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.