Directed evolution of tissue inhibitor of metalloproteinase 3 (TIMP-3) to develop novel Alzheimer’s disease (AD) therapeutics

摘要 控制阿尔茨海默病(AD)的有效疗法有限，因此有很强的需求 针对AD的新的致病生物标记物。梅津菌素超家族，包括基质金属蛋白酶 (MMPs)和一种去整合素和金属蛋白酶(ADAMS)，在生理和病理过程中发挥多方面的作用。 中枢神经系统的病理过程，因此是限制治疗的靶点 阿尔茨海默病的神经退行性变。例如，上调基质金属蛋白酶-9促进细胞凋亡，血脑屏障 紊乱和脱髓鞘，并通过蛋白分解参与淀粉样蛋白-β的处理途径 细胞外基质成分的降解和组织重塑，并增加tau的积累 在AD中扮演关键角色。但另一方面，ADAM-10的上调增加了可溶性淀粉样蛋白β的形成， 这有助于控制AD。因此，使用高度选择性的抑制剂来调整梅津星的蛋白分解活性- 阿尔茨海默病患者脑内的病理改变可能是一种很有前途的治疗策略。蛋白质抑制剂 与较小的合成广谱抑制分子相比，提供了更高的靶向基质金属蛋白酶-9的选择性 目标。金属蛋白酶组织抑制物(TIMPs)是MMPs和MMPs的主要内源性抑制物 ADAMS具有不同程度的结合亲和力。在所有TIMP中，TIMP-3与 触发不受欢迎的脱靶效应的细胞信号分子，如那些与肿瘤发生有关的分子， 提示其对基质金属蛋白酶依赖型疾病有较低的非靶点治疗效果。这使得TIMP-3成为 理想的蛋白质支架，用于设计改进的基质金属蛋白酶-9抑制剂。然而，TIMP-3也与其他 基质金属蛋白酶、亚当斯和生长因子。在这里，我们建议使用有理和随机的组合 开发以TIMP-3为基础的治疗神经退行性疾病的组合方法 高选择性靶向基质金属蛋白酶-9的研究进展。这些经过设计的基于TIMP的探测器将避免绑定 对AD具有神经保护作用的ADAM-10，以减少非靶点效应。这个项目的首要目标是 是设计和设计一种基于天然基质金属蛋白酶抑制剂的新的分子靶向疗法 高选择性的基质金属蛋白酶-9，通过结构研究和细胞培养了解其抑制机制 在治疗选择不充分的情况下改善神经退行性疾病预后的模型。

SUMMARY Limited efficient therapeutics are available to control Alzheimer's disease (AD), so there is a strong demand to target new pathogenic biomarkers of AD. The metzincin superfamily, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), play a multifaceted role in physiological and pathological processes in the central nervous system and therefore are therapeutic targets to limit neurodegeneration in AD. For instance, upregulation of MMP-9 promotes apoptosis, blood-brain barrier disorder, and demyelination and is involved in the amyloid-β processing pathway through proteolytic degradation of extracellular matrix components and tissue remodeling, and increasing tau accumulation which play key roles in AD. But on the other hand, upregulation of ADAM-10 increases soluble amyloid-β formation, which helps control AD. Thus, using highly selective inhibitors to tailor the proteolytic activity of metzincin- induced pathological changes in AD patients’ brains may be a promising therapeutic strategy. Protein inhibitors offer higher selectivity to target MMP-9 compared to smaller synthetic inhibitory molecules with broad-spectrum targets. Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous inhibitors of MMPs and ADAMs with varying degrees of binding affinity. Among all TIMPs, TIMP-3 has the lowest association with the cell signaling molecules triggering undesired off-target effect such as the ones responsible in tumorigenesis, suggesting that it has a low off-target therapeutic effect in MMP-dependent diseases. This makes TIMP-3 an ideal protein scaffold for engineering improved inhibitors for MMP-9. However, TIMP-3 also binds to other MMPs, ADAMs, and growth factors. Here, we propose to use a combination of rational and random combinatorial approaches to develop TIMP-3-based therapeutics to control neurodegenerative disease progression by targeting MMP-9 with high selectivity. These engineered TIMP-based probes will avoid binding to ADAM-10, which is neuroprotective in AD, to decrease off-target effects. The overarching goal of this project is to engineer and design a new molecularly targeted therapy based on natural MMP inhibitors that target MMP-9 with high selectivity, understand their mechanism of inhibition using structural studies and cell culture models to improve outcomes in neurodegenerative diseases with inadequate treatment options.