| Assay Method Information | |
| | JEV Protease Inhibition Assay |
| Description: | The enzyme inhibition assay was performed in 384-well flat bottom black polystyrene microplates (cat. N. 781900, Greiner,) in a reaction volume of 20 μl. Japanese encephalitis virus (JEV)-NS2B-NS3 protease (600 nM) was incubated with increasing inhibitor concentrations (0.097-100 M or 0.009-1 μM for the most active compounds) in assay buffer (50 mM Tris-HCl, pH 9.0, 0.1% BSA, 0.1% Triton X-100, 1% DMSO) for 10 minutes at 25° C. 10 μM of Pyr-RTKR-AMC substrate (Bachem, cat. N.4018149) was added and the reaction mixture incubated for additional 120 minutes at 25° C. Product formation was evaluated by fluorescence measurement (excitation 360 nm, emission 465 nM), using a SPARK TM10 Tecan instrument. Results were analyzed using Prism (GraphPad, San Diego, CA) and Vortex software (Dotmatics, Bioshops Stortford, UK). Dose-response curves were fitted by four-parameter logistic regression. |
| Affinity data for this assay | |
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