Mechanism & Inhibition of Collagenolytic Activity

描述（由申请人提供）：胶原蛋白三螺旋的溶解与影响身体各种成分结构完整性的各种疾病有关。胶原蛋白还在组织和细胞之间提供屏障;该屏障的破坏在肿瘤细胞侵袭和转移中起作用。基质金属蛋白酶（MMP）家族因其胶原蛋白溶解活性而被公认，因此一直是密集研究工作的主题，以阐明其作用机制并允许合理设计抑制剂。我们已经开发了构建三螺旋肽（THP）的方法，并已将这些合成蛋白质用于酶与胶原蛋白相互作用的研究。发现“三螺旋肽酶”活性在蛋白酶中比以前认为的更普遍。似乎胶原溶解活性的独特方面可能不是切割三螺旋的能力，而是正确结合和定向天然胶原分子的能力。这种范式转变可能对胶原溶解活性抑制剂的设计产生重大影响。为了进一步探索三螺旋肽酶活性的性质，将组装一系列THP底物，掺入已知的胶原水解位点并在一定范围的Tm值内变化。底物热稳定性与酶活性相关。在串联中，使用15 N标记的氨基酸的2D NMR实验将检查这些底物中的肽骨架的迁移率。将评价MMP、胰蛋白酶、嗜热菌蛋白酶、组织蛋白酶K和聚集蛋白聚糖酶水解THP的各个动力学参数和活化能。MMP-1、MMP-2、MMP-8和组织蛋白酶K突变体将用于确定这些酶中对三螺旋肽酶活性至关重要的区域。最后，选择性THP底物和新型THP抑制剂将被设计和测试。总体而言，三螺旋肽酶活性将系统地评价各种蛋白酶作为底物序列和构象灵活性的函数，并更好地理解胶原溶解活性的机制。

DESCRIPTION (provided by applicant): The dissolution of the collagen triple-helix has been implicated in a variety of diseases that effect the structural integrity of various components of the body. Collagen also provides a barrier between tissues and cells; destruction of this barrier plays a role in tumor cell invasion and metastasis. The matrix metalloproteinase (MMP) family has been recognized for their collagenolytic activities, and has thus been the subject of intense research efforts, in order to elucidate their mechanisms of action and allow for rational design of inhibitors. We have developed methodology for constructing triple-helical peptides (THPs) and have applied these synthetic proteins for the study of enzyme interactions with collagens. "Triple-helical peptidase" activity was found to be more widespread amongst proteases than previously believed. It appears that the unique aspect of collagenolytic activity may not be the ability to cleave a triple-helix, but rather the ability to bind and orient the native collagen molecule properly. This paradigm shift could have significant effects on the design of inhibitors against collagenolytic activity. To further explore the nature of triple-helical peptidase activity, a series of THP substrates will be assembled, incorporating known sites of collagen hydrolysis and varying over a range of Tm values. Substrate thermal stability will be correlated to enzyme activity. In tandem, 2D NMR experiments using 15N-labeled amino acids will examine the mobilities of the peptide backbone in these substrates. Individual kinetic parameters and activation energies will be evaluated for MMP, trypsin, thermolysin, cathepsin K, and aggrecanase hydrolysis of THPs. MMP-1, MMP-2, MMP-8, and cathepsin K mutants will be utilized to determine the regions within these enzymes critical for triple-helical peptidase activity. Finally, selective THP substrates and novel THP inhibitors will be designed and tested. Overall, triple-helical peptidase activity will be systematically evaluated for a variety of proteases as a function of substrate sequence and conformational flexibility, and the mechanism of collagenolytic activity will better understood.