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Osteoporosis is a common, age-related disease caused by imbalanced bone remodeling. Current treatments either shut down bone resorption or robustly stimulate bone formation. Here, we describe a novel compound that inhibits osteoclast activity without causing apparent disruptions to bone formation by targeting both c-FMS (i.e., osteoclast differentiation) and αvβ3 integrin (i.e., osteoclastic bone resorption) receptors. We show that human serum albumin (HSA)-conjugated M-CSF RGD protein (M-CSF RGD -HSA) effectively inhibits the activity of both receptors, with a three-fold higher serum half-life compared to the unconjugated M-CSF RGD . We then treated ovariectomized mice with different doses of M-CSF RGD -HSA, alendronate, or a monospecific control protein. The bispecific M-CSF RGD -HSA was superior to a monospecific control in alleviating bone loss and reducing osteoclast distribution and function. M-CSF RGD -HSA and alendronate effectively prevented ovariectomy-induced bone loss, but M-CSF RGD -HSA had a milder inhibitory effect on osteoclast distribution and activity. Moreover, alendronate halted bone formation, while M-CSF RGD -HSA-treated mice showed an increased level of serum amino-terminal propeptide of type I collagen, a bone formation marker. Our data indicate that the mild reduction in osteoclast activity facilitated by the bispecific M-CSF RGD -HSA allows the maintenance of certain levels of bone formation and may be superior to treatments that induce osteoclast depletion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (Scl N93A ), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that Scl N93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of Scl N93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (Scl N93A Fc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)