key: cord-0972992-f6yvolv3
authors: Gammeltoft, Karen A.; Zhou, Yuyong; Duarte Hernandez, Carlos R.; Galli, Andrea; Offersgaard, Anna; Costa, Rui; Pham, Long V.; Fahnøe, Ulrik; Feng, Shan; Scheel, Troels K. H.; Ramirez, Santseharay; Bukh, Jens; Gottwein, Judith M.
title: Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 In Vitro
date: 2021-08-17
journal: Antimicrobial agents and chemotherapy
DOI: 10.1128/aac.02680-20
sha: ff9f0f3261fafaf2cac56650f9098985f6a69ffd
doc_id: 972992
cord_uid: f6yvolv3

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC(50)) of ∼40 μM. Among the macrocyclic PIs, simeprevir had the highest (EC(50), 15 μM) and glecaprevir the lowest (EC(50), >178 μM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC(50)s of 72 and 23 μM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC(50) of 46 μM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC(50) PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC(50) boceprevir or 1-fold EC(50) simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC(50) remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

ABSTRACT Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC 50 ) of ;40mM. Among the macrocyclic PIs, simeprevir had the highest (EC 50 , 15mM) and glecaprevir the lowest (EC 50 , .178mM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had EC 50 s of 72 and 23mM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC 50 of 46mM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC 50 PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3-to 8-fold EC 50 boceprevir or 1-fold EC 50 simeprevir or grazoprevir, but not boceprevir, in combination with 0.4-to 0.8-fold EC 50 remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.

KEYWORDS coronavirus, antiviral, repurposing, combination treatment, synergy, S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus of the Coronaviridae family, which emerged in humans in 2019 most likely originating from a bat-borne virus (1) (2) (3) . SARS-CoV-2 causes coronavirus disease 2019 (COVID- 19) , a multisystemic disease with initial symptoms mostly localizing to the respiratory tract. At the end of April 2021, the COVID-19 pandemic had been responsible for .152 million infected, .3 million deaths, and an unknown number of individuals suffering from long-term health effects (4) (5) (6) (7) (8) .

Repurposing of drugs approved for other medical indications is promoted as a time-saving approach to the identification of urgently needed treatments. At present,

Differential potency of clinically relevant HCV protease and cofactor inhibitors against SARS-CoV-2 in vitro. To determine the potency of a panel of HCV PIs and an HCV NS4A inhibitor against SARS-CoV-2, we developed a cell-based short-term antiviral treatment assay in 96-well plates, adapting an assay previously developed to determine potency of HCV PIs against HCV (29, (37) (38) (39) (40) (41) (42) (43) . In this assay, replicate SARS-CoV-2infected cultures were treated with different dilutions of inhibitors. Following incubation, cultures were subjected to immunostaining for SARS-CoV-2 spike protein and automated counting of single spike protein-positive cells (31, 44) . Multiplicity of infection (MOI) and incubation time were chosen to avoid virus-induced cytopathogenic effects (CPEs) during the assay and to result in .1,000 single spike protein-positive cells in nontreated control cultures, as determined in pilot CPE and immunostaining assays (for details, see Materials and Methods). The potency of the inhibitors to reduce the number of spike protein-expressing cells compared to that of the nontreated controls was evaluated. Furthermore, cytotoxicity of inhibitors was determined by cell viability assays; in these assays, cell viability of .90% was confirmed for inhibitor concentrations used in treatment assays.

First, concentration-response studies were carried out in Vero E6 cells. Tested inhibitors were able to inhibit the virus with EC 50 values in the micromolar range, with exception of glecaprevir, voxilaprevir, and deldeprevir, where EC 50 values were not determined due to cytotoxicity of the drugs or antiviral activity of the diluent dimethyl sulfoxide (DMSO) at high drug concentrations ( Fig. 1 and Table 1 ; see also Fig. S2 ). The linear PIs boceprevir, telaprevir, and narlaprevir showed comparable potencies, with EC 50 values of ;40 mM. Among the macrocyclic PIs, simeprevir showed the highest potency, with an EC 50 of 15 mM. Furthermore, paritaprevir had an EC 50 of 22 mM, while grazoprevir and vaniprevir had EC 50 values of 42 and 51 mM, respectively. Finally, the EC 50 was 87 mM for danoprevir. For the acyclic PIs, faldaprevir (EC 50 , 23mM) was more potent than asunaprevir (EC 50 , 72mM).

To validate the immunostaining-based treatment assay, concentration-response assays quantifying CPE were carried out in Vero E6 cells for selected PIs. In this assay, replicate SARS-CoV-2-infected cultures were treated with different dilutions of inhibitors. MOI and incubation time were chosen to induce relatively strong virus-induced CPEs in nontreated control cultures upon termination of the assay (30% to 50% cell viability). In treated cultures, the potency of the inhibitors to inhibit virus-induced CPE compared to that in the nontreated control cultures was evaluated. In these assays, the tested PIs, boceprevir, simeprevir, grazoprevir, glecaprevir, and voxilaprevir, showed similar potency and EC 50 values as in the immunostaining-based assays (see Fig. S3 ).

To confirm the potency of PIs in human cells, selected PIs were studied in human Huh7.5 hepatoma cells. In these assays, the tested PIs, boceprevir, simeprevir, and grazoprevir, had similar concentration-response curves and EC 50 values as in Vero E6 cells (Fig. 2 , Table 1 ).

Finally, we confirmed differential potency of selected PIs in human A549 lung carcinoma cells transduced to express the SARS-CoV-2 entry receptor hACE2. In these A549-hACE2 cells, inhibitors showed slightly increased potency. Thus, boceprevir, simeprevir, and grazoprevir showed an ;2-fold decreased EC 50 compared to the EC 50 in Vero E6 cells. In line with this observation and in contrast to that in Vero E6 cells, the EC 50 value for voxilaprevir was determined in A549-hACE2 cells (10 mM) . Similarly to Vero E6 cells, no EC 50 was determined for glecaprevir in A549-hACE2 cells due to antiviral activity of DMSO at high inhibitor concentrations (Fig. 3) .

All inhibitors were diluted in DMSO. At the DMSO dilutions used for generation of specific data, no antiviral effect was observed in Vero E6, Huh7.5, or A549-hACE2 cells ( Fig. 1 to 3 and Fig. S2 ).

Cell viability assays were carried out for all studied drugs to determine their level of in vitro cytotoxicity and CC 50 values. In these assays, drug concentrations were used at FIG 1 Potency of a panel of HCV PIs and an HCV NS4A inhibitor against SARS-CoV-2 in Vero E6 cells. Vero E6 cells were seeded in 96-well plates and, the following day, infected with SARS-CoV-2 at an MOI of 0.002 followed by treatment with specified concentrations of the PIs boceprevir, telaprevir, narlaprevir, simeprevir, paritaprevir, grazoprevir, glecaprevir, voxilaprevir, vaniprevir, danoprevir, deldeprevir, asunaprevir, and faldaprevir as well as HCV NS4A inhibitor ACH-806, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. Data points (red dots) are means of counts from 7 replicate cultures 6 standard errors of the means (SEMs) and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration-response curves (red lines) were fitted and EC 50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay, as described in Materials and Methods. Data points (blue triangles) are means from 3 replicate cultures 6 SEMs and represent percent cell viability relative to mean absorbance from 12 replicate nontreated control cultures. Sigmoidal concentration-response curves were fitted and CC 50 values were determined as shown (Continued on next page) which no DMSO-induced cytotoxicity was observed ( Fig. 1 to 3 Based on these assays, in Vero E6 cells, the linear inhibitors had the highest selectivity indexes (SI = CC 50 /EC 50 ), with SIs of .28 for boceprevir, .11 for telaprevir, and 7.3 for narlaprevir (Table 1) . Of the macrocyclic inhibitors, paritaprevir and grazoprevir had the highest SIs (5.6 and 5.7, respectively), while simeprevir and vaniprevir had slightly lower SIs of 3.9 and 3.4, respectively. For glecaprevir, voxilaprevir, danoprevir, and deldeprevir, SIs could not be determined. For the acyclic inhibitors, SI values were 6.3 for faldaprevir and 3.7 for asunaprevir. Finally, SI values calculated based on assays in Huh7.5 and in A549-hACE2 cells were comparable to those based on assays in Vero E6 cells (Table 1) .

For the HCV NS4A inhibitor ACH-806, in Vero E6 cells, the EC 50 was 46 mM, CC 50 was .429 mM, and SI was .9.3 ( Fig. 1, Table 1 , and Fig. S4) .

HCV PIs showed differential interactions with remdesivir in short-term treatment assays. To study interactions between selected PIs and remdesivir, 96-well-based short-term immunostaining-based drug interaction assays were carried out. SARS-CoV-2-infected Vero E6 or A549-hACE2 cell cultures were treated with selected PIs singly, with remdesivir singly, or with a combination of PIs and remdesivir. Inhibitor dilution series were chosen based on determined EC 50 values (Table 1 ; Fig. S7 ). Inhibitors were only used at concentrations where no cytotoxicity or antiviral effects of DMSO were observed (see Fig. S2 , S8, and S9). For each inhibitor pair to be evaluated, 7 to 10 FIG 2 Potency of selected HCV PIs against SARS-CoV-2 was confirmed in Huh7.5 cells. Huh7.5 cells were seeded in 96-well plates and, the following day, infected with SARS-CoV-2 at an MOI of 0.02 followed by treatment with specified concentrations of the PIs boceprevir, simeprevir, and grazoprevir, as described in Materials and Methods. After 70 to 74 h of incubation, SARS-CoV-2infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. Data points (red dots) are means from 7 replicates 6 SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration-response curves (red lines) were fitted and EC 50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means from 3 replicate cultures 6 SEMs and represent percent cell viability relative to mean absorbance from 12 nontreated controls. Sigmoidal concentration-response curves were fitted and CC 50 values were determined, as shown in Fig. S5 . The blue dotted line represents the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to ,90%, according to Fig. S2 ; DMSO did not induce antiviral effects in the tested concentration ranges (Fig. S2 ). treatment conditions were chosen. Each treatment condition was defined by a given concentration of PI applied singly, a given concentration of remdesivir applied singly, and a combination of these same concentrations of a PI and remdesivir (see Table S2 ).

For each treatment condition, the residual infectivity of the culture receiving combination treatment was compared to the residual infectivity of the cultures receiving the FIG 3 Potency of selected HCV PIs against SARS-CoV-2 was confirmed in A549-hACE2 cells. A549-hACE2 cells were seeded in 96-well plates and, the following day, infected with SARS-CoV-2 at an MOI of 0.003 followed by treatment with specified concentrations of the PIs boceprevir, simeprevir, grazoprevir, glecaprevir, and voxilaprevir, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2-infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. Data points (red dots) are means from 7 replicates 6 SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from 14 replicate infected nontreated control cultures. Sigmoidal concentration-response curves (red lines) were fitted and EC 50 values were determined, as described in Materials and Methods. Cell viability data were obtained in replicate assays with noninfected cells using a colorimetric assay as described in Materials and Methods. Data points (blue triangles) are means from 3 replicate cultures 6 SEMs and represent percent cell viability relative to mean absorbance from 12 nontreated controls. Sigmoidal concentration-response curves were fitted and CC 50 values were determined, as shown in Fig. S6 . The red dotted lines represent the drug concentrations at which DMSO is expected to induce antiviral effects with reduction of residual infectivity to ,70%, according to Fig. S2 . The blue dotted lines represent the drug concentrations at which DMSO is expected to induce cytotoxicity with reduction of cell viability to ,90%, according to Fig. S2 .

Antimicrobial Agents and Chemotherapy corresponding single treatments. A graphical representation of the data is shown in Fig. 4 . All treatment conditions are detailed and the corresponding residual infectivity values are shown in Table S2 . For the linear inhibitors boceprevir and narlaprevir, under almost all treatment conditions, the effect of the combination did not exceed the effect of the most efficient single inhibitor, remdesivir (Fig. 4) . For example, under treatment condition 3, 17.4 mM boceprevir resulted in 79% residual infectivity and 1.7 mM remdesivir resulted in 47% residual infectivity. Combination treatment with 17.4 mM boceprevir and 1.7 mM remdesivir resulted in 74% residual infectivity. Thus, treatment with remdesivir alone proved equally or more efficient than a combination of a linear PI with remdesivir.

In contrast, for the macrocyclic inhibitors simeprevir, paritaprevir, and grazoprevir, under various treatment conditions, the effect of the combination exceeded the effect of both single inhibitors (Fig. 4) . For example, under treatment condition 3, 11.1 mM simeprevir resulted in 100% residual infectivity and 0.6mM remdesivir resulted in 83% residual infectivity. However, combination treatment with 11.1mM simeprevir and 0.6mM remdesivir resulted in 1% residual infectivity. Thus, treatment with the combination showed an added effect compared to that with the corresponding single drug treatments.

We confirmed the above-described differential interactions of linear versus macrocyclic PIs with remdesivir in human A549-hACE2 cells by carrying out similar assays with the selected PIs boceprevir, simeprevir, and grazoprevir ( Fig. 5 and Table S2 ).

To further define the nature of the interactions between the tested PIs and remdesivir, the above-described data sets were analyzed using the method of Chou and Talalay using CompuSyn software (45, 46) . This analysis revealed primarily antagonistic interactions between remdesivir and the linear PIs boceprevir and narlaprevir. In contrast, primarily synergistic interactions were observed between remdesivir and the macrocyclic PIs paritaprevir and grazoprevir (see supplemental text, Fig. S10 and S11, and Table S3 and S4).

CompuSyn software provided a suboptimal fit for the simeprevir plus remdesivir data sets, most probably due to the steep slope of the simeprevir concentrationresponse curve. Therefore, an alternative software, SynergyFinder 2.0, was applied to analyze alternative data sets generated as required for this analysis and as described in the supplemental text. This analysis showed overall synergistic interactions between simeprevir and remdesivir in Vero E6 and A549-hACE2 cells (supplemental text and Fig.  S12 and S13).

HCV PIs showed small differences in the barrier to escape of SARS-CoV-2. To investigate their barriers to escape, all PIs for which an EC 50 was determined were used for longer-term treatment of SARS-CoV-2-infected Vero E6 cells in culture flasks at the highest possible equipotent concentration (1-fold EC 50 ) according to predicted cytotoxicity ( Fig. 1 , Table 1 , and Fig. S4 ). In the nontreated control cultures, the infection spread to 50% of culture cells on day 1 and to 90% of culture cells on day 3 postinfection, as estimated by immunostaining for the SARS-CoV-2 spike protein (Fig. 6 ). Following day 3, typically massive virus-induced cell death was observed in these control cultures. For all PI-treated cultures (Fig. 6) , initial viral suppression was observed with 10% to 30% of infected culture cells on day 1 postinfection and treatment initiation. On day 3, only cultures treated with narlaprevir, grazoprevir, vaniprevir, asunaprevir, and faldaprevir showed viral suppression, with infections of 10% to 50% of culture cells, while in cultures treated with boceprevir, telaprevir, simeprevir, paritaprevir, and danoprevir, 90% of culture cells were infected. On day 5, virus spread to 90% of culture cells in grazoprevir-treated cultures, while cultures treated with vaniprevir and asunaprevir were closed due to massive cell death, assumed to be due to PI-induced cytotoxicity, possibly enhanced by SARS-CoV-2 infection. On day 7, in narlaprevir-and faldaprevir-treated cultures, 60% of culture cells were infected; these cultures were closed on day 9 due to massive cell death. Viral spread kinetics monitored by immunostaining were confirmed by the determination of SARS-CoV-2 RNA titers in cell culture supernatants using a reverse transcription-quantitative PCR (RT-qPCR) assay (Fig. 6 ). In the nontreated control culture, the number of SARS-CoV-2 genome copies increased by 4 orders of magnitude from 5.5 Â 10 6 /ml on day 1 to 9.7 Â 10 10 /ml on day 3 postinfection. Compared to these values, all treatments resulted in a small decrease in genome copies on days 1 and 3; however, none of the treatments was able to prevent viral spread as monitored by determination of viral RNA titers. Vero E6 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 at an MOI of 0.002 followed by treatment with serial dilutions of the linear PI boceprevir (BOC) or narlaprevir (NAR), the macrocyclic PI simeprevir (SIM), paritaprevir (PAR), or grazoprevir (GRA), polymerase inhibitor remdesivir (REM), or a combination of these PIs and remdesivir, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2-infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. For each inhibitor pair to be evaluated, 7 to 10 treatment conditions were used (indicated on x axes). Each treatment condition was defined by a given concentration of PI applied singly, a given concentration of remdesivir applied singly, and a combination of these same concentrations of PIs and remdesivir, as specified in Table S2 , resulting in 3 data points per treatment condition. Data points are means from 6 or 7 replicates 6 SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from infected nontreated control cultures. Sigmoidal concentration-response curves were fitted as described in Materials and Methods. The tested inhibitor concentrations did not impact cell viability (Fig. S8) . DMSO did not induce antiviral effects in the tested concentration ranges (Fig. S2 ).

To confirm the PI barrier to escape in human cells, similar longer-term treatments were carried out in A549-hACE2 cells. Compared to the nontreated culture with SARS-CoV-2 RNA titers of 3.4 Â 10 10 and 2.5 Â 10 10 genome copies/ml on days 3 and 5, respectively, treatment with 1-fold EC 50 of boceprevir, simeprevir, and grazoprevir had no to little effect on viral spread as monitored by determination of viral RNA titers (Fig.  7, left) . While A549-hACE2 cultures were also followed by immunostaining, in contrast to that for Vero E6 cells, percent infection was difficult to estimate following day 1 postinfection, when a higher percentage of culture cells had become infected.

In conclusion, 1-fold EC 50 of the tested PIs did not suppress SARS-CoV-2 in vitro. Boceprevir had the potential to completely suppress viral infection in vitro. As suboptimal viral suppression was observed under treatment with 1-fold EC 50 , we chose the PI with the highest SI to enable longer-term treatment at higher-fold EC 50 concentrations. Vero E6 cells infected with SARS-CoV-2 were treated with 1-, 1.5-, 2-, 2.5, 3-, and 5-fold EC 50 of boceprevir (Fig. 8) . Treatment with 1-and 1.5-fold EC 50 boceprevir only had a minor impact on viral spread on day 1 postinfection and treatment initiation, while 90% of culture cells became infected on day 3, as observed for nontreated control cells. Also, in cultures treated with 2-and 2.5-fold EC 50 , 90% of culture cells became infected on day 5. In contrast, treatment with 3-and 5-fold EC 50 resulted in sustained viral suppression with no evidence of infected cells in the culture treated with 3-fold EC 50 from day 3 and in the culture treated with 5-fold EC 50 from day 1 during follow-up periods of 9 and 17 days, respectively. In addition, from cultures treated FIG 5 Analysis of interactions of selected HCV PIs with remdesivir in A549-hACE2 cells. A549-hACE2 cells seeded in 96-well plates were infected the following day with SARS-CoV-2 at an MOI of 0.003 followed by treatment with serial dilutions of the linear PI boceprevir (BOC), the macrocyclic PI simeprevir (SIM) or grazoprevir (GRA), polymerase inhibitor remdesivir (REM), or a combination of these PIs and remdesivir, as described in Materials and Methods. After 46 to 50 h of incubation, SARS-CoV-2-infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein and quantified by automated counting, as described in Materials and Methods. For each inhibitor pair to be evaluated, 8 to 10 treatment conditions were used (indicated on x axes). Each treatment condition was defined by a given concentration of PI applied singly, a given concentration of remdesivir applied singly, and a combination of these same concentrations of PIs and remdesivir, as specified in Table S2 , resulting in 3 data points per treatment condition. Data points are means from 7 replicates 6 SEMs and represent percent residual infectivity, determined as percent SARS-CoV-2-positive cells relative to means of counts from infected nontreated control cultures. Sigmoidal concentrationresponse curves were fitted as described in Materials and Methods. DMSO was kept constant in all cultures. The tested inhibitor concentrations did not impair cell viability (Fig. S8) . DMSO did not induce antiviral effects in the tested concentration ranges (Fig. S2) . Antimicrobial Agents and Chemotherapy with 3-and 5-fold EC 50 on day 5 and day 3, respectively, replicate cultures receiving no treatment going forward were derived, which did not show any infected cells during a follow-up period of 10 days, suggesting that the infection was cured under these treatments. Viral spread kinetics monitored by immunostainings were confirmed by the determination of SARS-CoV-2 RNA titers in cell culture supernatants (Fig. 8) . In cultures treated with 1-to 2.5fold EC 50 boceprevir, viral RNA titers increased during the experiment; however, compared to FIG 6 Legend (Continued) dotted lines were inserted to highlight day 1 and 3 values of the nontreated culture, respectively. (Right) Replicate cultures were derived following cell splitting and treatment, immunostained for the SARS-CoV-2 spike protein (green) and counterstained with Hoechst dye (blue), and images were acquired, as described in Materials and Methods. Cultures summarized in this figure are derived from different experimental setups, each including an infected nontreated control culture, which showed viral spread comparable to that in the depicted representative culture. *, culture was terminated, or infection data not recorded, due to virus-induced cell death; **, culture was terminated due to druginduced cytotoxicity, possibly enhanced by viral infection. the nontreated control culture, this increase was decelerated in a concentration-dependent manner, and overall peak titers were lower. In contrast, in cultures treated with 3-and 5-fold EC 50 boceprevir, viral RNA titers decreased to values around the lower limit of quantification (LLOQ) during the experiment (Fig. 8) . In follow-up derived replicate cultures receiving no treatment, viral RNA titers were around the LLOQ.

To further confirm elimination of SARS-CoV-2 from cultures treated with 3-and 5fold EC 50 boceprevir, we carried out a 96-well based infectivity assay monitoring presence of infectious virus in all cell culture supernatants from the derived replicate cultures by inoculation of Vero E6 cell indicator cultures. Derived replicate cultures were chosen for this analysis, as they were not treated with antivirals, which might inhibit To investigate the potential of boceprevir to suppress SARS-CoV-2 infection in human cells, SARS-CoV-2-infected A549-hACE2 cells were treated with 1-, 2-, 3-, 5-, and 8-fold EC 50 boceprevir (Fig. 7, middle) . Virus RNA titrations revealed that 1-to 5-fold EC 50 boceprevir only had a minor impact on viral spread, while 8-fold EC 50 boceprevir resulted in viral suppression, with viral titers decreasing to values around the LLOQ during an observation period of 13 days. In addition, from the culture treated with 8fold EC 50 on day 7, a replicate culture receiving no treatment was derived, from which viral RNA titers were around the LLOQ during a period of 7 days.

Thus, 3-to 8-fold EC 50 boceprevir was able to suppress SARS-CoV-2 in vitro.

Simeprevir or grazoprevir in combination with remdesivir completely suppressed viral infection in vitro. To further study and confirm the interactions of PIs with remdesivir, three PIs with apparent differential interactions with remdesivir were selected for longer-term treatment of SARS-CoV-2-infected cells. Vero E6 cells infected with SARS-CoV-2 were treated with the PIs boceprevir, simeprevir, or grazoprevir singly, remdesivir singly, or either PI in combination with remdesivir, using 1-fold EC 50 PI and 0.4-fold EC 50 remdesivir. Inhibitor concentrations were selected to confer suboptimal effects in single treatments in order to rule out viral suppression in these treatments. Equipotent concentrations of PIs were used based on data shown in Fig. 1 and 6 and Table 1 . For remdesivir, potency was evaluated based on concentration-response curves obtained from data shown in Fig. 4 and Fig. S7 and, in addition, based on pilot longer-term treatment assays. Treatment with remdesivir, boceprevir, simeprevir, or grazoprevir singly, as well as treatment with boceprevir plus remdesivir, had no or only a minor impact on viral spread on day 1 postinfection and treatment initiation, while 70% to 90% of culture cells became infected on day 3, as observed for nontreated control cells (Fig. 9 ). In contrast, in cultures treated with simeprevir plus remdesivir or grazoprevir plus remdesivir, complete and sustained viral suppression was achieved with no evidence of infection from day 1 during a follow-up period of 15 days. In addition, to confirm complete viral suppression, from these cultures, replicate cultures receiving no treatment were derived on day 5. Replicate cultures derived from cultures treated with simeprevir plus remdesivir and with grazoprevir plus remdesivir did not show any infected cells during a follow-up period of 19 and 14 days, respectively.

Determination of SARS-CoV-2 RNA titers in cell culture supernatants confirmed viral spread kinetics monitored by immunostainings (Fig. 9) . Treatment with single inhibitors and boceprevir plus remdesivir had no or a minor impact on RNA titers compared to that in the nontreated control culture (Fig. 9 ). However, under treatment with simeprevir plus remdesivir or with grazoprevir plus remdesivir, RNA titers decreased to values around the LLOQ during the experiment. In derived replicate cultures receiving no treatment, viral RNA titers were around the LLOQ.

Elimination of SARS-CoV-2 from cultures treated with simeprevir plus remdesivir was confirmed by an infectivity assay monitoring the presence of infectious virus in all cell culture supernatants from the derived replicate cultures not receiving treatment.

To verify interactions of boceprevir, simeprevir, or grazoprevir with remdesivir in human cells, SARS-CoV-2-infected A549-hACE2 cells were treated with 1-fold EC 50 boceprevir, simeprevir, or grazoprevir singly, 0.8-fold remdesivir singly, or either PI in 

Antimicrobial Agents and Chemotherapy combination with remdesivir (Fig. 7, right) . Virus RNA titrations revealed that treatment with single inhibitors or boceprevir plus remdesivir only had a minor impact on viral spread, while treatment with simeprevir plus remdesivir or grazoprevir plus remdesivir resulted in viral suppression with viral RNA titers around the LLOQ. Thus, combination of simeprevir or grazoprevir with remdesivir was able to suppress SARS-CoV-2 in vitro.

In the present study, we provided a head-to-head comparison of the efficacy of a panel of clinically relevant HCV PIs, including all PIs approved for the treatment of chronic hepatitis C, against SARS-CoV-2 in cell-based assays. In short-term antiviral assays, PIs showed differential potencies, with EC 50 values between 15 mM (simeprevir) and .178 mM (glecaprevir) in Vero E6 cells and 9 mM (simeprevir) and .94 mM (glecaprevir) in A549-hACE2 cells. Detailed short-term synergy studies in both cell types using a PI subpanel revealed PI structure-dependent interactions with remdesivir, with linear inhibitors showing mostly antagonism and macrocyclic inhibitors showing mostly synergism. In longer-term Vero E6 and A549-hACE2 cell culture assays, at relatively low equipotent concentrations, PIs showed small differences regarding the barrier to viral escape. For boceprevir, a relatively high SI facilitated treatment with higher concentrations, revealing its potential to completely suppress viral infection. Furthermore, combination of simeprevir or grazoprevir with remdesivir suppressed viral infection at relatively low inhibitor concentrations, shown to be subtherapeutic in single treatments.

Even though cell lines do not entirely recapitulate in vivo conditions, they provide robust models for preclinical antiviral activity studies. Our findings are strengthened by the fact that similar results were obtained in three different cell lines, including two human cell lines, of which one was derived from human lung. Future studies to evaluate the efficacy of HCV PIs on SARS-CoV-2 in primary cells, organoids, animals, or humans would be of interest.

The sequence homology between different SARS-CoV-2 isolates is high. In comparison to the Wuhan reference isolate (GenBank accession number NC_045512), the isolate used in this study harbored six consensus amino acid changes (T85I in nsp2, P323L in nsp12, E309K and D614G in S, Q57H in ORF3a, and R209I in N); however, there were no changes in M pro or PL pro , the proposed main targets of the studied HCV PI (31) . EC 50 s against SARS-CoV-2 were in the micromolar range, with the lowest EC 50 s (9 and 15 mM for simeprevir in A549-hACE2 and Vero E6 cells, respectively) approaching the EC 50 of remdesivir (0.1 and 2.5 mM in A549-hACE2 and Vero E6 cells, respectively); EC 50 s of remdesivir were in line with previously reported results (30-32, 35, 36) . However, EC 50 s of PIs against SARS-CoV-2 were higher than the EC 50 s against HCV. Initially developed HCV PIs such as boceprevir and simeprevir were roughly 10-to 1,000-fold less potent, while optimized HCV PIs such as grazoprevir, glecaprevir, and voxilaprevir were roughly 1,000-to 100,000-fold less potent against SARS-CoV-2 than against different HCV isolates (29, (38) (39) (40) (41) (42) (43) (47) (48) (49) . This suggested that optimization of inhibition of the HCV protease counteracted broader activity against proteases of other RNA viruses, such as SARS-CoV-2.

Boceprevir showed the highest SIs (.61 and .28 in A549-hACE2 and Vero E6 cells, respectively), while simeprevir showed the lowest SIs (6.2 and 3.9 in A549-hACE2 and Vero E6 cells, respectively). Of note, some clinically relevant drugs such as digoxin have low therapeutic breadth, with SIs as low as 2 (50) , and HCV PIs have been proven safe in clinical practice. To estimate the clinical potential of inhibitors, comparisons of their EC 50 with clinically achievable plasma and tissue concentrations are more relevant than comparison with in vitro CC 50 values. For most HCV PIs, peak plasma concentrations (C max ) were significantly lower than the determined EC 50 values (see Table S5 in the supplemental material). The most favorable C max /EC 50 ratio was found for simeprevir (C max /EC 50 of ;1), followed by faldaprevir (C max /EC 50 of ;0.2) as well as boceprevir, telaprevir, and vaniprevir (C max /EC 50 of ;0.1) based on the EC 50 s obtained in Vero E6 cells. Furthermore, plasma concentrations of free and biologically active compounds are expected to be lower than reported C max values due to the high plasma protein binding of HCV PIs. Regarding tissue concentrations, for HCV PIs, mostly liver concentrations were reported and were 20-to 280-fold higher than plasma concentrations (43, (51) (52) (53) . In rats, following a single oral administration of simeprevir, concentrations in the intestine, which is permissive to SARS-CoV-2 infection (54) , were up to 128-fold higher than in the plasma, while concentrations in other tissues were roughly equal to plasma concentrations (55) . Therefore, it would be relevant to determine HCV PI lung concentrations in humans following multiple doses in steady state (56) . For remdesivir showing high in vitro efficacy, clinical efficacy might in part be limited by poor distribution to the lungs (31, 32, 56) . Poor lung distribution following systemic application of inhibitors might be overcome by improved formulations allowing topic application, such as inhalable formulations.

Inhibitor efficacy can be improved by combination treatments with synergistic and thus drug-saving effects as reported here for the combination of remdesivir with the macrocyclic PIs simeprevir, paritaprevir, or grazoprevir in Vero E6 cells and with simeprevir or grazoprevir in A549-hACE2 cells. Synergism was recently suggested for the combination of remdesivir with simeprevir (35, 36) or grazoprevir (36) in short-term assays in Vero E6 cells (35, 36) and human embryonic kidney (HEK293T) cells (36) . Our extensive results in Vero E6 and A549-hACE2 cells using short-term drug interaction assays demonstrated that the mode of interaction between PI and remdesivir depended on the PI structure. Thus, combination of remdesivir with the linear PIs boceprevir and narlaprevir showed mostly antagonism, while combination with macrocyclic PIs showed mostly synergism. In contrast to the previous studies (35, 36) , we confirmed these PI structure-dependent interactions with remdesivir in longer-term treatment assays in Vero E6 and A549-hACE2 cells, where the combination of remdesivir with simeprevir or grazoprevir, but not with boceprevir, resulted in added efficacy. This structure dependence might be explained by differences in viral targets. While the investigated HCV PIs were suggested to target M pro (30, (33) (34) (35) (36) , simeprevir was suggested to also target the SARS-CoV-2 polymerase (35) , and simeprevir, grazoprevir, and paritaprevir were suggested to also target PL pro (36, 62) . It should be noted that additional alternative viral targets, including nsp13 (helicase), nsp14 (exonuclease and methyltransferase), nsp15 (endoribonuclease), nsp16 (29-O-ribose methyltransferase), as well as structural proteins N (capsid) and spike, were suggested for paritaprevir, grazoprevir, and simeprevir by modeling studies (62, 74, (78) (79) (80) (81) (82) . Future detailed molecular studies are required to fully define the viral targets of different HCV PIs.

HCV PIs were designed and optimized to bind the HCV NS3 protease. In modeling studies, structural similarity between the HCV NS3 protease and SARS-CoV-2 M pro , including their active sites, was reported (36, 57, 68, 77) , despite a lack of overall sequence conservation. While both viral proteases are chymotrypsin-like proteases, the HCV NS3 protease has a larger and more shallow binding groove (34) . The HCV NS3 protease and PL pro do not show structural similarity (36) .

While carrying out and revising this study, several research articles addressing a potential effect of HCV PIs on SARS-CoV-2 were published. Using in silico modeling approaches, more than 20 studies predicted binding of different linear and macrocyclic HCV PIs to SARS-CoV-2 M pro (34, 36, . Fewer studies predicted binding of macrocyclic HCV PIs to PL pro (36, 62) . Furthermore, crystal structures of boceprevir, narlaprevir, and telaprevir bound to M pro were solved (34, 77, (83) (84) (85) . In addition, reports were published on inhibition of M pro by linear and macrocyclic HCV PIs (33-36, 83, 84) and of PL pro by macrocyclic and acyclic HCV PIs (36) .

Recently, five groups demonstrated the efficacy of different HCV PIs in Vero E6 cells (30, (33) (34) (35) (36) . Most EC 50 s reported in these studies were in the same range as those reported here; the slightly higher EC 50 values observed in our study are most likely caused by differences in experimental assay conditions. EC 50 s in human lung cells were only reported for simeprevir (EC 50 , 1mM) (35) and voxilaprevir (.10 mM), also using the A549-hACE2 cell line (30) . The similar PI EC 50 values in monkey Vero E6 cells, human Huh7.5 cells, and human A549-hACE2 cells reported in our study validate Vero E6 cells for the study of HCV PI efficacy against SARS-CoV-2. Furthermore, the short-term treatment assay based on the quantification of SARS-CoV-2 spike protein-expressing cells was validated here by an assay measuring virus-induced CPE.

Finally, we report antiviral activity of the HCV NS4A inhibitor ACH-806. Future studies are required to define the SARS-CoV-2 target of this compound and to investigate its potential to inform design of SARS-CoV-2 inhibitors.

In conclusion, we here provide a head-to-head comparison of the efficacy of a panel of clinically relevant HCV PIs against SARS-CoV-2, including detailed studies of interaction with remdesivir using different cell lines. Of the currently, clinically widely used HCV PIs, grazoprevir and voxilaprevir showed activity against SARS-CoV-2 in lung cells. For clinical use of HCV PIs with higher potency and higher plasma concentrations, such as simeprevir, production would need to be reinitiated, as was the case for remdesivir. While HCV PIs showed relatively low C max /EC 50 ratios assuming systemic administration, it remains possible that active concentrations will be attainable in relevant tissues. Furthermore, treatment efficacy might be increased by topical administration of improved formulations and by combination with remdesivir. As this study demonstrated structure-dependent differential interaction of HCV PIs with remdesivir, novel PIs should be tested for interaction with remdesivir if combination treatment is considered. While clinical studies would be needed to investigate if HCV PIs studied here could have direct clinical relevance, this study identifies compounds that could assist the development of further optimized PI molecules for future COVID-19 treatment regimes.

Cell cultivation. All cells were maintained at 37°C and 5% CO 2 . African green monkey kidney Vero E6 cells (gift from J. Dubuisson) and human Huh7.5 hepatoma cells (86) were maintained in Dulbecco's modified Eagle medium (Invitrogen, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco/ Invitrogen Corp., Carlsbad, CA, USA). A549-hACE2 cells (InvivoGen, Toulouse, France) were maintained in Dulbecco's modified Eagle medium nutrient mixture F-12 (Gibco, Paisley, UK) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.5 mg/ml puromycin (InvivoGen). Cells were split every 2 to 3 days with trypsin (Sigma) to maintain a subconfluent monolayer.

Virus isolate. The corona virus isolate SARS-CoV-2/human/Denmark/DK-AHH1/2020 was derived from a swab sample from a Danish patient that was passaged in Vero E6 cells. For the experiments presented here, we used a sequence-confirmed 2nd viral passage stock with an infectivity titer of 5.5 log 10 50% tissue culture infective dose (TCID50)/ml (31) .

Inhibitors. All inhibitors were purchased from Acme Bioscience (Palo Alto, CA, USA) and dissolved in DMSO (Sigma, St. Louis, MO, USA).

Short-term concentration-response antiviral treatment assays for evaluation of inhibitor potency. Ninety-six-well-based short-term antiviral treatment assays in Vero E6 cells, Huh7.5 cells, and A549-hACE2 cells were developed based on assays previously established for the determination of the potency of HCV PIs against HCV (29, (37) (38) (39) (40) (41) (42) (43) . Vero E6 and A549-hACE2 cells were seeded at 10,000 cells per well, and Huh7.5 cells were seeded at 9,000 cells per well in 96-well flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark). The following day, for Vero E6 cells, medium was changed to 50 ml fresh medium and cells were inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at an MOI of 0.002 by adding 50 ml virus stock diluted in medium to each well. Huh7.5 cells were inoculated at an MOI of 0.02, and A549-hACE2 cells were inoculated at an MOI of 0.003 (based on the infectivity titer determined in Vero E6 cells) by exchanging the medium with 50 ml virus stock diluted in medium. Following a 1-h incubation at 37°C and 5% CO 2 , infected cells were treated with a dilution series of inhibitors by adding 50 ml medium with inhibitor, resulting in the specified concentrations. Alternatively, cells were treated with a dilution series of DMSO alone serving as a control for the antiviral activity of DMSO. All concentrations of inhibitor were tested in 7 replicates; 14 infected and nontreated and 12 noninfected and nontreated replicates were included in each assay. For A549-hACE2 cells, the concentration of DMSO was kept constant in all cultures. Cells were subjected to immunostaining for the SARS-CoV-2 spike protein and evaluated as described below after incubation for 46 to 50 h for Vero E6 and A549-hACE2 cells or for 70 to 74 h for Huh7.5 cells.

Additionally, 96-well based concentration-response CPE assays were carried out in Vero E6 cells. Vero E6 cells were seeded at 10,000 cells per well in 96-well flat-bottom plates (Thermo Fisher Scientific). The following day, cells were inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at an MOI of 0.01 by exchanging the medium with 50 ml virus stock diluted in medium. After a 1-h incubation, infected cells were treated with a dilution series of inhibitor by adding 50 ml medium with inhibitor, resulting in the specified concentrations. All concentrations of inhibitor were tested in 4 replicates; 8 infected and nontreated and 16 noninfected and nontreated replicates were included in each assay. The concentration of DMSO was kept constant in all cultures. After 70 to 74 h of incubation at 37°C and 5% CO 2 , CPE was evaluated using viral ToxGlo assay (Promega, Madison, WI, USA) according to the manufacturer's guidelines. Relative light units (RLU) from infected and treated wells were related to the mean RLU from the 16 noninfected control wells. Data points are given as means from 4 replicates with standard errors of the means (SEMs). Sigmoidal concentration-response curves were fitted and EC 50 values were calculated using GraphPad Prism 8.0.0 applying the formula Y = Bottom 1 ((Top 2 Bottom)/ (1 1 10 (Log10EC50-X)*HillSlope )).

Immunostaining and evaluation of 96-well plates. Cells were fixed and virus was inactivated by submersion into methanol (J.T.Baker, Gliwice, Poland) for 20 min at room temperature. For immunostaining for the SARS-CoV-2 spike protein, plates were washed 2 times with phosphate buffered saline (PBS; Sigma, Gillingham, UK) containing 0.1% Tween 20 (Sigma, St Louis, MO) (PBS-Tween). Then, endogenous peroxidase activity was blocked by adding H 2 O 2 and incubating for 10 min followed by 2 more washes with PBS-Tween and blocking with PBS containing 1% bovine serum albumin (Roche, Mannheim, Germany) and 0.2% skim milk (Easis, Aarhus, Denmark) (PBSK) for 30 min. Next, plates were emptied and incubated with primary antibody, SARS-CoV-2 spike chimeric monoclonal antibody (number [no.] 40150-D004; Sino Biological, Beijing, China), diluted 1:5,000 in PBSK for 2 h at room temperature. Then, plates were washed 2 times with PBS-Tween and incubated for 1 h at room temperature with secondary antibody, F(ab9)2-goat anti-human IgG-Fc cross-adsorbed secondary antibody conjugated with horseradish peroxidase (HRP) (no. A24476; Invitrogen, Carlsbad, CA, USA) or goat F(ab9)2 anti-human IgG-Fc (HRP), preadsorbed (no. 98595; Abcam, Cambridge, UK), diluted 1:2,000 in PBSK. Finally, plates were washed 2 times with PBS-Tween, and SARS-CoV-2 spike protein-positive cells were stained using diaminobenzidine (DAB) substrate BrightDAB kit (no. BS04-110; Immunologic, Duiven, Netherlands) according to the manufacturer's guidelines. Plates were evaluated by automated counting of single SARS-CoV-2 spike protein-positive cells using an ImmunoSpot series 5 UV analyzer (CTL Europe GmbH, Bonn, Germany) (87) . The mean of counts from noninfected and nontreated wells, which was usually ,50, was subtracted from counts of infected wells. Counts from infected and treated wells were related to the mean count of the 14-replicate infected nontreated wells to calculate percent residual infectivity; mean counts of infected nontreated wells were 3,000 to 4,000 for Vero E6 cells, 1,000 to 2,000 for Huh7.5 cells, and 2,000 to 3,000 for A549-hACE2 cells. Data points are given as means from 7 replicates with SEMs. Sigmoidal concentration-response curves were fitted and EC 50 values calculated as described previously using GraphPad Prism 8.0.0 with a bottom constraint of 0 applying the formula Y = Top/(1 1 10 [Log10 EC50 2 X] Â Hill slope ) (43, 88) . Representative images from concentration-response antiviral treatment assays are shown by Zhou et al. (44) .

Short-term concentration-response antiviral treatment assays for analysis of interactions of PIs and remdesivir in Vero E6 and A549-hACE2 cells. Interactions of selected PIs in combination with remdesivir for inhibition of SARS-CoV-2 were investigated based on protocols previously established for HCV (38) . The experimental design was similar to that of the concentration-response antiviral treatment assays described above. In brief, Vero E6 cells or A549-hACE2 cells were seeded at 10,000 cells per well in 96-well flat-bottom plates, medium was changed to 50 ml fresh medium, and cells were inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at an MOI of 0.002 (Vero E6 cells) or an MOI of 0.003 (A549-hACE2 cells) by adding 50 ml virus stock diluted in medium to each well. Following a 1-h incubation at 37°C and 5% CO 2 , infected cells were treated with a dilution series of inhibitors by adding 50 ml medium with inhibitor resulting in the specified concentrations. Regarding inhibitor treatment, dilution series of selected PIs singly, remdesivir singly or a combination of PIs and remdesivir were used that were based on EC 50 values against SARS-CoV-2. Thus, for inhibitors and combinations of inhibitors, 1.15to 2-fold dilution series with at least 7 different dilutions were applied spanning the respective EC 50 values, aiming at achieving residual infectivity between 0% and 100%. For combination treatments, the same PI and remdesivir concentrations as used in single treatments were applied with a fixed ratio, except for the simeprevir plus remdesivir data set, where a nonconstant ratio was used. All treatment conditions were tested in 6 or 7 replicates, including 21 to 70 infected and nontreated replicates per experiment (with at least 7 replicates per experimental plate) and 12 noninfected and nontreated replicates per experimental plate. In experiments with A549-hACE2 cells, the concentration of DMSO was kept constant in all cultures. After 46 to 50 h of incubation, infected cells were visualized by immunostaining for the SARS-CoV-2 spike protein, and plates were evaluated by automated counting of single SARS-CoV-2 spike protein-positive cells, as described above.

Concentration-response cell viability assays. To evaluate cytotoxic effects of the inhibitors and DMSO, cell viability was monitored using the CellTiter 96 Aqueous one solution cell proliferation assay (Promega, Madison, WI, USA). Vero E6, Huh7.5, and A549-hACE2 cells were seeded in 96-well flat-bottom plates at 10,000, 9,000, and 10,000 cells per well, respectively, and the following day were treated with dilution series of inhibitors or combinations of inhibitors by adding 100 ml of medium containing inhibitors at the specified concentrations or DMSO alone at the specified dilutions. After 46 to 50 h for Vero E6 and A549-hACE2 cells and after 70 to 74 h for Vero E6 and Huh7.5 cells, cell viability was evaluated according to the manufacturer's guidelines. Vero E6 cells were tested with different incubation times to control for assays with 48 versus 72 h of incubation. In brief, 20 ml CellTiter 96 aqueous one solution reagent was added to each well, and plates were then incubated for 1 to 3 h at 37°C and 5% CO 2 . After incubation, for each well, absorbance at 492 nm was recorded by use of a FLUOstar OPTIMA 96-well plate reader (BMG, LABTECH, Offenburg, Germany). Each inhibitor concentration was tested in 2 to 4 replicate wells, and each experimental plate included 12 replicate nontreated control wells. Absorbance values from treated wells were related to the mean absorbance of the nontreated wells to estimate percent cell viability. Data points are given as means from 2 to 4 replicates with SEMs. Sigmoidal concentration-response curves were fitted and 50% cytotoxic concentration (CC 50 ) values were calculated using GraphPad Prism 8.0.0, with a bottom constraint of 0 applying the formula Y = Top/(1 1 10 [Log10 EC50 2 X] Â Hill slope ).

Viral cytopathogenic effect assay for determination of MOI for short-term treatment assays. To select a suitable MOI for short-term treatment assays not resulting in virus-induced CPE (immunostaining-based assay) or relatively strong CPE (CPE-based assay) during the assays, viral CPE assays were carried out. Cells were plated on 96-well flat-bottom plates as described for short-term treatment assays. The following day, cells were infected at different MOIs with SARS-CoV-2/human/Denmark/DK-AHH1/ 2020, using 100 ml per well with 4 replicates per MOI. Following the incubation times used in the shortterm treatment assays, CPE was evaluated using the viral ToxGlo assay (Promega) according to the manufacturer's guidelines. Relative light units (RLU) from infected cultures were related to the mean RLU from noninfected control cultures to detect CPE.

Longer-term antiviral treatment assays in SARS-CoV-2-infected Vero E6 and A549-hACE2 cells. Cells were seeded at 10 6 cells per flask in T25 flasks (Nunc) and, the following day, infected at an MOI of 0.00002 (Vero E6 cells) or an MOI of 0.0005 (A549-hACE2 cells) with SARS-CoV-2/human/Denmark/DK-AHH1/2020. Cells were treated with specified-fold EC 50 s of inhibitors on the day of infection by adding inhibitors together with the virus and again on day 1 postinfection. The cells were split and treated every 2 days with the specified concentrations of inhibitors, and the percentage of infected culture cells was evaluated by immunostaining for the SARS-CoV-2 spike protein and immunofluorescence imaging, as described below. In experiments with A549-hACE2 cells, the concentration of DMSO was kept constant in all cultures. Upon cell splitting, culture supernatants were harvested and stored at 280°C. Selected culture supernatants were used to determine viral RNA titers by RT-qPCR and to evaluate the presence of infectious virus by using an infectivity assay. For each experiment, a nontreated infected culture was included serving as a positive control for infection. Cultures were closed when massive cell death occurred, induced by viral infection and/or inhibitor treatment. Cell death was monitored with a light microscope.

Immunostaining and immunofluorescence imaging for evaluation of longer-term Vero E6 and A549-hACE2 cell cultures. In longer-term SARS-CoV-2-infected and PI-treated cultures, following cell splitting and treatment, replicate cell cultures were seeded in 8-well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA). The next day, cells were fixed, and virus was inactivated by submersion into methanol for 20 min. Chamber slides were washed twice with PBS-Tween and then incubated with primary antibody, SARS-CoV-2 spike chimeric monoclonal antibody (no. 40150-D004; Sino Biological, Beijing, China), diluted 1:1,000 in PBSK for 2 h at room temperature. Following 2 washes with PBS-Tween, chamber slides were incubated with secondary antibody, Alexa-Fluor 488 goat anti-human IgG (H1L) (no. A-11013; Invitrogen, Paisley, UK), diluted 1:500 and Hoechst 33342 (Invitrogen) diluted 1:1,000 in PBS-Tween for 20 min at room temperature. The percentage of SARS-CoV-2 spike protein-positive cells was evaluated by fluorescence microscopy (Axio Vert.A1; Zeiss, Jena, Germany), assigning the following designations: 0% infected cells (no cells infected), single infected cells, and 10% to 90% infected cells (in steps of 10%). The images were acquired with ZEN 3.0 software.

RT-qPCR assay for determination of SARS-CoV-2 RNA titers for evaluation of longer-term Vero E6 and A549-hACE2 cell cultures. For longer-term cultures, upon cell splitting and treatment, supernatant was harvested and stored at 280°C. Supernatant was mixed 1:3 with TRIzol LS (Life Technologies), and RNA was extracted with chloroform (Sigma) using 5PRIME phase gel lock heavy tubes (Quantabio). RNA was purified using an RNA clean and concentrator-5 kit (ZYMO Research) according to the manufacturer's guidelines, and RNA was eluted in nuclease-free water (Ambion). qPCRs were carried out using the TaqMan fast virus 1-step master mix (Thermo Fischer) with previously described primers and probes (89) : E_Sarbeco F (59-ACAGGTACGTTAATAGTTAATAGCGT-39), E_Sarbeco_R (59-ATATTGCAGCAGTACGCA CACA-39), and E_Sarbeco_P (6-carboxyfluorescein [FAM]-59-ACACTAGCCATCCTTACTGCGCTTCG-39-black hole quencher 1 [BHQ1]). Primers were used at 400 nM and probe was used at 200 nM together with 2.5 ml purified RNA. Cycling conditions were as follows: for reverse transcription, 10 min at 55°C, followed by 3 min at 95°C and 45 cycles of 95°C for 15 s and 58°C for 30 s using the LightCycler 96 System (Roche). For every assay, a negative control and RNA standards ranging from 10 to 10 5 RNA copies per ml (Twist Bioscience) were included. RNA titers (genome copies/milliliter) were calculated by interpolation of cycle threshold values of the standard curve generated using the standard panel and the LightCycler software. The LLOQ of the assay was calculated as mean of RNA titers in supernatants derived from noninfected control cultures plus 3 standard deviations.

Infectivity assay for evaluation of presence of infectious virus in supernatants from longerterm Vero E6 cell cultures. Vero E6 indicator cell cultures were seeded at 10,000 cells per well in 96well flat-bottom plates. The following day, the medium was exchanged with 100 ml of cell culture supernatants diluted 1:5 in cell culture medium. Culture supernatants were harvested every 2 to 3 days from replicate cultures not receiving treatment derived from longer-term Vero E6 cell cultures. Longer-term Vero E6 cell cultures treated with 3-or 5-fold EC 50 boceprevir (Fig. 8 ) or 1-fold EC 50 simeprevir in combination with 0.4-fold EC 50 remdesivir (Fig. 9) were investigated. For each supernatant, 4 replicate indicator cultures were inoculated. Twelve cultures inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at an MOI of 0.01 served as a positive control for infection, and 12 noninfected cultures served as negative controls. After 46 to 50 h of incubation at 37°C and 5% CO 2 , cells were subjected to immunostaining for the SARS-CoV-2 spike protein, and the number of single infected cells was evaluated by automated counting as described above.

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 2.1 MB.

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Identification of potential inhibitors of three key enzymes of SARS-CoV2 using computational approach

Whole genome analysis and targeted drug discovery using computational methods and high throughput screening tools for emerged novel coronavirus (2019-nCoV)

Discovery of potent SARS-CoV-2 inhibitors from approved antiviral drugs via docking and virtual screening

Structure-based virtual screening of phytochemicals and repurposing of FDA approved antiviral drugs unravels lead molecules as potential inhibitors of coronavirus 3C-like protease enzyme

Glecaprevir and maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy

Identification of 14 known drugs as inhibitors of the main protease of SARS-CoV-2

Repurposing simeprevir, calpain inhibitor IV and a cathepsin F inhibitor against SARS-CoV-2 and insights into their interactions with Mpro

Molecular investigation of SARS-COV-2 proteins and their interactions with antiviral drugs

Anti-HCV and anti-malaria agent, potential candidates to repurpose for coronavirus infection: virtual screening, molecular docking, and molecular dynamics simulation study

Comparative docking of SARS-CoV-2 receptors antagonists from repurposing drugs

Inhibition of SARS-CoV-2 main protease 3CL pro by means of a-ketoamide and pyridone-containing pharmaceuticals using in silico molecular docking

Destabilizing the structural integrity of COVID-19 by caulerpin and its derivatives along with some antiviral drugs: an in silico approaches for a combination therapy

Pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and FDA-approved inhibitors of SARS-CoV-2 main protease 3CL pro

Investigating the binding affinity, interaction, and structure-activity-relationship of 76 prescription antiviral drugs targeting RdRp and Mpro of SARS-CoV-2

Network analysis, sequence and structure dynamics of key proteins of coronavirus and human host, and molecular docking of selected phytochemicals of nine medicinal plants

Rational approach toward COVID-19 main protease inhibitors via molecular docking, molecular dynamics simulation and free energy calculation

Structural similarity of SARS-CoV2 Mpro and HCV NS3/4A proteases suggests new approaches for identifying existing drugs useful as COVID-19 therapeutics

In silico structure modelling of SARS-CoV-2 Nsp13 helicase and Nsp14 and repurposing of FDA approved antiviral drugs as dual inhibitors

Identification of potential inhibitors against SARS-CoV-2 by targeting proteins responsible for envelope formation and virion assembly using docking based virtual screening, and pharmacokinetics approaches

Identification of promising antiviral drug candidates against non-structural protein 15 (NSP15) from SARS-CoV-2: an in silico assisted drug-repurposing study

Identification of novel compounds against three targets of SARS CoV-2 coronavirus by combined virtual screening and supervised machine learning

An integrated drug repurposing strategy for the rapid identification of potential SARS-CoV-2 viral inhibitors

Malleability of the SARS-CoV-2 3CL Mpro active-site cavity facilitates binding of clinical antivirals

Broad-spectrum inhibition of coronavirus main Efficacy of HCV Protease Inhibitors against SARS-CoV-2 Antimicrobial Agents and Chemotherapy and papain-like proteases by HCV drugs

Structural plasticity of SARS-CoV-2 3CL M pro active site cavity revealed by room temperature X-ray crystallography

Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication

Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

We thank Bjarne Ørskov Lindhardt (Copenhagen University Hospital, Hvidovre) and Carsten Geisler (University of Copenhagen) for support. We thank Line Abildgaard Ryberg for discussion on structural interactions of M pro and PI, as well as Pia Pedersen, Lotte Mikkelsen, and Anna-Louise Sørensen (Copenhagen University Hospital, Hvidovre) for general laboratory assistance. We also thank Jean Dubuisson and Sandrine Belouzard for providing Vero E6 cells and Charles Rice for providing Huh7. We declare no conflict of interest.