Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Purified SARS-CoV-2 RdRp proteins expressed in baculovirus-infected insect cells ( Spodoptera frugiperda , Sf) analysed by SDS–PAGE and Coomassie staining. 7H8: nsp7-His 6 -nsp8, nsp12-HF: nsp12-His 6 -3xFlag, nsp12-F/7H8: nsp12-3xFlag/nsp7-His 6 -nsp8, nsp12-HF/7L8: nsp12-His 6 -3xFlag/nsp7-GGSGGS-nsp8, nsp12-HF/7/8: nsp12-His 6 -3xFlag/nsp7/nsp8. ( B ) Bacterially expressed and purified SARS-CoV-2 nsp7, nsp8 and nsp12 proteins analysed by SDS–PAGE and Coomassie staining. The proteins were expressed as 14His-SUMO fusion proteins in E. coli . 14His-SUMO was removed by a SUMO-specific protease during purification generating native N-termini. ( C ) Gel-based primer-extension assay to test RNA-dependent RNA synthesis using the RdRp preparation Sf nsp12-F/7H8. The substrate consists of a 10 nt RNA primer annealed to the 3′ end of a 35 nt RNA template. The 5′ end of the template strand is labelled with a Cy3 fluorophore. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. Formation of duplex RNA by RdRp was observed over time. Controls: a preformed Cy3-labelled dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Schematic diagram illustrating the FRET-based RdRp strand displacement assay. The RNA substrate is composed of a Cy3 fluorophore-containing template strand, an annealed primer and an annealed quencher strand with a 5′ flap. RdRp activity synthesises RNA by extending the primer strand and displaces the quencher strand. The displaced quencher strand can no longer anneal to fully synthesised duplex RNA leading to an increase in Cy3 fluorescent signal. ( E ) FRET-based strand displacement assay using the indicated concentrations of Sf nsp12-F/7H8.

Article Snippet: The following proteins were expressed in E. coli (Ec): 14His-SUMO-nsp7 (Ec 7, vector: pK27Sumo_His-SUMO-nsp7, Addgene ID: 169186), 14His-SUMO-nsp8 (Ec 8, vector: pK27Sumo_His-SUMO-nsp8, Addgene ID: 169187) and 14His-SUMO-nsp12 (Ec 12, vector: pK27Sumo_His-SUMO-nsp12, Addgene ID: 169188).

Techniques: Purification, Infection, SDS Page, Staining, Primer Extension Assay, Clear Native PAGE, Fluorescence, Activity Assay

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: RdRp was mixed at the indicated concentrations with 100 nM RNA substrate and 300 µM of each NTP and Cy3 fluorescence was recorded. Enzyme preincubation was performed where indicated at 22°C for 30 min at the indicated ratios (5 µM nsp12). ( A ) Sf nsp12-F/7H8. ( B ) Sf nsp12-HF/7L8. ( C ) Sf nsp12-HF after preincubation with Sf 7H8 (1 : 3 ratio). ( D ) Ec nsp12 after preincubation with Ec nsp7 and Ec nsp8 (1 : 3 : 6 ratio). ( E ) Ec nsp12 after preincubation with Sf 7H8 (1 : 3 ratio). ( F ) Sf nsp12-HF after preincubation with Ec nsp7 and Ec nsp8 (1 : 3 : 6 ratio).

Article Snippet: The following proteins were expressed in E. coli (Ec): 14His-SUMO-nsp7 (Ec 7, vector: pK27Sumo_His-SUMO-nsp7, Addgene ID: 169186), 14His-SUMO-nsp8 (Ec 8, vector: pK27Sumo_His-SUMO-nsp8, Addgene ID: 169187) and 14His-SUMO-nsp12 (Ec 12, vector: pK27Sumo_His-SUMO-nsp12, Addgene ID: 169188).

Techniques: Fluorescence

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A – C ) Strand displacement assay using insect cell expressed (Sf) nsp12-HF/7L8 at the indicated concentrations alone ( A ) or after pre-incubating at 22°C for 30 min with Ec nsp7 and Ec nsp8 (ratio 1 : 3 : 6) ( B ) or with Sf 7H8 (ratio 1 : 6) ( C ). As control, a condition without NTPs using 200 nM RdRp was included. ( D ) Optimisation experiment to decide on RdRp concentration using HTS conditions (100 nM strand displacement substrate, 300 µM NTPs). Sf nsp12-HF/7L8 was preincubated at 22°C for 30 min with Sf 7H8 at either 1 : 3 or 1 : 5 ratio as specified and tested in the strand displacement assay at the indicated concentrations. Under these conditions, the unquenched Cy3 template alone had an average fluorescence value of 50 × 10 3 a.u. ( E ) Reaction velocities extracted from the curves shown in ( D ).

Article Snippet: The following proteins were expressed in E. coli (Ec): 14His-SUMO-nsp7 (Ec 7, vector: pK27Sumo_His-SUMO-nsp7, Addgene ID: 169186), 14His-SUMO-nsp8 (Ec 8, vector: pK27Sumo_His-SUMO-nsp8, Addgene ID: 169187) and 14His-SUMO-nsp12 (Ec 12, vector: pK27Sumo_His-SUMO-nsp12, Addgene ID: 169188).

Techniques: Concentration Assay, Fluorescence

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( 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 [ , ] 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. ). 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. . 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.

Article Snippet: The following proteins were expressed in E. coli (Ec): 14His-SUMO-nsp7 (Ec 7, vector: pK27Sumo_His-SUMO-nsp7, Addgene ID: 169186), 14His-SUMO-nsp8 (Ec 8, vector: pK27Sumo_His-SUMO-nsp8, Addgene ID: 169187) and 14His-SUMO-nsp12 (Ec 12, vector: pK27Sumo_His-SUMO-nsp12, Addgene ID: 169188).

Techniques: Activity Assay, Incubation, Concentration Assay, Selection, Inhibition, In Vitro

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Concentration–response curves of selected compounds using the strand displacement assay. The experiment was performed using 150 nM RdRp, 100 nM RNA substrate and 300 µM of each NTP in the presence (+Triton) or absence (−Triton) of 0.01% Triton X-100. Quenching controls are shown in Supplementary Figure S5B. IC 50 values were calculated using Prism software. ( B,C ) Native gel-based assays using a primed RNA substrate as in . Reactions were started by mixing 300 nM RdRp complex (formed by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 at 1 : 3 ratio) with 50 nM RNA substrate and 1 mM NTPs. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. ( B ) RdRp reactions were incubated for the indicated amounts of time. ( C ) Validation of selected compounds using 30-min RdRp reactions. RdRp complex was incubated with 50 µM of the indicated compounds for 10 min before reactions were started by substrate addition. Controls: a preformed dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Chemical structures of selected RdRp inhibitors.

Article Snippet: The following proteins were expressed in E. coli (Ec): 14His-SUMO-nsp7 (Ec 7, vector: pK27Sumo_His-SUMO-nsp7, Addgene ID: 169186), 14His-SUMO-nsp8 (Ec 8, vector: pK27Sumo_His-SUMO-nsp8, Addgene ID: 169187) and 14His-SUMO-nsp12 (Ec 12, vector: pK27Sumo_His-SUMO-nsp12, Addgene ID: 169188).

Techniques: Concentration Assay, Software, Clear Native PAGE, Fluorescence, Incubation