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: 10545017
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10545017
• 关键词: Active Sites; 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; Data; Degenerative Disorder; 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; Outcome; Pathologic; Pathology; Peptide Hydrolases; Pharmaceutical Preparations; Physiological; Play; Process; Progression-Free Survivals; Protease Inhibitor; Protein Dynamics; Protein Engineering; Proteins; Publishing; Reagent; Research; 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; 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.

项目概要/摘要 基质金属蛋白酶（MMP）长期以来一直被认为是有前途的治疗靶点，但临床试验 早期 MMP 抑制剂在关节炎和癌症方面的研究结果令人失望。广谱基质金属蛋白酶 抑制剂产生严重的剂量限制性肌肉骨骼毒性，并且未能延长无进展生存期 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，因为一些 MMP 主要起保护作用，现在很清楚 不加区别地抑制所有 MMP 不可避免地会导致更差的结果。根据我们发布的和 根据初步数据，我们假设金属蛋白酶组织抑制剂（TIMP）、内源性调节剂 MMP 家族的成员，可被设计成高度选择性的 MMP 抑制剂，不会出现不良的脱靶现象 活动，以生产具有精确选择性的针对单个 MMP 的探针和治疗剂。在这个 应用程序中，我们将（a）优化选择性结合物定向进化的新方法以区分 在相关 MMP 和 adamalysin 蛋白酶的大家族中，(b) 揭示分子机制 识别控制 MMP-TIMP 结合特异性，以及 (c) 开发工程化 TIMP 工具箱 选择性地靶向具有高度增强的特异性的单个 MMP。为了实现这些目标，我们将使用 最先进的定向进化方法来设计 TIMP-1 支架，以精细区分 非常相似的 MMP，重新设计 N 端和 C 端 TIMP 结构域表位并利用协同性 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，该项目将阐明分子识别控制的基本原理 TIMP/MMP 选择性，并将产生针对单一 MMP 的设计 TIMP，具有高度增强的 特异性，具有开发为有用的分子探针和许多疾病的蛋白质治疗剂的潜力 MMP 失调引起的疾病。

项目成果

Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes.

• DOI: 10.1021/jacs.1c08707
• 发表时间: 2021-10-20
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Heyne M;Shirian J;Cohen I;Peleg Y;Radisky ES;Papo N;Shifman JM
• 通讯作者: Shifman JM

MMP1 drives tumor progression in large cell carcinoma of the lung through fibroblast senescence.

• DOI: 10.1016/j.canlet.2021.01.028
• 发表时间: 2021-06-01
• 期刊: Cancer letters
• 影响因子: 9.7
• 作者: Gabasa M;Radisky ES;Ikemori R;Bertolini G;Arshakyan M;Hockla A;Duch P;Rondinone O;Llorente A;Maqueda M;Davalos A;Gavilán E;Perera A;Ramírez J;Gascón P;Reguart N;Roz L;Radisky DC;Alcaraz J
• 通讯作者: Alcaraz J