Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

• 作者: Erik Martens Ghislain Opdenakker Jennifer Vandooren Nathalie Geurts Philippe E Van den Steen Piet Herdewijn Steven De Jonghe
• 来源期刊: 世界生物化学杂志：英文版（电子版）
• 学科: 生物学
• 关键词: Exosite INHIBITORS FLUOROGENIC SUBSTRATE GELATIN High-throughput screening assays Matrix metalloproteinase-9 SUBSTRATE specificity
• 年，卷（期）: 2011,（1）
• 页码范围: 14-24
• 页数: 11页
• 分类号: Q503

摘要

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.