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

项目摘要/摘要 长期以来，MMPs一直被认为是癌症治疗的潜在靶点，但药物的开发是为了靶向 这些酶一直没有成功。一个主要原因是选择性不足，因为大多数基质金属氧化物 抑制剂不能区分促进癌症进展的MMPs和其他预防癌症的MMPs 进步。我们最近开发了一种新的方法、专业知识和方法来进行多项工程 基于人类蛋白的更高选择性的基质金属蛋白酶抑制物-2组织抑制物 (TIMP2)。在我们最近发表的工作中，我们创造了TIMP2 N-末端的工程变体 结构域(N-TIMP2)对基质金属蛋白酶-9的选择性大大提高，这是一种关键的参与三重结构域的酶. 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 抑制剂显示出更强的阻断TNBC细胞侵袭的活性。我们建议进一步设计N- TIMP2用于增加对基质金属蛋白酶-9的选择性，并增强对第二种α3β1整合素的亲和力 TIMP2的天然靶点，通过TIMP2介导抑制肿瘤生长。我们将定义结构 工程N-TIMP2突变体选择性结合基质金属蛋白酶以实现更大分子的基础 我们将评估这些工程蛋白在多个方面的治疗潜力 TNBC的临床前补充模型。在目标1中，我们将使用结构性见解的组合， 计算设计和酵母表面展示(YSD)技术设计N-TIMP2，进一步优化 对基质金属蛋白酶-9的选择性和增强有益的整合素结合活性。在目标2中，我们将阐明 用X射线结晶学方法研究含有靶向和反靶MMPs的工程蛋白结构 工程选择性的结构基础，并促进工程的更大改进 Platform和我们的选择性基质金属蛋白酶-9抑制剂。在目标3中，我们将使用互补的小鼠原位移植， 转基因和患者来源的TNBC异种移植模型评价工程N-TIMP2的应用 变异体作为TNBC的治疗策略，并确定具有潜在潜在反应的候选生物标记物 将这种治疗方法引导到将从它中受益最大的患者。我们的建议既是概念上的 以及在产生新的蛋白质疗法的方法组合方面的技术创新。这个 拟议中的研究具有重要意义，因为它具有开发一种全新的 选择性抑制具有关键作用的靶点基质金属蛋白酶-9靶向治疗肿瘤的方法 在肿瘤生长、侵袭、转移和血管生成中起重要作用。

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.