A high throughput fluorogenic substrate for stromelysin (MMP-3).

Stromelysin, a member of the matrix metalloproteinase family of enzymes, has been implicated in the pathogenesis of tumor metastasis and inflammatory diseases such as rheumatoid arthritis. To screen prospective inhibitors of this protease, we developed a fluorogenic substrate with excitation and emission spectra compatible with commercially available 96-well plate readers. The substrate is based on the addition of 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] hexanoic acid (NBD) (EX467/EM534) and 7-dimethylaminocoumarin-4-acetate (DMC) (EX368/EM459) to the previously reported peptide substrate for stromelysin, Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH2. The new substrate, NBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys-(DMC)-NH2 is 95% quenched and the fluorescent product, Nva-Trp-Lys(DMC)-NH2 is easily detected (EX350/EM465). In competition assays the new fluorogenic substrate has a relative kcat/Km that is one half that of the parent peptide. The fluorophores NBD and DMC were chosen based on the high fluorescence yield of DMC and the overlap of the emission spectrum of DMC and excitation spectrum of NBD which results in an efficient energy transfer system in the intact substrate. These characteristics make this an excellent substrate for routine determination of in vitro activities of stromelysin inhibitors.

A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9).

Two members of the matrix metalloproteinase family of enzymes, interstitial collagenase and 92-kDa gelatinase, have been implicated in the pathogenesis of rheumatoid arthritis and tumor metastasis. In order to characterize the activities of these enzymes, we have developed a fluorogenic peptide substrate which is efficiently hydrolyzed by both enzymes. This substrate was developed based on the addition of the fluorescent tag, N-methyl-anthranilic acid (Nma), to several previously synthesized substrates that had been evaluated with respect to their turnover by interstitial collagenase. One substrate, Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(Nma)-NH2, had favorable solubility characteristics, was > 98% quenched, and produced a single cleavage product, Dnp-Pro-Cha-Gly, with a high fluorescence yield with both interstitial collagenase and 92-kDa gelatinase. Since the assay depends on measurement of increases in fluorescence, the position of the Nma group also proved to be important for optimization of the fluorescence signal. The assay is free from interference by organomercurial compounds and the cleavage product has excitation and emission spectra compatible with filters commonly available on commercial plate readers. The assay has been adapted to a 96-well format and provides a rapid screening protocol for the evaluation of inhibitors of these enzymes.