| Assay Method Information | |
| | Enzyme Assay |
| Description: | Assays to evaluate the inhibition of cathepsin L contained 25 pM cathepsin L (RD systems: 952-CY-010), 5 uM Z-LR-AMC fluorogenic peptide substrate, 100 nL of test compound in 100% DMSO, in a total of 10 uL of 20 mM KPO4, pH 6.0, 150 mM NaCl, 0.005% Tween20, 5 mM DTT in black low volume 384-well plates. The production of AMC (7-amino-4-methylcoumarin) was followed at 5 min intervals at 355 nm excitation, 460 nm emission in an Envision microplate reader (PerkinElmer). Reaction rates were determined by linear regression of the resulting progress curves. Rates were normalized to % inhibition, where 0% is equal to the rate in the presence of enzyme, and 100% is equal to the rate in the absence of enzyme. Nonlinear regression fits of the data to a one-site dose response curve were performed using XLFit (IDBS). Positive control, GC-376 (IC50=2.0 nM), was tested in parallel with the test compounds to ensure the accuracy of the assay. |
| Affinity data for this assay | |
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