Figure 4. In vitro validation of selected compounds identified as nsp13 inhibitors.

(A–F) Concentration–response curves of selected compounds determined using the FRET-based nsp13 helicase assay. The results shown here are part of the larger experiment shown in Supplementary Figure S6 (for B–E) or S7 (for A, F). The experiment was performed in the presence (+T20) or absence (−T20) of 0.02% Tween-20. FPA-124 (A), Suramin (B) and NF 023 (C) showed little or no detergent sensitivity, whereas Navitoclax (D), Linoleic acid (E) and SSYA10-001 (F) showed an increase in IC₅₀ when Tween-20 was included. IC₅₀ values were calculated with Prism software. (G) Schematic diagram illustrating the gel-based nsp13 helicase assay. A nucleic acid substrate with a 5’ overhang is generated by annealing a Cy3-labelled oligonucleotide (Cy3 strand) with a longer unlabelled oligonucleotide. A 10-fold excess of competitor oligonucleotide is used to capture the longer strand after nsp13 unwinds the substrate. (H) Validation of selected compounds in a gel-based nsp13 helicase assay visualizing inhibition of duplex unwinding. Compounds were incubated with 2 nM FH-nsp13 at 15 µM compound concentration for 10 min before reactions were started by addition of 2 mM ATP and 50 nM RNA substrate. After 5 min reaction products were analysed by native PAGE and visualization of Cy3 fluorescence. Controls: RNA substrate (Substrate only), Cy3 strand only (Cy3 only), RNA substrate after 5 min incubation at 95°C (Boiled).