Development of an assay suitable for high-throughput screening to measure matrix metalloprotease activity.

MMPs, part of a family of enzymes with >35 known members, play an important role in tissue remodeling and repair, in the biology of neoplasia, and during development. Hydroxamic and carboxylic acid inhibitors of these proteases have long been available, but their specificities are poor and there still exists a desire to find novel chemical structures, which could be modified to optimize specificity and biocompatibility. Established methods for measuring MMP activity are based on the cleavage of MCA-PLGL-A2pr(DNP)-AR, which provides a prompt fluorescent signal when cleaved; however, its absorption/emission properties (325/400 nm) are not best suited for HTS assays.

Evaluation of an imaging platform during the development of a FRET protease assay.

Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from biologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH(2) is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q.