Mechanism and Inhibition of Collagenolytic Activity

描述(申请人提供)：胶原蛋白作为结构支架和组织之间的屏障，因此胶原蛋白分解代谢(胶原酶分解)在正常生理中是一个严格调控的过程。反过来，在病理状态下，胶原的破坏或损伤在肿瘤的生长和侵袭、软骨退化或动脉粥样硬化斑块的形成和破裂中发挥作用。目前只鉴定了一小部分能够有效地处理胶原蛋白的三螺旋区域的蛋白酶。锌金属酶家族中的几个成员，特别是基质金属蛋白酶(MMPs)，具有胶原酶活性。多年来，人们一直在从机理上理解完整的胶原蛋白的断裂；这些研究的结果可能会导致真正具有选择性的基质金属蛋白酶抑制剂的开发。我们实验室开发了三螺旋多肽(THPS)作为基质金属蛋白酶的底物，目的是利用这些模型来研究胶原酶的分解行为，并开发选择性的基质金属蛋白酶抑制剂。在本项目的最新资助期(2008年4月1日至今)，我们已经通过实验得出了胶原酶分解的初始步骤，并确定了这一过程中涉及的特定残基，量化了特定胶原残基在基质金属蛋白酶底物特异性中的作用，开发了基于二级结合位点(Exosite)的选择性基质金属蛋白酶抑制剂，并展示了这些抑制剂在体内的应用。我们的研究还揭示了胶原酶降解MMPs有趣的、意想不到的行为，例如MMPs以略有不同的方向与三螺旋结合，人们可以使用外切点结合THPS来抑制给定酶的部分但不是全部的蛋白分解活性，这可以显著减少副作用，并且在三螺旋结构中没有观察到单链多肽的底物选择性。本文描述的研究计划集中于进一步开发三螺旋探针，用于梳理胶原酶降解的基质金属蛋白酶序列的特异性，识别选择性的基质金属蛋白酶抑制剂，并推进胶原酶溶解的机制。为了实现这些目标，我们建议(A)利用位置扫描组合文库来确定MMP族的THP序列偏好，(B)设计和表征MMP选择性、同三聚体和异三聚体三螺旋过渡态类似物，以及(C)利用最先进的核磁共振波谱技术探索胶原蛋白降解的后几步，并设计外位结合THPS。选定的抑制剂将在乳腺癌和黑色素瘤的小鼠模型中进行测试。最终，我们希望获得靶向与黑色素瘤和乳腺癌等癌症相关的那些蛋白水解酶的抑制剂(MMP1、MMP2、MMP9、MMP13和MT1-MMPs)，同时保留具有宿主有益功能的蛋白水解酶(MMP3和MMP8)。

DESCRIPTION (provided by applicant): Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity. A mechanistic understanding of the cleavage of intact collagens has been pursued for many years; the results of such studies could lead to the development of truly selective MMP inhibitors. Our laboratory developed triple-helical peptides (THPs) as MMP substrates, with the goal of using these models to dissect collagenolytic behavior and develop selective MMP inhibitors. In the most recent funding period of the present project (04/01/08-present), we have experimentally derived the initial steps of collagenolysis and identified specific residues involved in this process, quantified the roles of specific collagen residues in MMP substrate specificity, and developed selective MMP inhibitors based on secondary binding sites (exosites), and demonstrated the in vivo use of such inhibitors. Our studies also revealed intriguing, unexpected behaviors of collagenolytic MMPs, such as MMPs bind the triple-helix using slightly different orientations, one can use exosite binding THPs to inhibit some, but not all, proteolytic activities for a given enzyme, which could significantly reduce side effects, and substrate selectivity seen for single-stranded peptides is not observed in the triple-helix. The research plan described herein focuses on further development of triple-helical probes for teasing out collagenolytic MMP sequence specificities, identifying selective MMP inhibitors, and advancing the mechanism of collagenolysis. To achieve these goals we propose to (a) identify THP sequence preferences for the MMP family utilizing positional scanning combinatorial libraries, (b) design and characterize MMP selective, homotrimeric and heterotrimeric triple-helical transition-state analog inhibitors, and (c) explore the latter steps of collagenolysis usin state-of-the-art NMR spectroscopic techniques and design exosite binding THPs. Select inhibitors will be tested in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancers such as melanoma and breast carcinoma (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP-8).