Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay

Abstract

Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.

Biosensors for measuring matrix metalloproteinases: An emerging research field

“…Optical detection, in particular fluorescence based methods, is mainly used for this substrate cleavage approach [37]. MMPs activities can be quantified by measuring changes: 1) in the fluorescence signal from a pair of dyes that interact via resonance energy transfer [38]; 2) in the fluorescence intensity upon the presence or absence of an MMP inhibitor [39,40]; 3) in the fluorescence polarization [41].…”

Simultaneous quantification of exosomal MMP14 expression and proteolysis activity on a spherical dual-probe-based fluorescent nanosensor

“…Owing to their simplicity, high efficiency, and high sensitivity, biosensors based on different materials and probes have attracted much more attention in recent years. Activity based probes, 9,[16][17][18][19] substrate-based probes, [20][21][22] antibody probes, 9,[23][24][25] and aptamer probes [26][27][28] are commonly used in MMP biosensors. Peptide-and substrate-based sensors only react with proteolytically active MMPs, while labelled antibodyor aptamer-based sensors are used to detect the abundance of MMPs.…”