Novel approach for inhibition of MT1-MMP/gelatinase axis

描述（由申请人提供）：与癌症患者预后和生存不良相关的主要临床特征仍然是转移的发生。有证据表明，mt1 -MMP（一种膜锚定基质金属蛋白酶）在肿瘤转移和血管生成过程中起关键作用。mt1 - 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）的激活，是细胞外蛋白水解的主要介质，MMP-2（明胶酶a）是肿瘤转移的另一个关键酶。这项应用的长期目标是开发新的方法来特异性地抑制肿瘤组织中MT - 1-MMP和明胶酶的活性。作为一种膜系酶，mt1 - mmp经历自催化加工和外胞结构域脱落，这两个过程控制着细胞表面活性酶的数量。初步证据表明，可逆合成的MMP抑制剂抑制MT - 1-MMP的加工和脱落，导致活性酶在细胞表面的积累。矛盾的是，在TIMP-2存在的情况下，可逆的MMP抑制剂会增强MT1-MMP对MMP-2的激活。酶抑制动力学数据支持TIMP-2从mt1 -MMP活性位点取代可逆合成MMP抑制剂，促进MMP-2结合和激活的模型。这些观察结果代表了可逆合成MMP抑制剂调控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。我们的实验室开发了一种新的MMP抑制策略，包括基于机制的抑制剂，最近生产了第一个针对MMP-2和MMP-9的不可逆MMP抑制剂原型。在这里，我们建议采用综合和多学科的化学，生化和生物学方法来追求这种抑制MT - 1-MMP活性的方法。具体来说，我们将(1)为MT1-MMP和明胶酶生产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MT1-MMP加工，脱落，pro-MMP-2的激活和与TIMPs的相互作用以及(4)建立了基于机制的抑制剂对（ECM）降解和侵袭的有效性。预计这些结果将为合成MMP抑制剂调控MMPs创造一个新的范例，为靶向MMPs预防肿瘤生长和转移开辟了以前未探索的途径。

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.