新型异戊烯基二氢黄酮类组织蛋白酶K抑制剂的结合位点确证及其抗骨质疏松作用机制研究

Cathepsin K is a protease that highly expresses on osteoclast with the prominent effects on bone resorption, while it also expressed weakly in other tissues, such as heart, lung and skin. All current inhibitors that developed to block the active site of cathepsin K led to a completely inhibition of its hydrolytic activity. However, it not only affected the collagenase activity in bone but also influenced the none-collagen degradation in other tissues. Therefore many drug candidates were failed and stopped in clinical phases. Recently, some researches indicated exosite 1 is a special site of cathepsin K that could be selectively blocked by inhibitors on bone without off-targets side effects. In our previous study, a novel class of cathepsin K inhibitor: prenyl dihydroflavonoid, was discovered. It effectively suppressed bone resorption in vitro. It also interacted stably with exosite 1 by molecular dynamics simulation. Thus, in the current study, we hypothesized that two prenyl dihydroflavonoids：Kushennol F and Sophoraflavone G might exert its preventive effects on bone by direct binding to exosite 1 of cathepsin K and selective blocking the collagenase activity on bone. The ovariectomized osteoporotic mice model will be established, the anti-osteoporosis activity and collagenase selective inhibition on bone of these inhibitors will be evaluated. Moreover, the cathepsin K site-specific substrate degradation assay, the binding assay for compounds with site-directed mutagenesis protein using surface plasmon resonance (SPR) technique and bone resorbed assay using cathepsin K overexpression steady cell line and human bone marrow monocyte will be carried out in vitro. Our project will provide a strong evidence for developing exosite1-directed cathepsin K inhibitor on treatment of osteoporosis.

组织蛋白酶K是破骨细胞中表达量最高、溶骨活性最强的蛋白酶，但该酶也少量表达于心、肺、皮肤等组织。基于该酶活性位点开发抑制剂会全面抑制其活性，影响骨以外其他组织的非胶原蛋白降解，最终导致多个药物临床失败。最新研究发现结合于该酶外周位点exosite1的抑制剂，可选择性抑制骨胶原蛋白降解。申请人前期发现异戊烯基二氢黄酮类成分可抑制组织蛋白酶K活性和骨吸收，分子模拟显示它们可稳定结合于exosite1。由此提出假设：异戊稀基二氢类成分苦参醇F和槐黄烷酮G可能结合于exosite1，特异性抑制骨胶原蛋白降解，发挥抗骨质疏松作用。本项目拟建立去势骨质疏松小鼠模型，评价其药效及抑制骨胶原降解特异性。体外通过底物降解选择性测试，与位点突变蛋白结合的SPR分析，明确成分的结合位点。基于组织蛋白酶K过表达细胞株及人骨髓单核细胞的骨吸收诱导等实验，阐明其抗骨质疏松作用机制。本研究旨在为开发中药来源的新型组织蛋白酶K抑制剂治疗骨质疏松奠定基础。

组织蛋白酶K(CTSK)是治疗骨质疏松症的有效靶点，针对该靶点活性位点（active site）研发的抑制剂在临床试验中被证实疗效显著，但均由于安全性问题宣告失败。而选择性作用于CTSK非活性位点（exosite）抑制胶原降解的抑制剂被证实可有效避免不良反应发生。项目负责人前期研究发现中药骨碎补中一种戊烯基二氢黄酮类成分Sophoraflavanone G（SG）可潜在抑制CTSK进而抑制骨吸收。通过本项目的实施我们证实SG在高（150mg/kg/day）、低（50mg/kg/day）两个剂量均可有效抑制去势骨质疏松小鼠的骨丢失，增强骨密度，改善骨的微观结构，并显著增强股骨的三点弯曲生物力学性能。骨组织形态学计量显示SG抑制骨吸收显著大于骨形成。安全性评价显示：与雌激素不同，SG不增加小鼠子宫重量和内膜厚度。骨组织免疫组化结果显示SG不影响骨中CTSK的表达和定位，但可抑制骨中CTSK的酶催化底物降解活性。体外细胞实验显示：SG可显著抑制过表达CTSK的RAW264.7细胞以及原代骨髓单核细胞诱导破骨细胞分化成熟后的骨吸收活性。SG也可显著抑制原代破骨细胞介导的CTSK酶催化底物降解活性而不影响CTSK蛋白的表达水平。进一步我们利用基于配体诱导蛋白热稳定性的细胞热迁移分析实验（CETSA）证实了SG可与CTSK稳定结合。由于CTSK非活性位点（exosite）抑制剂主要表现为选择性抑制胶原蛋白降解而不影响非胶原蛋白。我们进一步通过证实SG可抑制人重组CTSK蛋白介导的I型胶原蛋白降解。此外，不同于CTSK活性位点抑制剂Odanacatib表现出了显著的非胶原蛋白TGF-β抑制活性，SG并不抑制人皮肤成纤维细胞中TGF-β的降解。综上，我们通过本项目的实施证实了SG具有显著的体内抗骨质疏松活性，并表现出较好的用药安全性。机制上发现SG可通过与CTSK蛋白稳定结合，抑制CTSK的胶原蛋白降解活性而不影响非胶原蛋白降解，进而发挥抑制破骨细胞骨吸收的活性。SG有望开发成为新的CTSK非活性位点抑制剂用于骨质疏松症的治疗。本项目发表SCI论文6篇，申请中国发明专利3项，获得授权2项，申请PCT国际发明专利1项。

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