title: Characterization of a tight-binding MMP-3 inhibitor using improved fluorescence spectroscopy techniques.
abstract: Accurate kinetic characterization of stromelysin (MMP-3) inhibitors is critical in the design of potent inhibitors of this enzyme. We have successfully modified a previously described assay [1] which used an internally quenched peptide substrate (Dnp-PYAYWMR) that, upon cleavage by MMP-3, produces the products, Dnp-PYA (quiet) and YWMR (a fluorophore at 360 nm). This improved assay uses purified human MMP-3 in the presence of either 5% methanol or 5% DMSO. Fluorescence intensities associated with total hydrolysis of substrate by enzyme have been successfully mimicked using a combination of the product peptides as a standard. We have determined a Km of 39.2 microM and Kcat/Km of 4.6 microM/h for MMP-3 (in 5% MeOH) using this peptide substrate. This assay was used successfully to characterize Ro 31-4724 ((N-[(N-[2-[(N-hydroxycarbamoyl)methyl]-4-methyl-valeryl]-L-leucyl ] - L-alanine ethyl ester) as a reversible, tightly binding, inhibitor with a Ki of 26 nm.
authors.affiliation: Department of Arthritis Research, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
authors.forename: M
authors.initials: M
authors.lastname: Finch-Arietta
authors.forename: W
authors.initials: W
authors.lastname: Johnson
authors.forename: L
authors.initials: L
authors.lastname: Lusch
authors.forename: N
authors.initials: N
authors.lastname: Fotouhi
authors.forename: R
authors.initials: R
authors.lastname: Walsky
authors.forename: A C
authors.initials: AC
authors.lastname: Hanglow
mesh_terms: D001665:Binding Sites; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
chemical_list: D006877:Hydroxamic Acids; D009842:Oligopeptides; C078187:Ro 314724; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3