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 s uccessfully modified a previously described assay [1] which used an in ternally quenched peptide substrate (Dnp-PYAYWMR) that, upon cleavage by MMP-3, produces the products, Dnp-PYA (quiet) and YWMR (a fluoropho re at 360 nm). This improved assay uses purified human MMP-3 in the pr esence of either 5% methanol or 5% DMSO. Fluorescence intensities asso ciated with total hydrolysis of substrate by enzyme have been successf ully mimicked using a combination of the product peptides as a standar d. We have determined a K(m) of 39.2 muM and K(cat)/K(m) of 4.6 muM/h for MMP-3 (in 5% MeOH) using this peptide substrate. This assay was us ed successfully to characterize Ro 31-4724 moyl)methyl]-4-methyl-valer yl]-L-leucyl]-L-alanine ethyl ester) as a reversible, tightly binding, inhibitor with a K(i) of 26 nm.