Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain.
| Autor: | Vandooren J; 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., Geurts N, Martens E, Van den Steen PE, Jonghe SD, Herdewijn P, Opdenakker G |
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| Jazyk: | angličtina |
| Zdroj: | World journal of biological chemistry [World J Biol Chem] 2011 Jan 26; Vol. 2 (1), pp. 14-24. |
| DOI: | 10.4331/wjbc.v2.i1.14 |
| Abstrakt: | 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 (k(cat)/k(M) ± 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. |
| Databáze: | MEDLINE |
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