Fluorescence-Based Peptidolytic Assay for High-Throughput Screening of MMP14 Inhibitors

The membrane-bound matrix metalloproteinase 14 (MMP14, also known as MT1-MMP) plays important roles in the remodeling of the extracellular matrix during various cellular processes such as cancer metastasis, angiogenesis, and wound healing through its proteolytic activity. There are no known MMP14-specific inhibitors to date, and hence identification of MMP14-specific inhibitors will be beneficial for finding potential therapeutics for various diseases, including cancer and inflammation. High-throughput screening (HTS) assays have become a common way to search for new small compounds, peptides, and natural products. Enzymatic assays are highly amenable to HTS because most enzyme activities are quantifiable with the effect of many small molecules of interest on a specific target enzyme. Here, we describe a fluorescence-based enzymatic assay that can be applied as a large-scale HTS and a follow-up enzyme kinetics assay to find MMP14-specific inhibitors.