Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain |
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Authors: | Vandooren Jennifer Geurts Nathalie Martens Erik Van den Steen Philippe E Jonghe Steven De Herdewijn Piet Opdenakker Ghislain |
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Affiliation: | Jennifer Vandooren, Nathalie Geurts, Erik Martens, Philippe E Van den Steen, Ghislain Opdenakker, Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, Minderbroederstraat 10, Leuven B-3000, Belgium;Steven De Jonghe, Piet Herdewijn, Medicinal Chemistry, Rega Institute for Medical Research, University of Leuven, Minderbroederstraat 10, Leuven B-3000, Belgium |
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Abstract: | 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)™-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a high-throughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of heterocyclic, 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 (barbiturate) 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 DQ™-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. |
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Keywords: | Exosite inhibitors Fluorogenic substrate Gelatin High-throughput screening assays Matrix metalloproteinase-9 Substrate specificity |
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