Engineering selective metalloproteinases inhibitors for developing new therapeutics for neurodegenerative diseases

针对神经退行性疾病的新的致病生物标记物是至关重要的，因为可用于控制此类疾病的有效疗法有限。梅津菌素超家族包括基质金属蛋白酶(MMPs)和去整合素和金属蛋白酶(ADAMS)，在中枢神经系统的生理和病理过程中发挥多方面的作用，因此是限制亨廷顿病、帕金森病(PD)和阿尔茨海默病(AD)等疾病神经退行性变的治疗靶点。鉴于认识到在神经退行性疾病中发挥核心作用的酶作为新的神经退行性疾病治疗药物的重要性，人们希望有高选择性的酶抑制剂。基质金属蛋白酶-9的过度表达在几种神经退行性疾病中起着重要作用，而ADAM-10有助于阻止AD的进展。金属蛋白酶组织抑制因子-3(TIMP-3)是一种天然的基质金属蛋白酶-9抑制物，具有微米级和亚纳摩尔亲和力。为了克服TIMP-3对不同类别的MMPs和ADAMS具有广泛的多特异性及其与生长因子相互作用的挑战，需要一种蛋白质工程方法来定制TIMP-3支架，以创造出一种优秀的神经退行性药物候选药物。 这项提议的首要目标是设计基于蛋白质的支架，这种支架可以高选择性地靶向特定的梅津菌素，而不会产生偏离靶点的效应，从而为神经退行性疾病创造新的治疗方法。在目标1中，我们建议通过定向进化和酵母表面展示来设计基于TIMPs的蛋白质支架，以提高对基质金属蛋白酶-9的结合选择性。在目标2中，我们将利用X射线结晶学研究这些TIMP-3变异体与基质金属蛋白酶-9和ADAM-10的相互作用机制，以了解抑制的潜在机制。在目标3中，我们将评估这些工程化的TIMP-3支架在神经退行性变模型中对基质金属蛋白酶-9的选择性提高。我们的长期目标是开发基于基质金属蛋白酶抑制剂或类似支架的选择性蛋白质疗法。这些研究将为神经退行性疾病和其他基质金属蛋白酶相关疾病的临床前体内模型和新的治疗策略奠定基础。

Targeting new pathogenic biomarkers of neurodegenerative diseases is critical as there are limited efficient therapeutics available to control such diseases. The metzincin superfamily, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), play multifaceted roles in physiological and pathological processes in the central nervous system and therefore are therapeutic targets to limit neurodegeneration in diseases such as Huntington disease, Parkinson’s disease (PD), and Alzheimer’s disease (AD). Given the significance of recognizing enzymes that play a central role in neurodegenerative disease as novel neurodegenerative therapeutics, enzyme inhibitors with high selectivity are desired. Overexpression of MMP-9 plays a significant role in several neurodegenerative disorders, while ADAM-10 helps block progression of AD. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a natural inhibitor of MMP-9 with pico- and subnanomolar affinity. To overcome the challenges of wide multispecificity of TIMP-3 for different classes of MMPs and ADAMs and its interaction with growth factors, a protein engineering approach is desired to tailor a TIMP-3 scaffold to create an outstanding neurodegenerative drug candidate. The overarching goal of this proposal is to engineer protein-based scaffolds that high selectively target a specific metzincin without off-target effects can create novel therapeutics for neurodegenerative diseases. In Aim 1, we propose to engineer protein scaffolds based on TIMPs to improve binding selectivity toward MMP-9 using directed evolution and yeast surface display. In Aim 2, we will study the mechanism of interaction of these TIMP-3 variants in complex with MMP-9 and ADAM-10 using X-ray crystallography to understand the underlying mechanism of inhibition. In Aim 3, we will evaluate these engineered TIMP-3 scaffolds with improved selectivity for MMP-9 in neurodegenerative models. Our long-term goal is to develop selective protein-based therapeutics based on MMP inhibitors or similar scaffolds. These studies will lay the foundation for preclinical in vivo models and novel therapeutic strategies for neurodegenerative and other MMP-related diseases.