From precision medicine to drug discovery: osteoblast-specific inhibition of Smurf1 activity promotes bone formation in distinctive rats with age-related osteoporosis

Age-related osteoporosis affects 20% of elderly population in mainland of China and Hong Kong. The only FDA-approved bone anabolic agent PTH also stimulates bone resorption after long-term use. Thus, it is urgent to develop alternative bone anabolic drugs for age-related osteoporosis.

Bone morphogenetic protein (BMP) signaling plays an essential role in osteoblast-mediated bone formation. Smurf1 ubiquitinates BMP downstream molecules and acts as a suppressor of BMP signaling. In 2002, recombinant human BMPs (rhBMPs) were approved for promoting local bone formation during spinal fusion. However, increasing evidence demonstrates large inter-individual variations in their bone anabolic potential for patients with age-related osteoporosis. Our data showed that the age-related osteoporotic individuals (human and rodents) could be classified into different subgroups based on distinct intraosseous BMP-2 levels and Smurf1 activity. One major subgroup showed a normal BMP-2 level and elevated Smurf1 activity (BMP-2normal/Smurf1elevated; BMP-2n/Smurf1e), whereas another major subgroup exhibited a decreased BMP-2 level and normal Smurf1 activity (BMP-2decreased/Smurf1normal; BMP-2d/Smurf1n). Serum osteocalcin with discriminated reduction extents could be a biomarker in stratifying the two subgroups. BMP-2n/Smurf1e subgroup of age-related osteoporotic human and rodents showed poor bone anabolic response to rhBMP-2 during spinal fusion when compared to BMP-2d/Smurf1n subgroup. Silencing Smurf1 enhanced BMP signaling and promoted osteogenic differentiation in primary osteoblasts isolated from BMP- 2n/Smurf1e subgroup, suggesting that Smurf1 inhibition in osteoblasts could be a precision medicine-based strategy to promote bone formation for the BMP-2n/Smurf1e subgroup of age- related osteoporosis. All the above results have been accepted by Science Translational Medicine.

By molecular docking-based virtual screening, we identified a chalcone derivative, i.e., 2-(4- cinnamoylphenoxy)acetic acid, which effectively targeted Smurf1 for inhibition. After local administration, the chalcone derivative promoted bone formation during spinal fusion for the BMP-2n/Smurf1e subgroup of age-related osteoporotic rats. To achieve the osteoblast-specific distribution of the chalcone derivative after systemic administration, we conjugated the chalcone derivative with an osteoblast-targeting and cell-penetrating oligopeptide (DSS)6. (DSS)6 facilitated the chalcone derivative inhibiting Smurf1 activity and enhancing in vitro osteogenic differentiation in primary osteoblasts isolated from rat BMP-2n/Smurf1e subgroup. In vivo, (DSS)6 facilitated the chalcone derivative accumulating in bone of the BMP- 2n/Smurf1e subgroup of age-related osteoporotic rats. Thus, we hypothesize that (DSS)6 could facilitate the chalcone derivative targeting osteoblasts and promoting systemic bone formation for the BMP-2n/Smurf1e subgroup in rats with age-related osteoporosis.

To test the above hypothesis, 6-month-old female rats will be ovariectomized to induce age- related osteoporosis according to our established protocol (Zhang G. Nat Med 2012; Wang X. Nat Med 2013). The following four specific aims will be examined in BMP-2n/Smurf1e subgroup of age-related osteoporotic rats in vivo:
1) Osteoblast-specific distribution of the (DSS)6-chalcone derivative conjugate;
2) Dose-response pattern and persistence time of the (DSS)6-chalcone derivative conjugate; 3) Bone anabolic action of the (DSS)6-chalcone derivative conjugate;
4) Toxicity of the (DSS)6-chalcone derivative conjugate.

The current proposal would advance the precision medicine-based bone anabolic strategy, i.e., osteoblast-specific inhibition of Smurf1 activity by the (DSS)6-chalcone derivative conjugate, for the BMP-2n/Smurf1e subgroup of age-related osteoporosis.