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. 2021 Jul 2;478(13):2425–2443. doi: 10.1042/BCJ20210200

Figure 4. High-throughput chemical library screen against SARS-CoV-2 RNA-dependent RNA polymerase.

Figure 4.

(A) Logistics of the screen. A custom chemical library consisting of over 5000 compounds was screened against RdRp activity using the FRET-based strand displacement assay in a 384-well format. RdRp was prepared by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 in a 1 : 3 ratio for 30 min at room temperature. RdRp was dispensed into compound-containing 384-well plates and incubated for 10 min. Reactions were started by the addition of a substrate mix and florescence monitored in 90 s intervals. (B,C) Results of the HTS screen performed at 1.25 µM (B) and 3.75 µM (C) compound concentration. The normalised reaction velocity plotted against the compound number is shown. Validated hits are shown in red. (D) Kinetic curves with >15% reduction in reaction velocity or >10% reduction in fluorescent signal at the endpoint were inspected manually. As example, kinetic data for compound GSK-650394 is shown (red curve, data from surrounding wells in black). (E) Summary of the HTS hit selection strategy. From over 5000 compounds tested in the screen, 64 were considered primary hits after manual inspection of HTS reactions, which showed a reduction in reaction velocity below 85%, or a reduction in endpoint signal below 90%. Out of these, 46 primary hits were eliminated as they likely represent nonspecific modes of enzymatic inhibition such as colloidal aggregation or interference with the substrate structure. As part of this analysis promiscuous compounds that were identified as hits in other SARS-CoV-2 HTS [46,48–52] were removed with the exception of five suramin and suramin-like compounds, which were also identified in the SARS-CoV-2 nsp13 helicase HTS (Zeng et al. [46]). In vitro validation of the effect of suramin and suramin-like compounds on the activity of SARS-CoV-2 helicase and SARS-CoV-2 RdRp can be found in Zeng et al. [46]. A total of 18 compounds (including five suramin and suramin-like compounds) were selected as hits, of which 14 were included in further in vitro validation in this work.