Abstract: The present disclosure is directed to methods of identifying a compound that binds to or interacts with a protein receptor involved in bone formation. Specifically, the disclosure is directed to methods of identifying a compound that regulates a Wnt pathway in a cell by binding to or interacting with cavities in proteins such as LRP5, LRP 6 and/or frizzled receptor and interfering with receptor binding to other proteins in a Wnt pathway. The present disclosure is further directed to methods and compositions that comprise an identified compound for treating or preventing a disease in a mammal in which Wnt pathway suppression plays a role.

Abstract: The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass (HBM). In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293 cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by over-expression of Wnt coreceptor LRP5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transducing canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at the late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b.

Abstract: The mechanism by which the high bone mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates the canonical Wnt signaling was investigated. The mutation was previously shown to reduce Dkk protein-1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for Dkk protein-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperon protein for LRP5/6 that is required for the coreceptors' transport to cell surfaces, resulting in less LRP5 molecules on the cell surface. Although the reduction in the level of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm.

Abstract: The mechanism by which the high bone mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates the canonical Wnt signaling was investigated. The mutation was previously shown to reduce Dkk protein-1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for Dkk protein-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperon protein for LRP5/6 that is required for the coreceptors' transport to cell surfaces, resulting in less LRP5 molecules on the cell surface. Although the reduction in the level of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm.

Abstract: The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass (HBM). In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293 cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by over-expression of Wnt coreceptor LRP5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transducing canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at the late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b.

Abstract: The mechanism by which the high bone mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates the canonical Wnt signaling was investigated. The mutation was previously shown to reduce Dkk protein-1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for Dkk protein-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperon protein for LRP5/6 that is required for the coreceptors' transport to cell surfaces, resulting in less LRP5 molecules on the cell surface. Although the reduction in the level of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm.

Abstract: The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass (HBM). In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293 cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by over-expression of Wnt coreceptor LRP5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transducing canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at the late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b.