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Published on: January 22, 2019
Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay
Ty J Lively1, Dale B Bosco2, Zahraa I Khamis1
1Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, FL, 32306, USA.
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.
Insights
Matrix metalloproteinase inhibitors (MMPIs) are crucial for studying and treating diseases linked to matrix metalloproteinases (MMPs). This method uses enzyme kinetics and fluorescence spectroscopy to measure MMPI potency accurately.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Matrix metalloproteinases (MMPs) are key enzymes regulating the extracellular matrix (ECM).
- Dysregulated MMP activity is implicated in diseases like cancer, neurodegeneration, stroke, and cardiovascular disease.
- Matrix metalloproteinase inhibitors (MMPIs) are developed as research tools and potential therapeutics.
Purpose of the Study:
- To detail a method for characterizing MMP inhibitors using enzyme kinetics.
- To provide a framework for assessing MMPI potency and efficacy.
Main Methods:
- Enzyme kinetics assays are employed to quantify MMP activity.
- Fluorescence spectroscopy is utilized with a Mca/Dpa quenched fluorogenic substrate.
- Inhibitor potency is determined by measuring the reduction in fluorescence intensity.
Main Results:
- The described technique allows for the characterization of various MMPs.
- MMPI potency is quantitatively assessed through enzyme inhibition assays.
- This method provides a reliable way to evaluate the effectiveness of MMPIs.
Conclusions:
- Enzyme kinetics and fluorescence spectroscopy offer a robust approach to characterize MMP inhibitors.
- This methodology is essential for advancing research into MMP-related diseases and developing new therapies.
