G protein-coupled receptor RXFP2 to control osteoblast differentiation and function. Expression of RXFP2 has beendemonstrated in human and mouse osteoblasts and osteocytes. Patients with RXFP2 mutations developosteopenia and osteoporosis. In mice, inactivation of RXFP2 causes a decrease in bone mass, mineralizingsurface, bone formation, and osteoclast surface compared with wild-type littermates. Treatment of osteoblastswith INSL3 induced their complete differentiation coupled with increased expression of osteoblast markers, aswell as the ability to mineralize the extracellular matrix. This establishes the INSL3 signaling pathway as apromising novel pharmacological target, especially because the INSL3 receptor is a cellular membrane GPCR.However, to date no small molecule RXFP2 agonists are known. The current application is designed to fill thisgap through high throughput screening (HTS) of a large small molecule compound library at NIH NCGC.RXFP2 activation by INSL3 causes an easily detectable increase in cAMP production. Using HEK293T cellsstably transfected with RXFP2, we have optimized a cAMP assay for quantitative HTS of RXFP2 agonists in a1536-well format. The assay will be used for the RXFP2 agonist screening campaign. After the primary screen,the active compounds will be tested in a series of secondary assays designed to identify specific INSL3receptor agonists. The secondary assays include a counterscreen against parental HEK293T cells, cellstransfected with related GPCRs, and a confirmation screen using an orthogonal cAMP detection method.Structure-activity relationship studies coupled with probe-receptor interaction modeling will be performed toimprove potency, efficacy and selectivity of the compounds. Tertiary cell-based assays that include osteoblastproliferation, differentiation, and mineralization effects, along with known target gene expression andproteomics studies will be used to select the most active compounds with preferred pharmacological profiles.Functional characterization of RXFP2 agonists will further verify their role in regulating bone formation. Thediscovery of INSL3 receptor agonists will provide a basis for their testing as novel, safeanabolic therapeutic drugs against osteoporosis and other diseases associated with low bone mass.
