Engineering tissue inhibitor of metalloproteinases-2 (TIMP-2) for triple negative breast cancer therapy

项目概要/摘要 MMPs长期以来一直被认为是癌症治疗的潜在靶点，但针对MMPs开发的药物 这些酶是不成功的。主要原因是选择性不足，因为大多数MMP 抑制剂不能区分促进癌症进展的MMPs和其他预防癌症的MMPs 进展我们最近开发了一种新的方法、专业知识和方法， 基于人蛋白质的更高选择性MMP抑制剂，金属蛋白酶组织抑制剂-2 （TIMP2）。在我们最近发表的工作中，我们已经创建了TIMP 2 N-末端的工程变体， 结构域（N-TIMP 2），对MMP-9的选择性大大提高，MMP-9是一种关键参与三重- 阴性乳腺癌（TNBC）进展和转移。在初步研究中，我们发现这个原型 抑制剂显示阻断TNBC细胞侵袭的增强活性。我们建议进一步设计N- TIMP-2对MMP-9的选择性增加以及对α3β1整合素的亲和力增强， TIMP 2的天然靶点，TIMP 2通过该靶点介导肿瘤生长的抑制。我们将定义结构 工程化的N-TIMP 2变体的选择性MMP结合的基础， 改进，我们将评估这些工程蛋白质的治疗潜力，在多个 补充TNBC的临床前模型。在目标1中，我们将结合使用结构性见解， 计算设计和酵母表面展示（YSD）技术来工程化N-TIMP 2，进一步优化 对MMP-9的选择性和增强有益的整联蛋白结合活性。在目标2中，我们将阐明 使用X射线晶体学分析具有靶向和抗靶向MMPs的工程化蛋白的结构，以揭示 工程选择性的结构基础，并促进我们工程的进一步改进 平台和选择性MMP-9抑制剂。在目标3中，我们将使用互补小鼠原位， TNBC的转基因和患者来源的异种移植物（PDX）模型，以评估工程化N-TIMP 2的效用 变异作为TNBC的治疗策略，并确定候选的生物标志物， 将这种治疗方法引导到最能从中受益的患者身上。我们的建议在概念上 并在技术上创新的方法组合，以产生新的蛋白质治疗。的 拟议的研究是非常重要的，因为它有很大的潜力，开发一个全新的 通过选择性抑制MMP-9靶向治疗TNBC的方法，MMP-9是一种经过充分验证的具有关键作用的靶标 在肿瘤生长、侵袭、转移和血管生成中。

PROJECT SUMMARY/ABSTRACT The MMPs have long been recognized as potential targets for cancer therapy, but drugs developed to target these enzymes have been unsuccessful. A primary reason has been inadequate selectivity, since most MMP inhibitors cannot discriminate among MMPs that drive cancer progression and other MMPs that prevent cancer progression. We have recently developed a new approach, expertise, and methodology for engineering much more highly selective MMP inhibitors based on a human protein, tissue inhibitor of metalloproteinases-2 (TIMP2). In our recently published work, we have created an engineered variant of the TIMP2 N-terminal domain (N-TIMP2) with greatly improved selectivity toward MMP-9, an enzyme critically involved in triple- negative breast cancer (TNBC) progression and metastasis. In preliminary studies, we find that this prototype inhibitor shows enhanced activity for blocking TNBC cellular invasion. We propose to further engineer N- TIMP2 for increased selectivity toward MMP-9 and also for enhanced affinity toward α3β1 integrin, a second natural target of TIMP2 through which TIMP2 mediates inhibition of tumor growth. We will define the structural basis for selective MMP binding of engineered N-TIMP2 variants to enable yet greater molecular improvements, and we will evaluate the therapeutic potential of these engineered proteins in multiple complementary preclinical models of TNBC. In Aim 1, we will use a combination of structural insights, computational design and yeast surface display (YSD) technology to engineer N-TIMP2, further optimizing selectivity toward MMP-9 and enhancing beneficial integrin binding activity. In Aim 2, we will elucidate structures of the engineered proteins with target and anti-target MMPs using X-ray crystallography, to uncover the structural basis for engineered selectivity and to facilitate yet greater refinements of our engineering platform and our selective MMP-9 inhibitors. In Aim 3, we will use complementary mouse orthotopic, transgenic, and patient-derived xenograft (PDX) models of TNBC to evaluate the utility of engineered N-TIMP2 variants as a therapeutic strategy in TNBC, and identify candidate biomarkers of response with potential for directing this therapeutic approach to patients who will most benefit from it. Our proposal is both conceptually and technically innovative in the combination of approaches toward generating novel protein therapeutics. The proposed research is highly significant because it has substantial potential to develop an entirely new approach for targeted treatment of TNBC by selectively inhibiting MMP-9, a well-validated target with key roles in tumor growth, invasion, metastasis, and angiogenesis.