Example 6
Individual inhibitors were dissolved in TCN buffer (50 mM Tris-HCl, 10 mM CaCl2, 150 mM NaCl2, 0.05% Brij 35 at pH 7.5) at appropriate dilutions then added to the wells of a microtiter plate (10 μL/well) in triplicate. Each well of test agent (and appropriate control wells) was then treated with activated MMP-2 or 9 (10 μL of a 40 nM solution in 50 mM Hepes, 10 mM CaCl2), 1% Brij 35 at pH 7.5; R&D Systems) followed by 30 μL TCN buffer and 150 μL the fluorogenic peptide substrate (Mca-PLGL-Dpa-AR-NH2; R&D Systems). The resulting mixtures were incubated 1 h at 27° C. then analyzed using a FL600 fluorescent plate reader (excitation=310/20; emission=420/50; optics=bottom; sensitivity=225) and KC4 Software.
