ABSTRACT
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. We describe an HTS-compatible method using the peptide substrate PLGLAARK, labeled at N- and C-termini with CyDye fluors Cy3 and Cy5Q, respectively, which is cleavable by MMP-1, -2, -3, -7, -9, and -13. HTS assays for MMP-13 and MMP-9 inhibitors were set up in approximately 20 microl in 384-well plates as a prompt fluorescence readout (excitation/emission = 540/570 nm) using the LEADseeker homogenous imaging system. These assays yielded IC(50) values comparable to standard methods, but with a faster, very sensitive, and normalized readout, thus conserving compound, enzyme (approximately 1.5 ng/well), and time (20 s read/plate). Data quality (Z' approximately 0.9) was such that hit-picking to -25% change in primary screening could be performed with confidence, and the subsequent rate of confirmation and validation in IC(50) determinations of the picked compounds was >60%. Parallel screening of related proteases also permitted immediate specificity comparisons, including evaluation of inactive or weakly active compounds.
