Novel approach for inhibition of MT1-MMP/gelatinase axis

描述（由申请人提供）：与癌症患者的不良预后和存活相关的主要临床特征仍然是转移的发展。MT 1-MMP是一种膜锚定的基质金属蛋白酶（MMP），在肿瘤的转移和血管生成过程中起重要作用。MT 1-MMP通过促进细胞外基质降解（ECM）和启动细胞表面MMP-2（明胶酶A）的活化（另一种肿瘤转移的关键酶），是细胞周围蛋白水解的主要介质。本申请的长期目标是开发特异性抑制癌组织中MT 1-MMP和明胶酶活性的新方法。作为一种膜系酶，MT 1-MMP经历自催化加工和胞外域脱落，这两个过程控制细胞表面活性酶的量。初步证据表明，可逆的合成MMP抑制剂抑制MT 1-MMP加工和脱落，导致活性酶在细胞表面上的积累。特别地，在TIMP-2存在下，可逆MMP抑制剂通过MT1-MMP增强MMP-2活化。酶抑制动力学数据支持这样的模型，其中TIMP-2从MT 1-MMP的活性位点置换可逆的合成MMP抑制剂，促进MMP-2结合和活化。这些观察结果代表了通过可逆合成MMP抑制剂调节MT 1-MMP/明胶酶轴的范例，并突出了当前MMP抑制方法的局限性。我们实验室开发的一种新的MMP抑制策略涉及基于机制的抑制剂，最近产生了第一个MMP-2和MMP-9的不可逆MMP抑制剂原型。在这里，我们建议采用综合性和多学科的化学，生物化学和生物学方法来抑制MT 1-MMP活性。具体而言，我们将（1）产生针对MT1-MMP和明胶酶的基于机制的不可逆抑制剂，（2）表征基于机制的抑制剂以建立抑制和特异性参数并评估其体外代谢命运，（3）研究基于机制的抑制剂对细胞中MT1-MMP/明胶酶活性的影响以确定其在MT 1-MMP加工、脱落、pro-MMP-2活化和与TIMP的相互作用，以及（4）建立基于机制的抑制剂对（ECM）降解和侵袭的有效性。预计这些结果将在通过合成MMP抑制剂调节MMPs方面创造一种新的范例，为靶向MMPs预防肿瘤生长和转移开辟以前未探索的途径。

DESCRIPTION (provided by applicant): The major clinical feature associated with poor prognosis and survival in cancer patients remains the development of metastasis. Evidence indicates that MT 1-MMP, a membrane-anchored matrix metalloproteinase (MMP), plays a key role in the process of tumor metastasis and angiogenesis. MT 1-MMP is a maj or mediator of pericellular proteolysis by promoting extracellular matrix degradation (ECM) and initiating the cell surface activation of MMP-2 (gelatinase A), another key enzyme for tumor metastasis. The long-term goal of this application is to develop novel approaches to specifically inhibit the activity of MT 1-MMP and gelatinases in cancer tissues. As a membrane-tethered enzyme, MT 1-MMP undergoes autocatalytic processing and ectodomain shedding, two processes that control the amount of active enzyme on the cell surface. Preliminary evidence shows that reversible synthetic MMP inhibitors inhibit MT 1-MMP processing and shedding resulting in the accumulation of active enzyme on the cell surface. Paradoxically, reversible MMP inhibitors enhance MMP-2 activation by MT1-MMP in the presence of TIMP-2. Enzyme inhibition kinetic data support a model in which TIMP-2 displaces the reversible synthetic MMP inhibitor from the active site of MT 1-MMP facilitating MMP-2 binding and activation. These observations represent a paradigm in the regulation of the MT 1-MMP/gelatinase axis by reversible synthetic MMP inhibitors and highlight the limitations of current approaches for MMP inhibition. A novel strategy for MMP inhibition involving mechanism-based inhibitors developed in our labs has recently produced the first prototype irreversible MMP inhibitor for MMP-2 and MMP-9. Here we propose to pursue this approach for inhibition of MT 1-MMP activity using a comprehensive and multidisciplinary chemical, biochemical and biological approach. Specifically, we will (1) produce mechanism-based irreversible inhibitors for MT1-MMP and gelatinases, (2) characterize the mechanism-based inhibitors to establish inhibition and specificity parameters and to assess their metabolic fate in vitro, (3) investigate the effects of mechanism-based inhibitors on MT1-MMP/gelatinase activity in cells to define their role in MT 1-MMP processing, shedding, pro-MMP-2 activation and interactions with TIMPs and (4) establish the effectiveness of mechanism-based inhibitors on (ECM) degradation and invasion. It is expected that the results would create a new paradigm in regulation of MMPs by synthetic MMP inhibitors, opening previously unexplored avenues for targeting MMPs in prevention of both tumor growth and metastasis.