Law Simon, Andrault Pierre-Marie, Aguda Adeleke H, Nguyen Nham T, Kruglyak Natasha, Brayer Gary D, Brömme Dieter
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Biochem J. 2017 Feb 20;474(5):851-864. doi: 10.1042/BCJ20160985.
Cathepsin K (CatK) is the predominant mammalian bone-degrading protease and thus an ideal target for antiosteoporotic drug development. Rodent models of osteoporosis are preferred due to their close reflection of the human disease and their ease of handling, genetic manipulation and economic affordability. However, large differences in the potency of CatK inhibitors for the mouse/rat vs. the human protease orthologs have made it impossible to use rodent models. This is even more of a problem considering that the most advanced CatK inhibitors, including odanacatib (ODN) and balicatib, failed in human clinical trials due to side effects and rodent models are not available to investigate the mechanism of these failures. Here, we elucidated the structural elements of the potency differences between mouse and human CatK (hCatK) using ODN. We determined and compared the structures of inhibitor-free mouse CatK (mCatK), hCatK and ODN bound to hCatK. Two structural differences were identified and investigated by mutational analysis. Humanizing subsite 2 in mCatK led to a 5-fold improvement of ODN binding, whereas the replacement of Tyr61 in mCatK with Asp resulted in an hCatK with comparable ODN potency. Combining both sites further improved the inhibition of the mCatK variant. Similar results were obtained for balicatib. These findings will allow the generation of transgenic CatK mice that will facilitate the evaluation of CatK inhibitor adverse effects and to explore routes to avoid them.
组织蛋白酶K（CatK）是哺乳动物中主要的骨降解蛋白酶，因此是抗骨质疏松药物开发的理想靶点。由于骨质疏松症的啮齿动物模型能很好地反映人类疾病情况，且易于处理、进行基因操作且经济实惠，所以它们是首选。然而，CatK抑制剂对小鼠/大鼠与人类蛋白酶直系同源物的效力存在很大差异，这使得无法使用啮齿动物模型。考虑到包括odanacatib（ODN）和balicatib在内的最先进的CatK抑制剂因副作用在人体临床试验中失败，而又没有啮齿动物模型来研究这些失败的机制，这一问题就更加突出。在这里，我们使用ODN阐明了小鼠和人类CatK（hCatK）之间效力差异的结构元件。我们测定并比较了无抑制剂的小鼠CatK（mCatK）、hCatK以及与hCatK结合的ODN的结构。通过突变分析确定并研究了两个结构差异。将mCatK中的亚位点2人源化导致ODN结合能力提高了5倍，而将mCatK中的Tyr61替换为Asp则产生了与ODN效力相当的hCatK。将这两个位点结合起来进一步提高了对mCatK变体的抑制作用。balicatib也得到了类似的结果。这些发现将有助于培育转基因CatK小鼠，这将有助于评估CatK抑制剂的不良反应并探索避免这些反应的途径。
