The level of ATP consumed by Nsp13 was quantified by measuring the amount of remaining ATP using a luciferase-based assay as previously described . The inhibitory effects of compounds were assessed in 384-well format (14 μL final volume) using reactions composed of 50 mM HEPES, pH 7.5, 5% Glycerol, 5 mM magnesium acetate, 5 mM DTT, 0.01% Triton X-100, 0.01% BSA, 0.1 nM Nsp13, 3.5 nM 30b PolyT ssDNA, 2.5 µM ATP, and 2% DMSO. Samples containing DMSO only (no compounds) were used as a control. Reactions were started by the addition of substrate and incubated for 60 min at room temperature. Then, 10 μL of the reactions were transferred into 384-well white plates containing 10 μL luciferase reagent (Cat# V6712; Promega, Madison, WI, USA) and incubated for another 20 min at room temperature. Compounds that were followed up for dose-response experiments were tested using the same luciferase reagent, and the data were analyzed using GraphPad Prism 9.