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.
Keywords: Exosite inhibitors; Fluorogenic substrate; Gelatin; High-throughput screening assays; Matrix metalloproteinase-9; Substrate specificity.