Characterization of a tight-binding MMP-3 inhibitor using improved fluorescence spectroscopy techniques.
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.
Finch-Arietta
M
M
Department of Arthritis Research, Hoffmann-La Roche, Inc., Nutley, NJ 07110.
Johnson
W
W
Lusch
L
L
Fotouhi
N
N
Walsky
R
R
Hanglow
A C
AC
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
D006877:Hydroxamic Acids; D009842:Oligopeptides; C078187:Ro 314724; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3