Mechanism and Inhibition of Collagenolytic Activity

描述(申请人提供)：胶原蛋白作为结构支架和组织之间的屏障，因此胶原蛋白分解代谢(胶原酶分解)在正常生理中是一个严格调控的过程。反过来，在病理状态下，胶原的破坏或损伤在肿瘤的生长和侵袭、软骨退化或动脉粥样硬化斑块的形成和破裂中发挥作用。目前只鉴定了一小部分能够有效地处理胶原蛋白的三螺旋区域的蛋白酶。锌金属酶家族中的几个成员，特别是基质金属蛋白酶(MMPs)，具有胶原酶活性。多年来，人们一直在从机理上理解完整的胶原蛋白的断裂；这些研究的结果可能会导致真正具有选择性的基质金属蛋白酶抑制剂的开发。我们实验室开发了三螺旋多肽(THPS)作为基质金属蛋白酶的底物，目的是利用这些模型来剖析胶原蛋白的分解行为。我们对THP底物的研究，以及其他研究小组之前的研究，已经导致了一个“构象熵移”假说，解释了MMPs如何在没有外部能源输入的情况下处理胶原蛋白。本文描述的研究计划重点是通过利用几种生物物理方法[核磁共振光谱、氢/氚交换质谱仪(HDX MS)、X射线结晶学和X射线吸收光谱(XAS)]结合定点突变和动力学分析来验证我们的胶原酶降解假说，以精确确定基质金属蛋白酶区域和残基在三螺旋结构的结合、解离和水解中的作用。将通过定点定向和组合方法创建THPS的变体，以获得在胶原酶降解MMPs中具有选择性的底物。基于机理研究结果，我们将利用定点文库和组合文库对含膦酸类THPS的抑制能力进行比较，以开发新型的、选择性的基质金属蛋白酶抑制剂。将利用MMPs和THP抑制剂的共结晶和HDX MS来评估这两个生物分子之间的相互作用位置，从而进一步优化先导化合物。选择的抑制剂将在血管生成模型以及乳腺癌和黑色素瘤小鼠模型中使用过表达靶向MMPs的细胞进行测试。最终，我们希望获得针对那些与癌症进展有关的蛋白水解酶(MMP2、MMP9和MT1-MMPs)的抑制剂，同时保留具有宿主有益功能的蛋白水解酶(MMP3和MMP8)。公共卫生评论：本研究旨在创造一类新的治疗剂，选择性地阻止降解蛋白质(蛋白酶)的肿瘤相关酶的作用。这些蛋白水解酶已被证明是癌症进展的重要因素，因此阻断它们的功能将损害癌症的扩散。

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. Our work with THP substrates, along with prior studies from other research groups, have led to a "conformational entropy shift" hypothesis explaining how MMPs process collagen without input from an external energy source. The research plan described herein focuses on testing our collagenolysis hypothesis by utilizing several biophysical approaches [NMR spectroscopy, hydrogen/deuterium exchange mass spectrometry (HDX MS), X-ray crystallography, and X-ray absorption spectroscopy (XAS)] in combination with site-specific mutagenesis and kinetic analyses to precisely determinate the roles of MMP regions and residues in the binding, unwinding, and hydrolysis of triple-helical structures. Variants of THPs will be created, by site-directed and combinatorial approaches, to obtain substrates that are selective within the collagenolytic MMPs. Based on the mechanistic results, we will compare the inhibitory capabilities of phosphinate-containing THPs using site-directed and combinatorial libraries to develop novel, selective MMP inhibitors. Co-crystallization and HDX MS of MMPs and THP inhibitors will be utilized to evaluate the sites of interaction between the two biomolecules, allowing for further optimization of lead compounds. Select inhibitors will be tested using cells overexpressing the targeted MMPs, in a model of angiogenesis, and in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancer progression (MMP-2, MMP-9, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP- 8). PUBLIC HEALTH REVELANCE: The present study is designed to create a novel class of therapeutic agents to selectively stop the action of tumor-associated enzymes that degrade proteins (proteases). These proteases have been shown to be important for cancer progression, and thus blocking their function will impair the spread of cancer.