Mechanism and Inhibition of Collagenolytic Activity

胶原蛋白分解活性的机制和抑制

• 批准号: 7465896
• 负责人: GREGG B FIELDS
• 金额: $ 7.6万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-05 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7465896
• 关键词: Active Sites; Arterial Fatty Streak; Behavior; Binding; Breast Carcinoma; Cartilage; Catabolism; Cells; Class; Collagen; Collagen Type I; Crystallization; Degenerative polyarthritis; Deuterium; Development; Endopeptidases; Energy-Generating Resources; Entropy; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Family; Gelatinase A; Gelatinase B; Goals; Hydrogen; Hydrolysis; Interstitial Collagenase; Kinetics; Knowledge; Laboratories; Lead; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Modeling; Mutagenesis; NMR Spectroscopy; Neutrophil Collagenase; Numbers; Object Attachment; Peptide Hydrolases; Peptides; Physiology; Play; Process; Progress Reports; Protein Overexpression; Proteins; Public Health; Relative (related person); Research; Role; Rupture; Site; Site-Directed Mutagenesis; Solubility; Spectrum Analysis; Stromelysin 1; Structure; Testing; Therapeutic Agents; Thermodynamics; Tissues; Variant; Work; X ray spectrometry; X ray spectroscopy; X-Ray Crystallography; Zinc; absorption; analog; angiogenesis; base; collagenase 3; combinatorial; design; human MMP14 protein; inhibitor/antagonist; insight; interstitial; matrix metalloproteinase 18; melanoma; member; metalloenzyme; mouse model; novel; preference; scaffold; small molecule; tumor; tumor growth; tumor progression

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.

描述（由申请人提供）：胶原蛋白作为结构支架和组织之间的屏障，因此在正常生理中，胶原分解代谢（胶原溶解）需要是一个严格调节的过程。反过来，病理状态下胶原蛋白的破坏或损伤在肿瘤生长和侵袭、软骨降解或动脉粥样硬化斑块形成和破裂中起作用。只有少数蛋白酶已被确定能够有效地处理胶原的三螺旋区域。锌金属酶家族的几个成员，特别是基质金属蛋白酶（MMPs），具有胶原溶解活性。对完整胶原蛋白裂解的机制理解已经进行了多年；这些研究的结果可能会导致真正选择性MMP抑制剂的开发。我们的实验室开发了三螺旋肽（THPs）作为MMP底物，目的是使用这些模型来解剖胶原溶解行为。我们对THP底物的研究，以及其他研究小组先前的研究，导致了一个“构象熵移”假设，解释了MMPs如何在没有外部能量输入的情况下处理胶原蛋白。本文描述的研究计划侧重于通过几种生物物理方法[核磁共振波谱，氢/氘交换质谱（HDX MS）， x射线晶体学和x射线吸收光谱（XAS）]结合位点特异性诱变和动力学分析来测试我们的胶原溶解假说，以精确确定MMP区域和残基在三螺旋结构的结合，解绕和水解中的作用。通过位点定向和组合方法，将创建THPs的变体，以获得在胶原溶解MMPs中具有选择性的底物。基于机制的结果，我们将比较含膦THPs的抑制能力，使用位点导向和组合文库来开发新的选择性MMP抑制剂。MMPs和THP抑制剂的共结晶和HDX质谱将用于评估两种生物分子之间相互作用的位点，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和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，我们希望获得针对与癌症进展相关的蛋白酶（MMP-2、MMP-9和MT1-MMP）的抑制剂，同时保留具有宿主有益功能的蛋白酶（MMP-3和MMP- 8）。公共卫生启示：目前的研究旨在创造一类新的治疗剂，选择性地停止降解蛋白质的肿瘤相关酶（蛋白酶）的作用。这些蛋白酶已被证明对癌症的进展很重要，因此阻断它们的功能将阻碍癌症的扩散。