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

项目摘要/摘要 基质金属蛋白酶(MMPs)长期以来一直被认为是有希望的治疗靶点，但临床试验 治疗关节炎和癌症的早期一代基质金属蛋白酶抑制剂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的肌肉骨骼毒性，并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。这是一次 关键问题，因为一些MMP主要起到保护作用，现在很明显 不分青红皂白地抑制所有MMP不可避免地会导致更糟糕的结果。基于我们发布的和 初步数据，我们假设金属蛋白酶组织抑制物(TIMPs)，内源性调节因子 作为基质金属蛋白酶家族的一员，可以被改造成高度选择性的基质金属蛋白酶抑制剂，而不会出现不受欢迎的脱靶现象 活性，生产针对单个MMPs的探针和疗法，具有精致的选择性。在这 应用，我们将(A)优化选择性结合物定向进化的新方法，以区分 在相关的MMPs和Adamalysin蛋白的大家族中，(B)揭示分子机制 管理MMP-TIMP结合特异性的识别，以及(C)开发一个工程TIMP工具箱，该工具箱 选择性地针对单个MMPs，具有高度增强的特异性。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 相似的MMPs，N-端和C-端TIMP结构域表位的重组和协同作用 在域之间。此外，我们将整合X射线结晶学和计算方法，以 阐明控制TIMP/MMPs亲和力和选择性的蛋白质结构和动力学特征 复合体。因此，本项目将阐明分子识别的基本原理 TIMP/MMP选择性，并将生产针对单个MMP的设计型TIMP，具有高度增强的功能 特异性，具有作为有用的分子探针和许多蛋白质疗法的潜力 由基质金属蛋白酶失调引起的疾病。

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.