Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture

Figures

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  Fig. 1. Prodrugs and their active triphosphate forms. Structures of the prodrugs Sofosbuvir (a), Remdesivir (b), Favipiravir (c), Tenofovir disoproxil (d), Molnupiravir (e), and AT-527 (f) (top) and their respective active triphosphate forms Sofosbuvir-5’-triphosphate, Remdesivir-5’-triphosphate, Favipiravir-ribofuranosyl-5’-triphosphate (Favipiravir-RTP), Tenofovir diphosphate, N4-hydroxycytidine-5’-triphosphate (NHC-TP), and 2’-fluoro-2’-methyl guanosine-5’-triphosphate (AT-9010, Gfm-TP) (bottom).

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  Fig. 2. 3D representation of the best docking poses for HCV NS5A inhibitors in the nsp14 exonuclease active site. The NS5A inhibitors were built and minimized in terms of energy by density functional theory (DFT). Docking was performed using GOLD 2020.2 software with ChemPLP as scoring function. Because of the diversity in NS5A inhibitors’ molecular weights, they were allowed to dock within 8 or 10 Å spheres in the SARS-CoV-2 nsp14 exonuclease active site, constructed in Supplementary Fig. S-2. The NS5A and catalytic amino acid residues are in stick representation. Mg++ and Zn++ are represented as green and indigo blue spheres, respectively.

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  Fig. 3. Inhibition of SARS-CoV-2 exonuclease activity by Pibrentasvir and Ombitasvir. A mixture of 500 nM RNA (sequence shown at the top of the figure) and 50 nM SARS-CoV-2 pre-assembled exonuclease complex (nsp14/nsp10) was incubated in buffer solution at 37 °C for 15 min in the absence (b, g) and presence of varying amounts of Pibrentasvir (c–e) and Ombitasvir (h, i). The RNA (a, f) and the products of the exonuclease reaction (b–e, g–i) were analyzed by MALDI-TOF MS. The signal intensity was normalized to the highest peak. The peak at 8160 Da corresponds to the intact RNA (8157 Da expected). In the absence of Pibrentasvir, exonuclease activity caused nucleotide cleavage from the 3′-end of the RNA as shown by the seven lower molecular weight fragments corresponding to cleavage of 1–7 nucleotides (b). Under our experimental conditions, ~7% intact RNA remained as indicated by the peak at 8167 Da (b). With increasing amounts of Pibrentasvir, exonuclease activity was reduced as shown by the reduced intensities of the fragmentation peaks and increased intact RNA peak (c–e). Similar results were observed with Ombitasvir (f–i).

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  Fig. 4. Inhibition of SARS-CoV-2 exonuclease activity by Pibrentasvir for Sofosbuvir (S) and Remdesivir (R) terminated RNA. A mixture of 500 nM RNAs (sequences shown at the top of the figure) and 50 nM SARS-CoV-2 pre-assembled exonuclease complex (nsp14/nsp10) were incubated in buffer solution at 37 °C for 15 min in the absence (b, e) and presence of 20 µM Pibrentasvir (c, f). The intact RNAs (a, d) and the products of the exonuclease reactions (b–f) were analyzed by MALDI-TOF MS. The signal intensity was normalized to the highest peak. In the absence of Pibrentasvir, exonuclease activity caused nucleotide cleavage from the 3′-end of the RNA as shown by the lower molecular weight fragments corresponding to cleavage of 1–7 nucleotides (b, e). When 20 µM Pibrentasvir was added, exonuclease activity was reduced as shown by the reduced intensities of the fragmentation peaks and increased intact RNA peaks (c, f).

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  Fig. 5. Inhibition of SARS-CoV-2 exonuclease activity by Pibrentasvir for natural RNA and RNA with delayed termination by Remdesivir (R). A mixture of 400 nM RNAs (sequences shown at the top of the figure) and 50 nM SARS-CoV-2 pre-assembled exonuclease complex (nsp14/nsp10) were incubated in buffer solution at 37 °C for 15 min in the absence (b, e) and presence of 20 µM Pibrentasvir (c, f). The intact RNAs (a, d) and the products of the exonuclease reactions (b, c, e, f) were analyzed by MALDI-TOF MS. The signal intensity was normalized to the highest peak. The peak at 9776 Da corresponds to the intact RNA (9773 Da expected) and the peak at 9812 Da corresponds to the intact Remdesivir delayed RNA termination product (9797 Da expected). The small peak at 10158 Da corresponds to mismatched incorporation of an additional G; this is likely due to the low fidelity of SARS-CoV-2 RdRp11. In the absence of Pibrentasvir, exonuclease activity caused nucleotide cleavage from the 3′-end of the RNA as shown by the lower molecular weight fragments corresponding to cleavage of 1–11 nucleotides (b, e). When 20 µM Pibrentasvir was added, exonuclease activity was reduced as shown by the reduced…

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  Fig. 6. Inhibition of SARS-CoV-2 exonuclease activity by Pibrentasvir for natural RNA and Tenofovir (Tfv) terminated RNA. A mixture of 500 nM RNAs (sequences shown at the top of the figure) and 50 nM SARS-CoV-2 pre-assembled exonuclease complex (nsp14/nsp10) were incubated in buffer solution at 37 °C for 15 min in the absence (b, e) and presence of 20 µM Pibrentasvir (c, f). The intact RNAs (a, d) and the products of the exonuclease reactions (b–f) were analyzed by MALDI-TOF MS. The signal intensity was normalized to the highest peak. In the absence of Pibrentasvir, exonuclease activity caused nucleotide cleavage from the 3′-end of the natural RNA as shown by the lower molecular weight fragments corresponding to cleavage of 1–8 nucleotides (b). However, for Tfv-terminated RNA, only minor cleavage was observed (e). When 20 µM Pibrentasvir was added, exonuclease activity was reduced as shown by the reduced intensities of the fragmentation peaks and the increased peak height of the intact RNA (c, f).

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  Fig. 7. Antiviral activity of combinations of SARS-CoV-2 polymerase and exonuclease inhibitors. Calu-3 cells, at a density of 5 × 105 cells/well in 48-well plates, were infected with SARS-CoV-2 at a MOI of 0.1, for 1 h at 37 °C. An inoculum was removed and cells were washed and incubated with fresh DMEM containing 10% FBS and the indicated concentration of Remdesivir (RDV) (a), Sofosbuvir (b), Tenofovir (c), and Favipiravir (d), alone and in combination with the HCV NS5A inhibitors. Supernatants were assessed after 48–72 h. Viral replication in the culture supernatant was measured as PFU/mL by titering in Vero E6 cells. Results are displayed as virus titers. The data represent means ± SEM of three independent experiments.

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  Fig. 8. Synergistic effects of combinations of RdRp and NS5A inhibitors in inhibiting SARS-CoV-2 replication. Calu-3 cells (5 x 105 cell/well) were infected with SARS-CoV-2 at a MOI of 0.1. After a 1 h of inoculation period, cells were treated with Remdesivir (RDV) (a), Sofosbuvir (SFV) (b), Tenofovir (TFV) (c), or Favipiravir (d) (FPV) with or without Pibrentasvir (PIB), Ombitasvir (OMB), or Daclatasvir (DCV); culture supernatant was harvested 2–3 days post-infection and titered in Vero E6 cells by PFU. Results were calculated as the percentage of inhibition and plotted online using SyngergyFinder2.0 (https://synergyfinder.fimm.fi/). Scores were determined after applying the highest single agent (HSA) algorithm. Scores are interpreted as follows: synergistic effect >10, additive effect <10 and > −10, antagonistic effect < −10.