| Assay Method Information | |
| | Inhibition Assay |
| Description: | MMP-13 inhibitor activity of the MMP inhibitors of the present invention was measured using the method of Knight (Knight, C. G. et. al, FEBS LETT. 296 (3), (1992), 263-266), using an assay buffer comprising of 50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 5 mM CaCl2 and 1 uM ZnSO4. A concentration of MMP inhibitor of the present invention was tested (1 microMolar) in duplicate runs. Catalytic domain of MMP-13 (human recombinant) enzyme was then added to the compound solution. The mixture of enzyme and compound in assay buffer was then thoroughly mixed and incubated for 60 minutes at 37° C. Upon the completion of incubation, the assay was then started by the addition of 10 uM of fluorescent substrate Mca-P-L-G-L-Dpa-A-R-NH2. The fluorescent product, McaPLG, was then measured at an excitation wavelength of 355 nm and emission wavelength of 405 nm using an automatic plate multireader at 37° C. A positive control was also run separately using the broad spectrum MMP inhibitor GM6001 as a control compound. Any inhibition <50% is considered not active under these assay conditions. Table 22 summarizes the results of the single concentration inhibition study. The time-dependent increase in fluorescence is measured at the 355 nm excitation and 405 nm emission by automatic plate multireader. The IC50 values for MMP-13 inhibition are then calculated from the initial reaction rates. |
| Affinity data for this assay | |
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