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 aK m of 39.2 μM andK cat/K m of 4.6 μM/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 aK i of 26 nm.
Similar content being viewed by others
References
S. Netzel-Arnett, S. K. Mallya, H. Nagase, H. Birkedal-Hansen, and H. E. Van-Wart,Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Anal. Biochem.195, 86–92 (1991).
J. F. Woessner,Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J.5, 2145–2154 (1991).
M. Dixon and E. C. Webb,Enzymes 3rd Edition, pp. 361–368 Longman, London (1979).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Finch-Arietta, M., Johnson, W., Lusch, L. et al. Characterization of a tight-binding MMP-3 inhibitor using improved fluorescence spectroscopy techniques. Agents and Actions 39 (Suppl 1), C189–C191 (1993). https://doi.org/10.1007/BF01972762
Issue Date:
DOI: https://doi.org/10.1007/BF01972762