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）：胶原蛋白是组织之间的结构支架和障碍，因此，胶原蛋白分解代谢（胶原溶解）必须是正常生理学的严格调节过程。反过来，病理状态期间胶原蛋白的破坏或损害在肿瘤生长和侵袭，软骨降解或动脉粥样硬化斑块的形成和破裂中起作用。仅确定了少数蛋白酶能够有效地处理胶原蛋白的三螺旋区域。锌金属酶家族的几个成员，特别是基质金属蛋白酶（MMP）具有胶原式活性。对完整胶原蛋白的裂解的机械理解已经进行了多年。此类研究的结果可能导致真正选择性MMP抑制剂的发展。我们的实验室将三螺旋肽（THP）作为MMP底物开发，目的是使用这些模型来剖析胶原式行为。我们与THP底物的工作以及其他研究小组的先前研究导致了“构象熵转移”的假设，该假设解释了MMP的过程胶原蛋白如何而没有外部能源的输入。本文描述的研究计划着重于通过使用几种生物物理方法[NMR光谱，氢/氘交换质谱法（HDX MS），X射线晶体学和X射线吸收光谱（XAS）]与位点确定的分析相结合，从三螺旋结构的结合，放松和水解中的残基。将通过站点定向和组合方法创建THP的变体，以获得在胶原式MMP中具有选择性的底物。基于机械结果，我们将使用位置定向和组合文库来比较含磷酸THP的抑制能力，以开发新型的选择性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，从而进一步优化铅化合物。在血管生成模型以及乳腺癌和黑色素瘤的小鼠模型中，将使用过表达靶向MMP的细胞测试选择抑制剂。最终，我们希望获得针对涉及癌症进展的蛋白酶（MMP-2，MMP-9和MT1-MMP）的抑制剂，同时将蛋白酶保留具有宿主 - 脱表函数的蛋白酶（MMP-3和MMP-8）。公共卫生的启示：本研究旨在创建一种新型的治疗剂，以选择性地停止降解蛋白质（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，因此阻止其功能会损害癌症的传播。