墨旱莲组分中组织蛋白酶K非活性位点抑制剂研究  

Study on exosite inhibitor of cathepsin K from Herba Ecliptae fractions

作　　者：蒋益萍[1] 吴岩斌[2] 秦路平[1] 张巧艳[1] 辛海量[1] Dieter Bromme 薛黎明[1,3,4] 

机构地区：[1]中国人民解放军第二军医大学药学院,上海200433 [2]福建中医药大学中西医结合研究院,福建福州350108 [3]加拿大不列颠哥伦比亚大学 [4]上海市疾病预防控制中心化学品毒性检定所,上海200336

出　　处：《药学学报》2017年第6期936-942,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81503493;81403162)

摘　　要：组织蛋白酶K(CTSK)是抗骨质疏松药物的关键靶标。CTSK活性位点抑制剂能抑制胶原蛋白降解活性,同时抑制CTSK降解其他蛋白的水解活性,存在较大的不良反应,CTSK非活性位点(exosite)抑制剂具有抑制骨胶原降解活性且不影响活性位点(active site)的生物活性。本研究通过毕赤酵母表达系统表达出重组CTSK,进一步通过N端正丁基琼脂糖凝胶柱和SP-琼脂糖凝胶柱分离,得到高纯度有活性的重组CTSK。利用所得CTSK考察筛选了中药墨旱莲不同提取物及化学成分对CTSK的胶原降解活性和对CTSK与Z-FR-MCA底物结合的抑制作用,明确了墨旱莲的正丁醇部位为理想的潜在非活性位点活性部位,从中提取的墨旱莲皂苷IX为潜在的非活性位点抑制剂。Cathepsin K （CTSK） is considered a critical pharmaceutical target in the treatment of osteoporosis. CTSK exerts proteolytic activities against regulatory proteins besides its collagenase function, which may account for some of the adverse reactions when blocked by active site-directed inhibitors. Exosite inhibitors that can discriminate between the therapeutic collagenase and other biological activities of CTSK specifically inhibit the collagenase activity of CTSK without interfering with the other proteolytic activities of the protease. Active recombinant CTSK was expressed in Pichia pastoris, and purified by n-butyl sepharose and SP sepharose column chromatography. Herba Ecliptae is a common traditional Chinese medicine in the treatment of bone diseases. Collagenase assay and benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin （Z-FR-MCA） substrate assay based on CTSK are applied to verify the exosite inhibitors, n-Butanol extract of Herba Ecliptae are the most active fraction and eclalbasaponin IX isolated from n-butanol fraction is the potential exosite inhibitor of CTSK.

关 键 词：组织蛋白酶K 墨旱莲 活性位点抑制剂 非活性位点抑制剂 正丁醇部位 墨旱莲皂苷IX 

分 类 号：R285[医药卫生—中药学]