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的关节疾病，对社会造成了重大的社会经济负担。该疾病的特征是通过蛋白酶活性升高降解主要的细胞外基质分子、聚集蛋白和胶原蛋白而破坏关节软骨，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。涉及的关键酶是基质金属蛋白酶（MMPs）和具有血栓反应蛋白基序的ADAMTSs。它们的活性受内源性组织金属蛋白酶抑制剂（TIMPs）的调控。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ADAMTSs的野生型TIMP-3则没有效果。这表明高选择性抑制剂对治疗发展至关重要。为实现这一目标，提出了两个主要目标。首先是利用生化、生物物理和结构方法了解TIMP-3变异选择性的分子基础，并以此进一步开发高鉴别的金属蛋白酶抑制剂。具体而言，我们将合理设计不同抑制主要两种聚集酶ADAMTS-4和-5的TIMP-3变体，并筛选人抗体噬菌体展示文库，寻找聚集酶和胶原酶的“外源抑制剂”。外源性抑制剂的原型将通过突变进一步完善。聚聚糖酶外源抑制剂戊聚糖聚硫酸钙（CaPPS）增强TIMP-3与聚聚糖酶相互作用的机制也将被阐明。第二个主要目的是通过体外和体内OA模型评估现有的和新开发的金属蛋白酶抑制剂的功效，并验证这些抑制剂在人类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，通过结合免疫学和蛋白质组学方法分析这些抑制剂保护的细胞外基质成分，来描述它们在阻断OA进展方面的差异作用的分子基础。新开发的外源性抑制剂和TIMP变体将通过体外软骨外植体培养系统和内侧半月板韧带交易诱导的OA小鼠模型来测试其保护软骨免受降解的功效。有效的抑制剂也将在培养的人类软骨中进行测试，以进一步验证其功效。利用现有的抑制剂，我们将鉴定和表征一种新的TIMP-1敏感的聚集酶，因为它被认为是人类的潜在治疗靶点。这些目标的实现将为OA的治疗干预提供新的原则。

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.