A fluorescence-based high throughput-screening assay for the SARS-CoV RNA synthesis complex

Abstract

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.

Keywords: Coronavirus; Replication complex; Screening; Small molecule.

Graphical abstract

Fig. 1

Setting up of the SARS-CoV-RTC activity experimental conditions based on a fluorescent readout. (A)Poly(A) template variation with SARS nsp12 in complex with nsp7L8. Velocity values of SARS nsp12 (150 nM) in complex with nsp7L8 (1.5 μM) was measured for various concentrations in Poly (A) template (5; 10; 20; 40; 60; 80; 100; 150; 200; 350; 500 nM). Using the Prism software, velocity values of each condition were determined by calculating the slope of the linear phase of the kinetic and then plotted against the Poly(A) template concentration by using Michaelis-Menten fitting. Data were the results of three independent experiments. (B) Nucleotide variation with SARS nsp12 in complex with nsp7L8. Velocity values of SARS nsp12 (150 nM) in complex with nsp7L8 (1.5 μM) was measured for various concentrations in UTP (50;80;100;150;200;350;500 ;650;800 and 1000 nM). Using the Prism software, velocity values of each condition were determined by calculating the slope of the linear phase of the kinetic and then plotted against the UTP concentration by using Michaelis-Menten equation (full line) or Hill equation (dot line) to determine the apparent Km (UTP) of the SARS-CoV-RTC. Data were the results of three independent experiments. (C) Variation in SARS nsp12 in complex with nsp7L8. Velocity values obtained during a time course with 350 nM Poly (A) and 500 μM UTP were plotted against different concentrations in SARS nsp12 in complex with nsp7L8. The concentration ratio nsp12:nsp7L8 was conserved at 1:10. Curve was fitted according the Hill equation.

Fig. 2

SARS nsp12 in complex with nsp7L8 polymerase activity on a Picogreen kinetic assay. The polymerase activity of 100 nM DV2-NS5 pol (●), 150 nM nsp12 in complex with 1.5 μM nsp7L8 (▲), 150 nM nsp12 alone (Δ) or 1.5 μM nsp7L8 alone (□) were measured in a time course (0;2;5;7.5;10;20;30 and 60 min). The produced doubled strand RNA was detected by adding an intercalant reagent (Picogreen®) and by measuring the fluorescence emission at 530 nM. Each assay was performed three times (mean value ± SD).

Fig. 3

Quantification of SARS-CoV-RTC enzymatic activity inhibitors. Increasing concentrations of Hinokiflavone (▲), Amentoflavone (Δ), 3’dUTP (○), Quercetin (◼) and Apigenin (□) were incubated with 150 nM nsp12, 1.5μ M nsp7Lnsp8, 500 μM UTP, 350 nM Poly (A) at 30 °C during 20 min. The Inhibitory concentrations 50 (IC₅₀s) were then calculated using graphPad Prism equation (Experiments were done twice in triplicate; Mean value ± SD).

Fig. 4

Number of compounds from PCL on the screening of SARS-CoV RTC, according to their inhibitory potential. Based on their efficiency on the SARS-CoV RTC assay, the number of compounds with more of 30 %; 40 %; 50 %; 60 %; 70 %; 80 %; 90 % and 100 % inhibition were evaluated. The exact value was indicated in white above each bar graph. (Only compounds with more 30 % inhibition of the polymerase activity in the assay were represented. Frequent Hitter and fluorescent compounds were excluded). The Z’ value was calculated based on the ten control wells on each microplate, resulting in an overall Z’ score of 0,8 ± 0,06.

Fig. 5

Quantification of SARS-CoV-RTC enzymatic activity inhibitors belonging to the anthracycline and tetracyclin chemical families. (A) Anthracyclin chemical family. Increasing concentrations of Prestw-385 (▲), Prestw-1752 (◼), Prestw-1224 (□), Prestw-438 (●) and Prestw-487 (○) were used to determine the IC₅₀s of each compound. Hinokiflavone (Δ) was indicated as a control. (B) Tetracycline chemical family. Increasing concentrations of Prestw-456 (♦), Prestw-1799 (◊), Prestw-1000 (_*), Prestw-145 (X) and Prestw-140 () were used to determine the IC₅₀s of each compound. Compounds were incubated with 150 nM nsp12, 1.5 μM nsp7Lnsp8, 500 μM UTP, 350 nM Poly (A) at 30 °C during 20 min. The IC_50s were then calculated using graphPad Prism equation (Experiment were done twice in triplicate; Mean value ± SD). IC₅₀: concentration for 50 % inhibition. The IC₅₀ of Prestw-140 was approximate.

Fig. A1

Complete amino acid sequence of nsp7L8. The fusion protein nsp7-nsp8 was generated by inserting a GSGSGS linker sequence, underlined, between the nsp7 and nsp8 coding sequences, and is named nsp7L8.

Fig. A2

Characterization of the purified recombinant SARS nsp12 and nsp7L8 proteins. 5 μg of SARS nsp12 and 5 μg of nsp7L8 were loaded on a 10 % and 12 % SDS-PAGE gel respectively, stained by Coomassie blue. (MW, molecular size markers).