| Assay Method Information | |
| | Dissociation Constants (KD) Measurements and Enzyme Inhibition Assay (IC50) |
| Description: | The NMR experiments were performed to determine the binding affinity (KD) for the MMP-12 catalytic domain. The alteration of the chemical shifts induced on 2D 1H-15N HSQC upon the titration with the test compound was analyzed. The fit of the experimental data as a function of ligand concentration provided dissociation constant. The inhibition potency (IC50) was measured by fluorescent enzymatic assays. The compounds were evaluated for their ability to inhibit the hydrolysis of fluorescence-quenched peptide substrate. The enzyme was incubated with increasing concentration of inhibitor and the fluorescence (ex=328 nm; em=393 nm) was measured for 3 min after the addition of the substrate using a Varian Eclipse fluorimeter. Fitting of hydrolysis rate as a function of inhibitor concentration provided IC50. |
| Affinity data for this assay | |
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