title: Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain.
abstract: OBJECTIVE
  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).
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Vandooren
authors.forename: Nathalie
authors.initials: N
authors.lastname: Geurts
authors.forename: Erik
authors.initials: E
authors.lastname: Martens
authors.forename: Philippe E
authors.initials: PE
authors.lastname: Van den Steen
authors.forename: Steven De
authors.initials: SD
authors.lastname: Jonghe
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.forename: Ghislain
authors.initials: G
authors.lastname: Opdenakker