Mechanism and Inhibition of Collagenolytic Activity

胶原蛋白分解活性的机制和抑制

• 批准号: 7597224
• 负责人: GREGG B FIELDS
• 金额: $ 29.57万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 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 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7597224
• 关键词: Active Sites; Arterial Fatty Streak; Behavior; Binding; Breast Carcinoma; Cartilage; Catabolism; Cells; Collagen; Collagen Type I; Crystallization; Degenerative polyarthritis; Deuterium; Development; 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; Peptide Hydrolases; Peptides; Physiology; Play; Process; Progress Reports; Proteins; Public Health; Relative (related person); Research; Roentgen Rays; Role; Rupture; Site; Site-Directed Mutagenesis; Solubility; Spectrum Analysis; Stromelysin 1; Structure; Testing; Therapeutic Agents; Thermodynamics; Tissues; Variant; Work; 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; overexpression; 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（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（胶原溶解）需要成为正常生理学中严格调节的过程。反过来，病理状态期间胶原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三螺旋区的胶原蛋白。锌金属酶家族的几个成员，特别是基质金属蛋白酶（MMPs），具有胶原蛋白溶解活性。多年来，人们一直在研究完整胶原蛋白裂解的机制;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（THP）作为MMP底物，目的是使用这些模型来剖析胶原溶解行为。我们对THP底物的研究，沿着其他研究小组先前的研究，已经导致了“构象熵变”假说，解释了MMP如何在没有外部能量源输入的情况下加工胶原蛋白。本文所述的研究计划集中于通过利用几种生物物理方法[NMR光谱学、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，以精确地确定MMP区域和残基在结合、解旋、和三螺旋结构的水解。将通过位点定向和组合方法产生THP的变体，以获得在胶原蛋白溶解MMP内具有选择性的底物。基于机制的结果，我们将比较使用定点和组合库的含膦酸酯的THP的抑制能力，以开发新的选择性MMP抑制剂。MMP和THP抑制剂的共结晶和HDX MS将用于评估两种生物分子之间的相互作用位点，从而进一步优化先导化合物。选择抑制剂将使用过表达靶向MMPs的细胞在血管生成模型以及乳腺癌和黑色素瘤的小鼠模型中进行测试。最终，我们希望获得靶向参与癌症进展的蛋白酶（MMP-2、MMP-9和MT 1-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）。公共卫生部门：本研究旨在创建一类新型治疗药物，以选择性地阻止降解蛋白质（蛋白酶）的肿瘤相关酶的作用。这些蛋白酶已被证明对癌症进展很重要，因此阻断它们的功能将损害癌症的扩散。