Mechanism and Inhibition of Collagenolytic Activity

描述（由申请人提供）：胶原蛋白作为结构支架和组织之间的屏障，因此在正常生理中，胶原分解代谢（胶原溶解）需要是一个严格调节的过程。反过来，病理状态下胶原蛋白的破坏或损伤在肿瘤生长和侵袭、软骨降解或动脉粥样硬化斑块形成和破裂中起作用。只有少数蛋白酶已被确定能够有效地处理胶原的三螺旋区域。锌金属酶家族的几个成员，特别是基质金属蛋白酶（MMPs），具有胶原溶解活性。对完整胶原蛋白裂解的机制理解已经进行了多年；这些研究的结果可能会导致真正选择性MMP抑制剂的开发。我们的实验室开发了三螺旋肽（THPs）作为MMP底物，目的是使用这些模型来解剖胶原溶解行为并开发选择性MMP抑制剂。在本项目最近的资助期（08年4月1日至今），我们通过实验导出了胶原溶解的初始步骤，并确定了参与该过程的特定残基，量化了特定胶原残基在MMP底物特异性中的作用，开发了基于二级结合位点（外源位点）的选择性MMP抑制剂，并证明了这些抑制剂的体内使用。我们的研究还揭示了令人感兴趣的、意想不到的胶原溶解MMPs的行为，例如MMPs以略微不同的方向结合三螺旋，人们可以使用外源性结合THPs来抑制某些（但不是全部）特定酶的蛋白水解活性，这可以显著减少副作用，并且在三螺旋中没有观察到单链肽的底物选择性。本文所述的研究计划侧重于进一步开发三螺旋探针，以梳理胶原溶解MMP序列特异性，识别选择性MMP抑制剂，并推进胶原溶解机制。为了实现这些目标，我们建议(a)利用定位扫描组合文库确定MMP家族的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抑制剂，以及(c)使用最先进的核磁共振波谱技术探索胶原溶解的后期步骤并设计外源结合THP。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，我们希望获得针对与黑色素瘤和乳腺癌等癌症相关的蛋白酶（MMP-1、MMP-2、MMP-9、MMP-13和MT1-MMP）的抑制剂，同时保留具有宿主有益功能的蛋白酶（MMP-3和MMP-8）。

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).