Engineering selective inhibition of metalloproteinases by tissue inhibitors of metalloproteinases (R01 GM132100 RESUB - *TIMPs)

通过金属蛋白酶组织抑制剂对金属蛋白酶进行工程选择性抑制（R01 GM132100 RESUB - *TIMP）

• 批准号: 10319170
• 负责人: Evette S Radisky
• 金额: $ 31.3万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319170
• 关键词: Address; Affinity; Arthritis; Autoimmune Diseases; Binding; Biological Assay; Biological Models; Biology; C-terminal; Cardiovascular Diseases; Catalytic Domain; Complement; Complex; Computer Analysis; Computer Models; Crotalus adamanteus proteinase II; Crystallization; Data; Development; Directed Molecular Evolution; Discrimination; Disease; Disease model; Diversity Library; Dose-Limiting; Drug Targeting; Endopeptidases; Engineering; Enzymes; Epitopes; Family; Foundations; Free Energy; Generations; Goals; Individual; Inflammatory; Inhibition of Matrix Metalloproteinases Pathway; Kinetics; Length; Libraries; Malignant Neoplasms; Maps; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Mediating; Metalloproteases; Methodology; Methods; Modeling; Molecular; Molecular Probes; Morphogenesis; Musculoskeletal; Mutagenesis; Mutation; N-terminal; Names; Outcome; Pathologic; Pathology; Peptide Hydrolases; Pharmaceutical Preparations; Physiological; Play; Process; Progression-Free Survivals; Protease Inhibitor; Protein Dynamics; Protein Engineering; Proteins; Publishing; Reagent; Roentgen Rays; Role; Scaffolding Protein; Shapes; Specificity; Structural Models; Structure; Surface; Therapeutic; Tissue Engineering; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tissues; Toxic effect; Variant; Work; X-Ray Crystallography; Yeasts; Zinc; base; design; early phase clinical trial; inhibitor; molecular dynamics; molecular recognition; next generation; novel; protein complex; protein folding; scaffold; screening; synergism; therapeutic protein; therapeutic target; therapeutically effective; tool

PROJECT SUMMARY/ABSTRACT Matrix metalloproteinases (MMPs) have long been regarded as promising therapeutic targets, but clinical trials of early-generation MMP inhibitors in arthritis and cancer proved disappointing. Broad-spectrum MMP inhibitors produced serious dose-limiting musculoskeletal toxicity and failed to extend progression-free survival in cancer trials, partly due to the inability of the inhibitors to distinguish among different MMPs. This was a critical problem, because some MMPs serve a primarily protective function, and it is now clear that indiscriminate inhibition of all MMPs inevitably leads to poorer outcomes. Based on our published and preliminary data, we hypothesize that tissue inhibitors of metalloproteinases (TIMPs), endogenous regulators of the MMP family, can be engineered into highly selective MMP inhibitors free of undesired off-target activities, to produce probes and therapeutics targeting individual MMPs with exquisite selectivity. In this application, we will (a) optimize novel methodology for directed evolution of selective binders to discriminate within the large families of related MMPs and adamalysin proteases, (b) uncover mechanisms of molecular recognition that govern MMP-TIMP binding specificity, and (c) develop a toolbox of engineered TIMPs that selectively target individual MMPs with highly enhanced specificity. To accomplish these goals, we will use state-of-the-art directed evolution approaches to engineer the TIMP-1 scaffold for fine discrimination between closely similar MMPs, reengineering N- and C-terminal TIMP domain epitopes and exploiting cooperativity between domains. Additionally, we will integrate X-ray crystallographic and computational approaches to elucidate the protein structural and dynamic features that govern affinity and selectivity of TIMP/MMP complexes. This project will thus elucidate fundamental principles of molecular recognition governing TIMP/MMP selectivity, and will produce designer TIMPs targeting single MMPs with highly enhanced specificity, with potential for development as useful molecular probes and as protein therapeutics for the many diseases driven by MMP dysregulation.

项目摘要/摘要 基质金属蛋白酶（MMP）长期以来一直被视为有希望的治疗靶标，但临床试验 事实证明，关节炎和癌症的早期MMP抑制剂令人失望。广谱MMP 抑制剂产生严重的剂量限制肌肉骨骼毒性，无法延长无进展生存 在癌症试验中，部分原因是抑制剂无法区分不同的MMP。这是一个 关键问题，因为某些MMP主要具有保护功能，现在很明显 对所有MMP的不加区分抑制不可避免地导致结果较差。根据我们出版的 初步数据，我们假设金属蛋白酶（TIMP），内源性调节剂的组织抑制剂 MMP家族可以设计成高度选择性的MMP抑制剂，没有不希望的脱靶 活动，以精致的选择性产生针对单个MMP的探针和治疗剂。在这个 应用，我们将（a）优化新的方法论，以歧视选择性粘合剂的定向演变 在相关MMP和Adamalysin蛋白酶的大家族中，（b）发现分子的机制 识别管理MMP-TIMP绑定特异性的认可，以及（c）开发一个工具箱的工具箱 选择性地靶向具有高度增强特异性的单个MMP。为了实现这些目标，我们将使用 最先进的定向进化方法，以设计TIMP-1脚手架，以判断 紧密相似的MMP，重新设计的N-和C端TIMP域表位并利用合作性 在域之间。此外，我们将整合X射线晶体学和计算方法 阐明控制TIMP/MMP亲和力和选择性的蛋白质结构和动态特征 复合物。因此，该项目将阐明控制分子识别的基本原理 TIMP/MMP选择性，并将产生针对具有高度增强的单个MMP的设计师TIMP 特异性，具有开发作为有用的分子探针的潜力，并且是许多人的蛋白质疗法 MMP失调驱动的疾病。