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志物是至关重要的，因为控制这类疾病的有效治疗方法有限。metzincin超家族，包括基质金属蛋白酶（MMPs）和崩解素和金属蛋白酶（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）等疾病的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型神经退行性治疗的重要性，需要具有高选择性的酶抑制剂。MMP-9的过表达在多种神经退行性疾病中发挥重要作用，而ADAM-10有助于阻止AD的进展。组织金属蛋白酶抑制剂-3 （TIMP-3）是MMP-9的天然抑制剂，具有皮摩尔和亚纳摩尔亲和力。为了克服TIMP-3对不同类型的MMPs和ADAMs的广泛多特异性及其与生长因子的相互作用的挑战，需要采用蛋白质工程方法来定制TIMP-3支架以创建出色的神经退行性候选药物。

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.