Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2in Vitro

Abstract

Antivirals targeting SARS-CoV-2 could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PI) against SARS-CoV-2 and their interactions with remdesivir, the only antiviral approved for treatment of COVID-19. HCV PI showed differential potency in VeroE6 cell-based antiviral assays based on detection of the SARS-CoV-2 Spike protein. Linear PI boceprevir, telaprevir and narlaprevir had 50% effective concentrations (EC50) of ≈40 μM. Among macrocyclic PI simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir and deldeprevir, simeprevir had the highest (EC50 15 μM) and glecaprevir the lowest (EC50 >178 μM) potency. Acyclic PI asunaprevir and faldaprevir had EC50 of 72 and 23 μM, respectively. ACH-806, an HCV NS3 protease co-factor NS4A inhibitor, had EC50 of 46 μM. For selected PI, potency was similar in human hepatoma Huh7.5 cells. Selectivity indexes, based on antiviral and cell viability assays, were highest for linear PI. In combination with remdesivir, linear PI boceprevir and narlaprevir showed antagonism, while macrocyclic PI simeprevir, paritaprevir and grazoprevir showed synergism with drug reduction indexes of up to 27 for simeprevir. Treatment of infected cultures with equipotent concentrations (1-fold EC50) of HCV PI revealed minor differences in barrier to SARS-CoV-2 escape. Complete viral suppression was achieved treating with ≥3-fold EC50 boceprevir or combination of 1-fold EC50 simeprevir with 0.4-fold EC50 remdesivir, not leading to significant viral suppression in single treatments. Considering potency, human plasma concentrations and synergism with remdesivir, simeprevir seemed the most promising compound for optimization of future antiviral treatments of COVID-19.

Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system

“…We confirmed the high efficacy of nirmatrelvir against original SARS-CoV-2 in VeroE6 monkey kidney and A549-hACE2 human lung cells ( 14 , 15 ), recording median effective concentrations (EC 50 ) of 4.4 and 0.08 μM, respectively (fig. S1).…”

“…VeroE6 cells (gift from J. Dubuisson) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Paisley, UK) as described ( 15 , 26 , 27 ). A549-hACE2 cells (InvivoGen, Toulouse, France) were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (Gibco, Paisley, UK) as described ( 15 , 26 , 27 ).…”

“…VeroE6 cells (gift from J. Dubuisson) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Paisley, UK) as described ( 15 , 26 , 27 ). A549-hACE2 cells (InvivoGen, Toulouse, France) were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (Gibco, Paisley, UK) as described ( 15 , 26 , 27 ). Both culture media were supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml; Gibco/Invitrogen Corporation, Carlsbad, CA, USA).…”

“…To evaluate cytotoxic effects of the studied inhibitors, cell viability assays were done as described ( 15 , 26 , 27 ) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). VeroE6 or A549-hACE2 cells, plated the previous day at 10,000 cells per well in 96-well flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark), were treated with the specified concentrations of inhibitors, including three replicate wells per concentration and 10 nontreated control wells.…”

COVID-19: A need for new rather than repurposed antiviral drugs

“…Emetine, used for the treatment of amoebiasis, has been shown to inhibit MERS-CoV and SARS-CoV-2 in vitro 25,45 . The hepatitis C virus proteases grazoprevir and danoprevir have also shown anti-SARS-CoV-2 activity with EC 50 s below 100 μM 46 , while danoprevir is also investigated in four clinical trials. Information relevant for clinical use of these compounds are summarised in Supplementary Table 3.…”