title: A stromelysin assay for the assessment of metalloprotease inhibitors on human aggregated proteoglycan.
abstract: Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.
authors.affiliation: Research Department, CIBA-GEIGY Corp., Summit, NJ 07901.
authors.forename: J R
authors.initials: JR
authors.lastname: Doughty
authors.forename: R L
authors.initials: RL
authors.lastname: Goldberg
authors.forename: V
authors.initials: V
authors.lastname: Ganu
authors.forename: R A
authors.initials: RA
authors.lastname: Melton
authors.forename: S I
authors.initials: SI
authors.lastname: Hu
authors.forename: G
authors.initials: G
authors.lastname: Di Pasquale
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002358:Cartilage, Articular / Q000378:metabolism; D002809:Chondroitin Sulfates; D004797:Enzyme-Linked Immunosorbent Assay; D005269:Femur; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000276:immunology; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D010618:Phenanthrolines / Q000494:pharmacology; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013373:Substance P / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D010618:Phenanthrolines; D011509:Proteoglycans; D011994:Recombinant Proteins; D013373:Substance P; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; C008890:phosphoramidon; C025205:1,10-phenanthroline