从精准医学到药物发现：查耳酮衍生物抑制Smurf1活性，对特定老年骨质疏松小鼠亚群脊柱融合术中局部骨形成的影响研究

Age-related osteoporosis always accelerates the development of spinal degenerative diseases. Spinal fusion, which is widely applied in treatment of these diseases, requires osteoinductive factors to promote local bone formation. Bone morphogenetic protein (BMP) signaling is essential for osteoblastic bone formation. Recombinant human BMPs (rhBMPs) have been approved to promote local bone formation during spinal fusion. However, emerging evidence demonstrates large inter-individual variations in therapeutic action of rhBMPs. Smurf1 ubiquitinates BMP transducers (Smad1/5/8) for proteasomal degradation and is a bone formation suppressor. Recently, we found that age-related osteoporotic individuals (human and mice) could be classified into different subgroups based on distinct intraosseous BMP-2 levels and Smurf1 activity. Interestingly, one major subgroup demonstrated a decreased BMP-2 level and normal Smurf1 activity (termed as BMP-2d/Smurf1n), whereas another major subgroup had a normal BMP-2 level and elevated Smurf1 activity (termed as BMP-2n/Smurf1e). Moreover, BMP-2n/Smurf1e subgroup showed poor local bone anabolic response to rhBMP-2 during spinal fusion when compared to BMP-2d/Smurf1n subgroup. Then, we performed virtual screening and identified a chalcone derivative as the Smurf1 inhibitor. In vitro data showed that the chalcone derivative effectively interacted with Smurf1 WW1-WW2 domains, inhibited Smurf1 activity, increased BMP signaling and promoted in vitro osteogenic differentiation in osteoblasts from BMP-2n/Smurf1e subgroup. In this project, we will further examine the in vivo effects of the chalcone derivative on Smurf1 activity, BMP signaling and local bone formation during spinal fusion and also evaluate in vivo toxicity of the chalcone derivative in BMP-2n/Smurf1e subgroup of mice with age-related osteoporosis. This project will provide a precision medicine-based local bone anabolic strategy and the chalcone derivative will be a novel agent for spinal fusion in BMP-2n/Smurf1e subgroup of individuals with age-related osteoporosis.

骨质疏松加速脊柱退行性疾病的发生。脊柱融合作为主要疗法，需借助诱导因子促进局部骨形成。BMP通路调节骨形成。rhBMPs已被用于脊柱融合，但其促骨潜能有较大个体差异。Smurf1可抑制BMP通路。我们发现老年骨质疏松个体根据骨内BMP-2和Smurf1活性分成不同亚群。一个亚群有低的骨内BMP-2和正常Smurf1活性，并对rhBMP-2响应显著，另一个亚群有正常的骨内BMP-2和高Smurf1活性（BMP-2n/Smurf1e），但对rhBMP-2无明显响应。我们筛选了新型Smurf1抑制分子：查耳酮衍生物。该衍生物体外可作用并抑制Smurf1活性，促进BMP-2n/Smurf1e亚群成骨细胞分化。在此，我们将研究该衍生物对BMP-2n/Smurf1e亚群小鼠Smurf1活性、BMP通路、局部骨形成、脊柱融合的影响及其安全性。本项目中的查耳酮衍生物有望成为基于精准医学的促脊柱融合药物。

骨质疏松加速脊柱退行性疾病的发生。脊柱融合作为主要疗法，需借助诱导因子促进局部骨形成。BMP通路调节骨形成。rhBMPs已被用于脊柱融合，但其促骨潜能有较大个体差异。Smurf1可抑制BMP通路。我们发现老年骨质疏松个体根据骨内BMP-2和Smurf1活性分成不同亚群。一个亚群有低的骨内BMP-2和正常Smurf1活性，并对rhBMP-2响应显著，另一个亚群有正常的骨内BMP-2和高Smurf1活性（BMP-2n/Smurf1e），但对rhBMP-2无明显响应。我们筛选了新型Smurf1抑制分子：查耳酮衍生物。该衍生物体外可作用并抑制Smurf1活性，促进BMP-2n/Smurf1e亚群成骨细胞分化。在本项目中，我们对BMP-2n/Smurf1e亚群骨质疏松脊柱融合小鼠模型进行查耳酮衍生物局部给药。我们发现给药后小鼠脊柱融合率和血清骨钙素与对照相比有显著升高。X线观察、显微CT和骨形态计量学证据显示查耳酮衍生物显著提高了小鼠脊柱融合区的骨量和骨形成。我们从治疗后的小鼠L4-L6椎体之间分离原代成骨细胞，结果显示查耳酮衍生物治疗后的小鼠有较低的Smurf1活性、升高的Smad1磷酸化水平、Runx2活化及osteocalcin mRNA水平。而且，生化和血液学检测表明查尔酮衍生物无明显毒性。本项目中的查耳酮衍生物有望成为基于精准医学的促脊柱融合药物。