Mechanism and Inhibition of Collagenolytic Activity

胶原蛋白分解活性的机制和抑制

• 批准号: 7737312
• 负责人: GREGG B FIELDS
• 金额: $ 23.6万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737312
• 关键词: Adamts5 protein; Affect; Amino Acid Sequence; Arterial Fatty Streak; Arthritis; Back; Behavior; Binding; Biological Assay; Breast Carcinoma; Cartilage; Catabolism; Cells; Circular Dichroism; Class; Classification; Cleaved cell; Collagen; Connective Tissue; Crystallization; Deuterium; Development; Discrimination; Disease; Disintegrins; Drug Delivery Systems; Endopeptidases; Energy-Generating Resources; Entropy; Enzyme Interaction; Enzymes; Esters; Family; Gelatinase A; Gelatinase B; Generations; Goals; Hydrogen; Hydrolysis; Individual; Interstitial Collagenase; Investigation; Kinetics; Knowledge; Laboratories; Lead; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Metalloproteases; Methodology; Methods; Modeling; Molecular Conformation; NMR Spectroscopy; Nature; Neutrophil Collagenase; Numbers; Object Attachment; Peptide Hydrolases; Peptides; Phase; Phase III Clinical Trials; Physiology; Play; Process; Protein Overexpression; Proteins; Public Health; Relative (related person); Reporting; Research; Role; Rupture; Site; Site-Directed Mutagenesis; Solid; Specificity; Spectrum Analysis; Stromelysin 1; Structure; Testing; Therapeutic Agents; Thermolysin; Thinking; Tissues; Trypsin; Tumor Cell Invasion; Variant; Vertebral column; Work; X ray spectrometry; X-Ray Crystallography; Zinc; absorption; aggrecanase; aggrecanase-1; analog; angiogenesis; base; cathepsin K; collagenase 3; combinatorial; design; enzyme activity; feeding; human MMP14 protein; inhibitor/antagonist; matrix metalloproteinase 26; melanoma; member; metalloenzyme; metastatic process; mouse model; mutant; novel; novel strategies; phosphonate; programs; protein aminoacid sequence; research study; scaffold; synthetic construct; synthetic protein; thermostability; triple helix; tumor; tumor growth; tumor progression; two-dimensional

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）：胶原蛋白充当结构支架和组织之间的屏障，因此胶原蛋白分解代谢（胶原蛋白分解）被要求是正常生理学中严格调节的过程。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、软骨退化或动脉粥样硬化斑块形成和破裂中发挥作用。只有少数蛋白酶已被鉴定出能够有效加工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），具有胶原溶解活性。多年来，人们一直在追求对完整胶原蛋白裂解的机制理解。此类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，目的是利用这些模型来剖析胶原蛋白溶解行为。我们对 THP 底物的研究以及其他研究小组的先前研究得出了“构象熵变”假说，解释了 MMP 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侧重于通过利用几种生物物理方法 [NMR 光谱、氢/氘交换质谱 (HDX MS)、X 射线晶体学和 X 射线吸收光谱 (XAS)] 结合位点来测试我们的胶原蛋白溶解假说。 -特异性诱变和动力学分析，精确确定 MMP 区域和残基在三螺旋结构的结合、解旋和水解中的作用。将通过定点和组合方法创建 THP 变体，以获得在胶原蛋白溶解 MMP 中具有选择性的底物。根据机理结果，我们将使用定点和组合文库比较含次膦酸盐的 THP 的抑制能力，以开发新型选择性 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。将使用过度表达靶向 MMP 的细胞在血管生成模型以及乳腺癌和黑色素瘤小鼠模型中测试选定的抑制剂。最终，我们希望获得针对与癌症进展有关的蛋白酶（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）。公众健康启示：本研究旨在创造一类新型治疗剂，以选择性地阻止肿瘤相关酶降解蛋白质（蛋白酶）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。