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

基本信息

批准号：
8055541
负责人：
KEITH BREW
金额：
$ 50.26万
依托单位：
FLORIDA ATLANTIC UNIVERSITY
依托单位国家：
美国
项目类别：
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/8055541
关键词：
Affect Age Animal Disease Models Animal Model Antibodies Binding Biochemical Calcium Cartilage Cartilage Matrix Clinical Collaborations Collagen Complex Crotalus adamanteus proteinase II Degenerative polyarthritis Disease Economic Burden Elderly Engineering Enzymes Extracellular Matrix Funding Germany Goals Health 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 Screening procedure Societies Specificity Stromelysin 1 Structure System TNF-alpha converting enzyme Testing Therapeutic Agents Therapeutic Intervention Tissue Engineering Tissue Inhibitor of Metalloproteinase-1 Tissue Inhibitor of Metalloproteinase-3 Tissue Inhibitor of Metalloproteinases Transgenic Mice Tumor Necrosis Factor-alpha Use Effectiveness Variant aggrecan 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 socioeconomics therapeutic development therapeutic target

项目摘要

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（由申请人提供）：骨关节炎（OA）是影响大多数老年人的最普遍的关节疾病，对社会造成了重大的社会经济负担。该疾病的标志是升高蛋白酶活性的关节软骨分解，这些蛋白酶活性降低了主要的细胞外基质分子，Aggrecans和胶原蛋白，但目前尚无有效的OA治疗方法，除了关节替代手术外。我们的长期目标是找到特殊抑制这些蛋白酶并将其应用于OA的临床干预的方法。所涉及的关键酶是具有血小板传播基序（ADAMTSS）的基质金属蛋白酶（MMP）和Adamalysins。它们的活性受金属蛋白酶（TIMP）的内源性组织抑制剂调节。我们最近的研究表明，TIMP-3变体有选择地抑制脂肪酶阻止动物模型中OA的进展，而野生型TIMP-3广泛抑制MMP和ADAMTS无效。这表明高度选择性抑制剂对于治疗性发育至关重要。为了实现这一目标，提出了两个主要目标。首先是了解使用生化，生物物理和结构方法的TIMP-3变体中选择性的分子基础，并使用它来进一步发展高度区分金属蛋白酶的抑制剂。具体而言，我们将在合理设计TIMP-3变体中差异抑制主要的两种脂肪酶Adamts-4和-5，并筛选人类抗体噬菌体噬菌体显示库，用于“凝集酶和胶原酶”的“外部抑制剂”。原型外部抑制剂将通过突变进一步完善。果胶酶的外部抑制剂（CAPPS）的机制也将增强TIMP-3和脂肪酶之间的相互作用。第二个主要目的是使用体外和体内OA模型评估可用和新开发的金属蛋白酶抑制剂的功效，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，以通过分析由这些免疫学和蛋白质组方法组合来划分受这些抑制剂保护的细胞外基质成分来描述其差异对阻断OA进展的分子基础。新开发的外部抑制剂和TIMP变体将通过体外软骨外植体培养系统和内侧半月菌韧带交易引起的OA的体内小鼠模型来保护软骨免受降解的功效。有效的抑制剂还将在培养物中的人体软骨中进行测试，以进一步验证其功效。使用可用的抑制剂，我们将识别并表征新型的TIMP-1-敏感脂肪酶，因为它被认为是人类的潜在治疗靶标。实现这些目标将为开发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25岁以上美国约2100万人的疾病，随着人口年龄的增长，巨大的经济成本正在迅速增长。该项目旨在开发和评估使用工程蛋白和其他分子的新型OA治疗方法，这些方法专门阻止了负责降解软骨降解软骨的酶。我们将使用疾病的动物模型和从关节置换手术中得出的疾病动物模型和人类组织来研究它们的作用和评估其有效性的机制。