CTSL Activity Assay Using Z-FR-AMC

A Cytation 5 (BioTek) plate-reader was used to analyze CTSL enzymatic activity against a dipeptide substrate Z-FR-AMC (R&D Systems ES009). CTSL for this experiment was purchased from BPS Biosciences as part of the Cathepsin L Inhibitor screening assay kit (Cat. # 79591). The kit buffer was substituted with a similar buffer: 100 mM MES-NaOH pH 5.5, 7.5 mM DTT, and 150 mM NaCl. 2-Fold serial dilutions of CQ were preincubated with CTSL for 25 min on ice. The final enzyme concentration was 0.016 ng/μL, the final substrate concentration was 5 μM, and the reaction was run at 25 °C for 30 min. Three technical replicates were run for each reaction, and the experiment was performed three times. AMC fluorescence (substrate cleavage) was monitored by excitation at 340 nm and emission at 445 nm (F₄₄₅). Proper controls were run for all components, as CQ fluorescence has potential to impact fluorescence spectra. Percent activity was calculated by comparing F₄₄₅ for DMSO treated CTSL to the varying CQ concentrations. IC₅₀ was calculated using Graphpad Prism analysis using log(inhibitor) vs response nonlinear regression analysis.