Development of small molecule probes of matrix metallo protease function

描述(申请人提供)：基质金属蛋白酶(MMPs)是一大类酶，已被发现调节一系列人类疾病的发病机制，尤其是癌症。它们在肿瘤生长过程中对基质降解的潜在作用最初使它们成为开发新的化疗药物的有希望的靶点。然而，由于对该蛋白家族的特定成员在癌症病理中的作用缺乏全面的了解，导致大量临床试验失败，没有新的基质金属蛋白酶药物进入市场。限制更好地确定金属蛋白酶功能的努力的一个令人困惑的问题是，金属蛋白酶家族总体上很大，并且缺乏高选择性的抑制剂来对特定的金属蛋白酶家族成员进行时间控制的抑制。此外，从基因上敲除特定的蛋白水解酶的努力受到许多此类蛋白水解酶的基本性质和相关家庭成员补偿的可能性的限制。因此，需要高风险和非传统的方法来选择性地抑制和成像单个基质金属蛋白酶的定位动态，以开始了解每个基质金属蛋白酶家族成员在人类疾病(如癌症)发病中的特定功能作用。这份提案概述了我们开发一种新的策略来开发小分子的计划，这种小分子可以用于在复杂的生物系统中选择性地抑制单个基质金属蛋白酶，并动态地对其进行成像。该方法基于特定的基质金属蛋白酶靶标的工程，使其在活性部位附近含有活性半胱氨酸残基，可用于通过含有活性电泳体的探针进行直接共价修饰。然后，可以使用与活性部位的金属结合并能够共价连接到工程半胱氨酸残基上的抑制剂来选择性地靶向Cys突变的MMPs。这允许选择性共价抑制和标记单个基质金属蛋白酶靶标。在这个项目完成时，我们计划将该方法验证为多个基质金属蛋白酶靶点，这样它就可以应用于旨在进一步解剖基质金属蛋白酶生物学的长期项目。 与公共健康相关：这个项目概述了开发一种通用方法来设计基质金属蛋白酶(MMPs)的计划，以便可以使用基于小分子活性的探针选择性地抑制它们并进行成像。这种方法一旦应用于复杂的活体系统，就可以研究单个的基质金属蛋白酶在疾病病理中的作用。

DESCRIPTION (provided by applicant): The matrix metallo proteases (MMPs) are a large family of enzymes that have been found to regulate the pathogenesis of a wide rage of human diseases, most notably cancer. Their potential role in matrix degradation during tumor growth originally made them promising targets for the development of new chemotherapy drugs. However, an overall lack of understanding of the roles of specific members of this protease family in cancer pathology resulted in a large number of failed clinical trials and no new MMP drugs entering the market. One of the confounding issues that limited efforts to better define MMP function is the overall large size of the metallo protease family and a lack of highly selective inhibitors for temporally controlled inhibition of specific MMP family members. Furthermore, efforts to genetically knock-out specific proteases have been limited by the essential nature of many of these proteases and the potential for compensation by related family members. Thus, high risk and non-traditional methods to both selectively inhibit and image to dynamics of localization of individual MMP proteases will be required to begin to understand specific functional roles of each MMP family member in the pathogenesis of human diseases such as cancer. This proposal outlines our plans to develop a novel strategy to develop small molecules that can be used to both selectively inhibit and also dynamically image individual MMP proteases in the context of a complex biological system. This method is based on the engineering of specific MMP targets to contain a reactive cysteine residue near the active site that can be used for direct covalent modification by a probe containing a reactive electrophile. Inhibitors that bind to the metal in the active site and have the ability to covalently link to the engineered cysteine residue can then be used to selectively target the Cys-mutant MMPs. This allows selective covalent inhibition and labeling of a single MMP target. At the completion of this project we plan to have the method validated for multiple MMP targets such that it can then be applied to longer term projects designed to further dissect MMP biology. PUBLIC HEALTH RELEVANCE: This project outlines plans to develop a general method to engineer matrix metallo proteases (MMPs) such that they can be selectively inhibited and imaged using small molecule activity based probes. This methodology, once applied to complex in vivo system, will allow studies of the involvement of individual MMP proteases in disease pathology.