Development of small molecule probes of matrix metallo protease function

描述（由申请人提供）：基质金属蛋白酶（MMPs）是一个大家族的酶，已被发现调节广泛的人类疾病的发病机制，尤其是癌症。它们在肿瘤生长过程中基质降解中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，由于缺乏对该蛋白酶家族特定成员在癌症病理中的作用的全面了解，导致大量临床试验失败，也没有新的MMP药物进入市场。限制更好地定义MMP功能的一个混淆问题是金属蛋白酶家族的总体大小和缺乏高选择性抑制剂来暂时控制特定MMP家族成员的抑制。此外，基因敲除特定蛋白酶的努力受到许多这些蛋白酶的本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抑制和成像单个MMP蛋白酶的定位动态，以便开始了解每个MMP家族成员在人类疾病（如癌症）发病机制中的特定功能作用。该提案概述了我们开发一种新策略的计划，以开发可用于在复杂生物系统中选择性抑制和动态成像单个MMP蛋白酶的小分子。该方法基于对特定的MMP靶标进行工程改造，使其在活性位点附近含有活性半胱氨酸残基，该残基可通过含有活性亲电试剂的探针进行直接共价修饰。抑制剂与活性位点的金属结合，并具有与工程半胱氨酸残基共价连接的能力，然后可用于选择性靶向cys突变型MMPs。这允许选择性共价抑制和标记单个MMP靶标。在该项目完成后，我们计划对多个MMP目标进行方法验证，以便将其应用于旨在进一步解剖MMP生物学的长期项目。

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 Their potential role in matrix degradation during tumor growth originally made them promising targets for the development of new chemotherapy drugs.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.