STRUCTURE, FUNCTION AND APPLICATION OF METALLOPROTEINASE INHIBITORS IN OSTEOARTHR

金属蛋白酶抑制剂的结构、功能及其在骨关节炎中的应用

• 批准号: 7651722
• 负责人: KEITH BREW
• 金额: $ 51.61万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7651722
• 关键词: Affect; Age; Animal Disease Models; Animal Model; Antibodies; Binding; Biochemical; Calcium; Cartilage; Cartilage Matrix; Catalytic Domain; Clinical; Collaborations; Collagen; Complex; Crotalus adamanteus proteinase II; Degenerative polyarthritis; Disease; Economic Burden; Elderly; Engineering; Enzymes; Extracellular Matrix; Funding; Germany; Goals; Human; In Vitro; Institutes; Interstitial Collagenase; Intervention; Joints; Libraries; Ligaments; Matrix Metalloproteinases; Medial; Metalloproteases; Methods; Molecular; Mus; Mutation; Operative Surgical Procedures; Pentosan Polysulfate; Peptide Hydrolases; Phage Display; Play; Population; Protein Engineering; Proteins; Proteomics; Replacement Arthroplasty; Research; Role; Screening procedure; Societies; Specificity; Stromelysin 1; Structure; System; Testing; Therapeutic Agents; Therapeutic Intervention; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinases; Transgenic Mice; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Use Effectiveness; Variant; aggrecanase; arthropathies; articular cartilage; base; collagenase; collagenase 3; design; economic cost; effective therapy; efficacy testing; human tissue; in vivo; in vivo Model; inhibitor/antagonist; mouse model; mutant; novel; novel strategies; novel therapeutics; overexpression; prevent; prototype; public health relevance; therapeutic development; therapeutic target

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the most prevalent joint disease affecting most of the elderly, imposing a major socio- economic burden on society. The hallmark of the disease is the breakdown of articular cartilage by elevated proteinase activities that degrade major extracellular matrix molecules, aggrecans and collagens, but currently there are no effective treatments for OA, except joint replacement surgery. Our long-term objective is to find ways to specifically inhibit these proteinases and apply them for clinical intervention in OA. The key enzymes involved are matrix metalloproteinases (MMPs) and adamalysins with thrombospondin motif (ADAMTSs). Their activities are regulated by the endogenous tissue inhibitors of metalloproteinases (TIMPs). Our recent studies have shown that TIMP-3 variants that selectively inhibit aggrecanases prevent the progression of OA in an animal model, whereas wild-type TIMP-3 that broadly inhibits MMPs and ADAMTSs was not effective. This suggests that highly selective inhibitors are essential for therapeutic development. To achieve this goal two major aims are proposed. The first is to understand the molecular basis for selectivity in our TIMP-3 variants using biochemical, biophysical and structural methods, and to use this to further develop highly discriminating inhibitors of metalloproteinases. Specifically, we will rationally design TIMP-3 variants that differentially inhibit the main two aggrecanases, ADAMTS-4 and -5, and screen human antibody phage display libraries for "exosite inhibitors" of aggrecanases and collagenases. The prototype exosite inhibitors will be further refined by mutation. The mechanism by which calcium pentosan polysulfate (CaPPS), an exosite inhibitor of aggrecanases, enhances the interactions between TIMP-3 and aggrecanases will be also elucidated. The second major aim is to evaluate the efficacy of available and newly developed metalloproteinase inhibitors using in vitro and in vivo models of OA and to validate target enzymes in human OA using these inhibitors. To this end, we will continue characterizing TIMP-3 and [-1A]TIMP-3 transgenic mice to delineate the molecular basis of their differential effects on blocking OA progression by analyzing extracellular matrix components protected by these inhibitors using a combination of immunological and proteomic approaches. Newly developed exosite inhibitors and TIMP variants will be tested for their efficacy to protect cartilage from degradation using an in vitro cartilage explant culture system and the in vivo mouse model of OA induced by medial meniscotibial ligament transaction. Effective inhibitors will be also tested in human cartilage in culture to further validate their efficacy. Using available inhibitors we will identify and characterize a novel TIMP-1- sensitive aggrecanase as it is considered as a potential therapeutic target in humans. Accomplishment of these goals will provide new principles for developing therapeutic interventions for OA. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is a disease that afflicts approximately 21 million people in the US over the age of 25, generating an enormous economic cost that is growing rapidly as the population ages. This project is aimed at developing and evaluating a novel approach for OA treatment employing engineered proteins and other molecules that specifically block the enzymes responsible for degrading cartilage in OA. We will investigate the mechanisms through which they act and assess their effectiveness using animal models of the disease and human tissues derived from joint replacement surgery.

描述(申请人提供)：骨关节炎(OA)是影响大多数老年人的最常见的关节疾病，给社会带来重大的社会经济负担。该病的特点是关节软骨因蛋白水解酶活性升高而破坏，从而降解主要的细胞外基质分子、侵袭物和胶原蛋白，但目前除关节置换手术外，尚无有效的治疗方法。我们的长期目标是找到特异性抑制这些蛋白水解酶的方法，并将其应用于骨关节炎的临床干预。其中涉及的关键酶是基质金属蛋白酶(MMPs)和凝血酶敏感蛋白(ADAMTS)。它们的活性受内源性金属蛋白酶组织抑制因子(TIMPs)的调节。我们最近的研究表明，在动物模型中，选择性抑制聚集酶的TIMP-3变体可以阻止OA的进展，而广泛抑制MMPs和ADAMTS的野生型TIMP-3并不有效。这表明高选择性的抑制剂对于治疗的发展是必不可少的。为了实现这一目标，提出了两个主要目标。首先是使用生化、生物物理和结构方法了解我们的TIMP-3变异体选择性的分子基础，并利用这一点进一步开发高度区分的金属蛋白酶抑制剂。具体地说，我们将合理设计TIMP-3变体，对两种主要的聚集酶ADAMTS-4和-5产生不同的抑制作用，并筛选人抗体噬菌体展示文库以寻找聚集聚集酶和胶原酶的外切抑制物。原型外排体抑制剂将通过突变进一步提纯。此外，还将阐明聚戊聚糖聚硫酸钙(CAPS)增强TIMP-3和聚集聚糖酶之间相互作用的机制。第二个主要目的是利用体外和体内的骨性关节炎模型评价现有的和新开发的金属蛋白酶抑制剂的有效性，并使用这些抑制剂来验证人类骨性关节炎的靶酶。为此，我们将继续鉴定TIMP-3和[-1A]TIMP-3转基因小鼠，通过结合免疫学和蛋白质组学方法分析受这些抑制剂保护的细胞外基质成分，描绘它们在阻止OA进展方面的不同作用的分子基础。新开发的Exosite抑制剂和TIMP变异体将通过体外软骨移植培养系统和体内小鼠半月板胫骨内侧韧带损伤模型来测试它们保护软骨免受降解的有效性。有效的抑制剂也将在培养的人类软骨中进行测试，以进一步验证它们的有效性。利用现有的抑制剂，我们将鉴定和表征一种新的对TIMP-1敏感的聚集聚糖酶，因为它被认为是人类潜在的治疗靶点。这些目标的实现将为开发骨关节炎的治疗干预措施提供新的原则。与公共卫生相关：骨关节炎(OA)是一种疾病，在美国约有2100万25岁以上的人受到折磨，产生了巨大的经济成本，随着人口老龄化，这种成本正在迅速增长。该项目旨在开发和评估一种新的治疗骨性关节炎的方法，该方法使用工程蛋白和其他分子来特异性地阻断骨性关节炎中负责降解软骨的酶。我们将利用疾病的动物模型和来自关节置换手术的人体组织来研究它们的作用机制，并评估它们的有效性。