Engineering selective metalloproteinases inhibitors for developing new therapeutics for neurodegenerative diseases

靶向神经退行性疾病的新的致病性生物标志物是至关重要的，因为可用于控制此类疾病的有效治疗剂有限。metzincin超家族，包括基质金属蛋白酶（MMP）和去整合素和金属蛋白酶（亚当斯），在中枢神经系统的生理和病理过程中发挥多方面的作用，因此是限制疾病如亨廷顿病、帕金森病（PD）和阿尔茨海默病（AD）中神经变性的治疗靶标。鉴于识别在神经变性疾病中起核心作用的酶作为新型神经变性治疗剂的重要性，需要具有高选择性的酶抑制剂。MMP-9的过表达在几种神经退行性疾病中起重要作用，而ADAM-10有助于阻断AD的进展。金属蛋白酶组织抑制剂-3（TIMP-3）是一种天然的MMP-9抑制剂，具有皮科和亚纳摩尔的亲和力。为了克服TIMP-3对不同类型的MMP和亚当斯的广泛多特异性及其与生长因子的相互作用的挑战，需要蛋白质工程方法来定制TIMP-3支架以产生出色的神经退行性药物候选物。 该提案的总体目标是设计基于蛋白质的支架，其高度选择性地靶向特定的metzincin而没有脱靶效应，可以为神经退行性疾病创造新的治疗方法。在目标1中，我们提出了基于TIMPs的工程蛋白支架，以提高对MMP-9的结合选择性，使用定向进化和酵母表面展示。在目标2中，我们将使用X射线晶体学研究这些TIMP-3变体与MMP-9和ADAM-10复合物的相互作用机制，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TIMP-3支架在神经退行性模型中对MMP-9具有改善的选择性。我们的长期目标是开发基于MMP抑制剂或类似支架的选择性蛋白质治疗剂。这些研究将为神经退行性疾病和其他MMP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.