TIMP Engineering and Application to Arthritis

TIMP 工程及其在关节炎中的应用

• 批准号: 6611952
• 负责人: KEITH BREW
• 金额: $ 39.42万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611952
• 关键词: SDS polyacrylamide gel electrophoresis; antiarthritic agent; articular cartilage; calorimetry; chondroitin sulfates; clinical research; collagenase; drug screening /evaluation; electrospray ionization mass spectrometry; enzyme activity; enzyme linked immunosorbent assay; enzyme therapy; human tissue; laboratory mouse; mass spectrometry; matrix assisted laser desorption ionization; metalloendopeptidases; osteoarthritis; pathologic process; polymerase chain reaction; protein binding; protein engineering; proteoglycan; rheumatoid arthritis; tissue inhibitor of metalloproteinases; western blottings

DESCRIPTION (provided by applicant): The matrix metalloproteinases (MMPs) are zinc metalloproteinases that degrade components of the extracellular matrix. They play major roles in diseases including arthritis, cancer and atherosclerosis. The activities of MMPs are regulated by endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs)-1, to -4. Our long-range goals are to understand how TIMPs inhibit MMPs and to use this information to engineer variant TIMPs that selectively inhibit individual or chosen groups of metalloproteinases. These targeted TIMPs will be tested for efficacy in alleviating the progression of diseases associated with increased degradation of the extracellular matrix. Our recent finding that TIMP-3 is a potent inhibitor of two aggrecanases (ADAMTS-4 and ADAMTS-5), key enzymes in the degradation of the cartilage proteoglycan, aggrecan, leads us to further investigate and test TIMP-3, and other TIMP variants, as inhibitors for preventing the progression of arthritis. To achieve these goals we will elucidate the structural basis of TIMP specificity for aggrecanases and MMPs and engineer TIMPs that are targeted for metalloproteinases involved in cartilage degradation. These inhibitors will be tested for effectiveness in ex vivo and in vivo models of rheumatoid arthritis (RA) and osteoarthritis (OA). The Specific Aims are: (1) to investigate the mechanism of inhibition of aggrecanases by TIMP-3 and generate specific inhibitors of these enzymes; (2) to produce TIMP variants that selectively inhibit collagenases (MMP-1 and -13), gelatinase A (MMP-2), or MT1-MMP; (3) identify and characterize low-molecular weight peptide inhibitors employing non-toxic variants of sarafotoxin, an analogue of the unique inhibitory region of TIMPs; (4) characterize the structural and physical basis of strong and specific metalloproteinase binding in TIMPs; (5) test recombinant TIMP-3 and other wild-type and variant TIMPs for their ability to prevent cartilage breakdown using the cartilage explant system; (6) test the efficacy of TIMP-3 and TIMP variants as potential blockers of cartilage degradation in the collagen-induced arthritis model of RA, and in the STR/ort mouse OA model; and (7) identify metalloproteases that act in articular cartilage breakdown during the progression of OA in humans. These studies will produce mechanistic and structural information about the interactions of TIMPs and metalloproteinases and new insights into therapeutic approaches for arthritis.

描述（由申请人提供）：基质金属蛋白酶（MMP）是降解细胞外基质成分的锌金属蛋白酶。它们在关节炎、癌症和动脉粥样硬化等疾病中发挥着重要作用。 MMP 的活性受到内源性抑制剂、金属蛋白酶组织抑制剂 (TIMP)-1 至 -4 的调节。我们的长期目标是了解 TIMP 如何抑制 MMP，并利用这些信息设计变体 TIMP，选择性抑制单个或选定的金属蛋白酶组。将测试这些靶向 TIMP 在缓解与细胞外基质降解加剧相关的疾病进展方面的功效。我们最近发现 TIMP-3 是两种聚集蛋白聚糖酶（ADAMTS-4 和 ADAMTS-5）的有效抑制剂，这两种聚集蛋白聚糖酶是软骨蛋白多糖聚集蛋白聚糖降解的关键酶，这促使我们进一步研究和测试 TIMP-3 和其他 TIMP变体，作为预防关节炎进展的抑制剂。为了实现这些目标，我们将阐明软骨聚集蛋白聚糖酶和 MMP 的 TIMP 特异性的结构基础，并设计针对参与软骨降解的金属蛋白酶的 TIMP。这些抑制剂将在类风湿性关节炎（RA）和骨关节炎（OA）的离体和体内模型中测试其有效性。具体目标是：（1）研究TIMP-3抑制蛋白聚糖酶的机制，并产生这些酶的特异性抑制剂； (2) 产生选择性抑制胶原酶（MMP-1和-13）、明胶酶A（MMP-2）或MT1-MMP的TIMP变体； (3) 使用 sarafotoxin（TIMP 独特抑制区域的类似物）的无毒变体来鉴定和表征低分子量肽抑制剂； (4) 表征 TIMP 中强且特异性的金属蛋白酶结合的结构和物理基础； (5) 使用软骨外植体系统测试重组 TIMP-3 和其他野生型和变体 TIMP 防止软骨破坏的能力； (6) 在胶原诱导的RA关节炎模型和STR/ort小鼠OA模型中测试TIMP-3和TIMP变体作为软骨退化的潜在阻断剂的功效； (7) 鉴定在人类 OA 进展过程中参与关节软骨破坏的金属蛋白酶。这些研究将产生有关 TIMP 和金属蛋白酶相互作用的机制和结构信息，以及对关节炎治疗方法的新见解。