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

项目总结/摘要 基质金属蛋白酶（MMPs）一直被认为是有希望的治疗靶点，但临床试验 早期MMP抑制剂在关节炎和癌症中的应用结果令人失望。广谱MMP 抑制剂产生严重的剂量限制性肌肉骨骼毒性，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 这是一个关键问题，因为一些MMPs主要起保护作用，现在很清楚， 不加区别地抑制所有MMP不可避免地导致较差的结果。根据我们发布的和 初步数据，我们假设，组织金属蛋白酶抑制剂（TIMPs），内源性调节剂， 可以被工程化为高度选择性的MMP抑制剂，而不含不希望的脱靶 活性，以产生具有精确选择性的靶向单个MMP的探针和治疗剂。在这 应用，我们将（a）优化新的方法，用于选择性结合剂的定向进化，以区分 在相关的MMP和adamalysin蛋白酶的大家族中，（B）揭示了分子调节的机制， 控制MMP-TIMP结合特异性的识别，和（c）开发工程化TIMP的工具箱， 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MMPs，重组N-和C-末端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， 阐明了支配TIMP/MMP亲和力和选择性的蛋白质结构和动力学特征 配合物因此，本项目将阐明分子识别的基本原理， TIMP/MMP的选择性，并将产生设计师TIMP靶向单一MMP，具有高度增强的 特异性，具有作为有用的分子探针和作为蛋白质治疗剂的发展潜力， MMP失调导致的疾病。

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.