A next-generation orally administered precise sclerostin inhibitor in osteogenesis imperfecta

Sclerostin represents a promising therapeutic target for bone anabolic therapies. Romosozumab, the first FDA-approved sclerostin inhibitor, blocks sclerostin loop2-LRP5/6 interaction by targeting loop2, which effectively treats osteoporosis. However, increased cardiovascular risks in clinical trials limit its use. Recently, we found that sclerostin could antagonize bone anabolic through sclerostin loop3-LRP4 interaction, and protect the cardiovascular system through sclerostin loop2-LRP8 interaction in macrophages, respectively. We proposed a precise sclerostin inhibition strategy that specifically blocks loop3-LRP4 interaction while maintaining loop2-LRP8 interaction to promote bone formation without elevating cardiovascular concerns.

We have identified four key residues within loop3 for loop3-LRP4 interaction used by sclerostin to suppress osteogenic potential in MC3T3-E1 cells (osteoblasts). We then designed a small-molecular inhibitor (PA-Hit B) targeting loop3, which was predicted to bind above four key residues. In vitro, PA-Hit B completely blocked loop3-LRP4 interaction and demonstrated a superior inhibitory potency against sclerostin's suppressive effect on osteogenic potential than romosozumab. As expected, PA-Hit B maintained loop2-LRP8 interaction and did not influence inflammatory cytokines in macrophages, whereas romosozumab did. In osteogenesis imperfecta (OI, Col1a2+/G610C) mice, PA-Hit B promoted bone formation in a dose-dependent manner, outperforming romosozumab. To be noted, PA-Hit B had a long half-life (12h) and high oral bioavailability (62.11%) in healthy rats. Importantly, as a small molecule, PA-Hit B is a promising candidate for developing the next-generation orally administered precise sclerostin inhibitor.

Thus, we hypothesized that PA-Hit B could exert bone anabolic potential without elevating cardiovascular risk in OI mice via specifically blocking loop3-LRP4 interaction. Our in vitro mechanistic studies showed that PA-Hit B promoted osteogenic potential in MC3T3-E1 cells via specifically blocking loop3-LRP4 interaction. Consistently, in vivo data from a small sample size of OI mice indicated that PA-Hit B promoted bone formation via the same mechanism.

Four objectives will be achieved to further test the hypothesis. In Objectives 1 & 2, we will evaluate, in a larger sample size of OI mice, whether PA-Hit B exerts its bone anabolic potential via specifically blocking loop3-LRP4 interaction. This will be evaluated through pretreatment with blocking peptide designed based on key residues within loop3, and by replacing PA-Hit B with its variant, respectively. In Objective 3, we will assess whether PA-Hit B increases cardiovascular concerns in hSOSTTg.Col1a2+/G610C.ApoE-/- mice. In Objective 4, we will investigate its long-term safety profile in normal rats.

This project aims to discover a next-generation orally administered precise sclerostin inhibitor and understand how it works in OI.