STRUCTURE, FUNCTION AND APPLICATION OF METALLOPROTEINASE INHIBITORS IN OSTEOARTHR

描述（由申请人提供）：骨关节炎（OA）是影响大多数老年人的最常见关节疾病，给社会带来了重大的社会经济负担。该疾病的标志是通过升高的降解主要细胞外基质分子、聚集蛋白聚糖和胶原蛋白的蛋白酶活性破坏关节软骨，但目前除了关节置换手术外，没有有效的治疗OA的方法。我们的长期目标是找到特异性抑制这些蛋白酶的方法，并将其应用于OA的临床干预。参与的关键酶是基质金属蛋白酶（MMPs）和具有血小板反应蛋白基序的金刚溶解素（ADAMTSs）。它们的活性受内源性金属蛋白酶组织抑制剂（TIMPs）的调节。我们最近的研究表明，选择性抑制聚集蛋白聚糖酶的TIMP-3变体在动物模型中预防OA的进展，而广泛抑制MMP和ADAMTS的野生型TIMP-3则无效。这表明高选择性抑制剂对于治疗开发是必不可少的。为了实现这一目标，提出了两个主要目标。第一个是使用生物化学，生物物理学和结构方法了解TIMP-3变体选择性的分子基础，并使用此进一步开发高度识别的金属蛋白酶抑制剂。具体而言，我们将合理设计TIMP-3的变体，差异抑制主要的两个聚集蛋白聚糖酶，ADAMTS-4和-5，并筛选人抗体噬菌体展示库的聚集蛋白聚糖酶和胶原酶的“外位点抑制剂”。原型外部位点抑制剂将通过突变进一步改进。戊聚糖多硫酸钙（CaPPS），一种外位点抑制剂的聚集蛋白聚糖酶，增强TIMP-3和聚集蛋白聚糖酶之间的相互作用的机制也将阐明。第二个主要目的是使用OA的体外和体内模型评价现有和新开发的金属蛋白酶抑制剂的疗效，并使用这些抑制剂验证人OA中的靶酶。为此，我们将继续表征TIMP-3和[-1A]TIMP-3转基因小鼠，通过使用免疫学和蛋白质组学方法的组合分析受这些抑制剂保护的细胞外基质成分，来描述其阻断OA进展的差异效应的分子基础。将使用体外软骨外植体培养系统和内侧胫前韧带损伤诱导的OA体内小鼠模型测试新开发的外位点抑制剂和TIMP变体保护软骨免于降解的功效。有效的抑制剂也将在培养的人软骨中进行测试，以进一步验证其功效。使用现有的抑制剂，我们将确定和表征一种新的TIMP-1敏感的聚集蛋白聚糖酶，因为它被认为是一个潜在的治疗目标，在人类。这些目标的实现将为开发OA的治疗干预提供新的原则。公共卫生相关性：骨关节炎（OA）是一种疾病，在美国约有2100万25岁以上的人患有这种疾病，随着人口老龄化，这种疾病产生了巨大的经济成本。该项目旨在开发和评估一种新的OA治疗方法，该方法采用工程蛋白和其他分子，专门阻断负责降解OA软骨的酶。我们将研究它们的作用机制，并使用该疾病的动物模型和关节置换手术获得的人体组织评估其有效性。

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.