In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

Abstract

Background Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. Methods SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19. Results Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Conclusions Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

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

“…Besides remdesivir, several other clinically approved nucleoside analogue compounds are currently under study for treating COVID-19 [11]. Molnupiravir [12,13] and AT-527 [14] are the most promising candidates so far, while ritonavir [15], ribavirin [16], favipiravir [17] and sofosbuvir [6,18] have not demonstrated significant antiviral effect against SARS-CoV-2 in the laboratory or clinical settings.…”

Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics

“…We previously demonstrated that the FDA approved HCV NS5A inhibitors, Daclatasvir and Velpatasvir, and to a lesser extent the NS5A inhibitors Elbasvir and Ledipasvir, can inhibit the SARS-CoV-2 exonuclease 29 . Of particular interest, Daclatasvir and Velpatasvir inhibit both the SARS-CoV-2 polymerase and exonuclease 20,21 . Here, we showed that two additional NS5A inhibitors, Pibrentasvir and Ombitasvir, also inhibit the exonuclease, and have the highest inhibitory activity based on our molecular assay.…”

“…The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, shown to be required for the processive polymerase activity of nsp12, were cloned and purified as described 11,20…”

“…10 µL of 10 µM RNA template-loop-primers (5'-UUUUCAUCGCGUAGUUUUCUACGCG-3' for RDV-TP extension; 5'-UUUUCUACGCGUAGUUUUCUACGCG-3' for SOF-TP extension) in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8) 11,20 in 1 × reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. 10 µL of 10 µM RNA template-loop-primers (5'-UUUUUACAUCGCGUAGUUUUCUACGCG-3' for RDV-TP + UTP extension) in 1 × RdRp reaction buffer was first annealed by heating to 75 °C for 3 min and cooling to room temperature.…”

“…The SARS-CoV-2 genome is 3-fold larger than that of HCV, and the analogous NS5A functions are likely distributed among proteins nsp1 to nsp16 of the coronavirus 19 . We have previously shown that the NS5A inhibitors Daclatasvir and Velpatasvir inhibit both the SARS-CoV-2 polymerase and exonuclease activities 20,21 . The combination of Velpatasvir with Remdesivir was shown to have a synergistic effect relative to Remdesivir alone in inhibiting SARS-CoV-2 replication 22 .…”