Vandooren J, Geurts N, Martens E, Steen PEVD, Jonghe SD, Herdewijn P, Opdenakker G. Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain. World J Biol Chem 2011; 2(1): 14-24 [PMID: 21537473 DOI: 10.4331/wjbc.v2.i1.14]
Corresponding Author of This Article
Ghislain Opdenakker, MD, PhD, Professor, Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, Minderbroederstraat 10, Leuven B-3000, Belgium. ghislain.opdenakker@rega.kuleuven.be
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World J Biol Chem. Jan 26, 2011; 2(1): 14-24 Published online Jan 26, 2011. doi: 10.4331/wjbc.v2.i1.14
Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain
Jennifer Vandooren, Nathalie Geurts, Erik Martens, Philippe E Van den Steen, Steven De Jonghe, Piet Herdewijn, Ghislain Opdenakker
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
Author contributions: Vandooren J performed the majority of the experiments and wrote the majority of the manuscript; Geurts N, Martens E and Van den Steen PE performed the experiments and assisted in writing the manuscript; De Jonghe S and Herdewijn P provided the ChemBridge, ChemDiv and InterBioScreen compound library and were involved in study design; Opdenakker G co-ordinated the study and was involved in study design and writing and editing the manuscript.
Supported by A postdoctoral fellowship (Van den Steen PE) and a research assistantship (Geurts N) of the Fund for Scientific Research, Belgium (FWO-Vlaanderen)
Correspondence to: Ghislain Opdenakker, MD, PhD, Professor, Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, Minderbroederstraat 10, Leuven B-3000, Belgium. ghislain.opdenakker@rega.kuleuven.be
Telephone: +32-16-337341 Fax: +32-16-337340
Received: September 17, 2010 Revised: November 18, 2010 Accepted: November 25, 2010 Published online: January 26, 2011
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.
