key: cord-0872807-t66xvg38 authors: Muturi, Elishiba; Hong, Wei; Li, Junhua; Yang, Wan; He, Jin; Wei, Hongping; Yang, Hang title: Effects of simeprevir on the replication of SARS-CoV-2 in vitro and in transgenic hACE2 mice date: 2021-12-17 journal: Int J Antimicrob Agents DOI: 10.1016/j.ijantimicag.2021.106499 sha: 00f55cc335e36a5384f8b59600eea6ced34af789 doc_id: 872807 cord_uid: t66xvg38 In a bid to contain the current COVID-19 (coronavirus disease 2019) pandemic, various countermeasures have been applied. To date, however, there is a lack of an effective drug for the treatment of COVID-19. Through molecular modelling studies, simeprevir, a protease inhibitor approved for the management of hepatitis C virus infection, has been predicted as a potential antiviral against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of COVID-19. Here we assessed the efficacy of simeprevir against SARS-CoV-2 both in vitro in Vero E6 cells and in vivo in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model. The results showed that simeprevir could inhibit SARS-CoV-2 replication in Vero E6 cells with a half-maximal effective concentration (EC50) of 1.41 ± 0.12 μM. In a transgenic hACE2 mouse model of SARS-CoV-2 infection, intraperitoneal administration of simeprevir at 10 mg/kg/day for 3 consecutive days failed to suppress viral replication. These findings collectively imply that simeprevir does not inhibit SARS-CoV-2 in vivo and therefore do not support its application as a treatment against COVID-19 at a dosage of 10 mg/kg/day. COVID-19 (coronavirus disease 2019) is a highly infectious respiratory disease caused by a novel coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). The first COVID-19 outbreak was reported in China in December 2019 and it has since spread rapidly resulting in a global pandemic [1] . The fight against COVID-19 has been one filled with challenges, including the failure of experimental drugs. The most recent challenge is the emergence of variant strains that are capable of undermining the protective effect of vaccines that are being administered [2] . Hence, even with the availability of vaccines, there is still a pressing need for an effective anti-SARS-CoV-2 drug. Remdesivir was the first drug to be approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Several clinical trials were conducted, but discordant findings were reported regarding its clinical efficacy [3] . Recently, analysis of real-world data from three retrospective studies on the treatment and outcome of remdesivir against COVID-19 have demonstrated that remdesivirtreated hospitalised patients have a significantly reduced risk of mortality compared with matched controls (https://www.gilead.com/news-and-press/press-room/pressreleases/2021/6/gileads-veklury-remdesivir-associated-with-a-reduction-in-mortalityrate-in-hospitalized-patients-with-covid19-across-three-analyses-of-large-ret). Monoclonal antibody therapies, including casirivimab + imdevimab and bamlanivimab + etesevimab, have gained increasing attention, especially for use against variant strains [4] . The most recent advancement involves three investigational drugs (monulpiravir, paxlovid and favipiravir) that have performed well in clinical trials and shown promising results in reducing the risk of hospitalisation and death [5, 6] . Despite the availability of 6 numerous treatment approaches, there is still no definitive treatment for SAR-CoV-2 infection. To rapidly develop an antiviral drug, researchers have resorted to repurposing preexisting FDA-approved drugs. Simeprevir is a second-generation protease inhibitor approved for the management of hepatitis C virus (HCV) infection. The anti-HCV activity of this drug is mediated by inhibition of viral NS3/4A protease, thus preventing viral maturation by impeding protein synthesis [7] . In silico structural modelling studies predicted that simeprevir inhibits SARS-CoV-2 by targeting the viral main protease (M pro ) [8] , RNA-dependent RNA polymerase (RdRp, also called Nsp12) [9] and Nsp13 (NTPase/helicase) [10] . M pro cleaves viral polyproteins to generate non-structural proteins including Nsp12 and Nsp13, which are essential components of the viral replication-transcription complex. Molecular dynamics simulation analysis also established that simeprevir could inhibit interaction of the viral spike protein with the receptor angiotensin-converting enzyme 2 (ACE2) by binding to side chains of residues in the binding pocket of the receptor-binding domain (RBD), suggesting that the drug may be a multitarget inhibitor [11] . Although there is a great deal of evidence supporting the anti-SARS-CoV-2 efficacy of simeprevir in vitro [12] [13] [14] , its in vivo potency has not yet been fundamentally evaluated. In the present study, the antiviral effects of simeprevir against SARS-CoV-2 were evaluated both in vitro and in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model. 7 Vero E6 cells (an African green monkey kidney cell line) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% fetal bovine serum, 100 g/mL streptomycin and 100 U/mL penicillin at 37 °C and 5% CO 2 . SARS-CoV-2 strain (nCoV-2019BetaCoV/Wuhan/WIV04/2019) was propagated in Vero E6 cells and was titrated by standard plaque assay following the standard procedure. Simeprevir We evaluated the cytotoxicity of simeprevir in Vero E6 cells using a Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China). Cells were seeded at a density of 5 × 10 3 cells/well in 96-well plates and were incubated overnight at 37 °C and 5% CO 2 . Cells were exposed to a series of simeprevir concentrations (0-100 M) for another 24 h. The contents of the wells were then replaced with fresh medium containing 10% CCK-8 solution and were incubated at 37 °C for 1.5 h. The optical density at 450 nm was measured using a Synergy H1 microplate reader (BioTek, USA). 8 An antiviral assay was performed to assess the antiviral effects of simeprevir on the replication of SARS-CoV-2. Vero E6 cells were seeded at a density of 1 × 10 5 To evaluate the effect of simeprevir on protein expression levels in SARS-CoV-2infected Vero E6 cells, western blot analysis was performed. Vero E6 cells were infected with SARS-CoV-2 in the presence of 0, 3 and 10 M of simeprevir. Cells were lysed with RIPA lysis buffer (Thermo Scientific) and were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 24 hpi. Protein bands were electroblotted onto nitrocellulose membranes (Bio-Rad, Shanghai, China). The membranes were blocked with phosphate-buffered Salome (PBS) containing 0.1% Tween 20 and 5% skimmed milk. Proteins were detected by probing the membranes 9 with a mixture of rabbit anti-SARS-CoV-2 nucleocapsid protein (NP) polyclonal antibody/rabbit anti-SARS-CoV-2 spike protein (SP) polyclonal antibody and rabbit anti-GAPDH polyclonal antibody (Beyotime) for 1 h at room temperature. Membranes were then washed in PBS containing 0.05% Tween 20 for 20 min. Blots were detected with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Beyotime) followed by chemiluminescence reagents. Signals were detected by exposure to X-ray film for 1-3 min. To further verify the inhibitory impact of simeprevir on the expression of SARS-CoV-2 antigens, an indirect immunofluorescence assay was performed. Vero E6 cells were First, the cytotoxicity of simeprevir in Vero E6 was examined by CCK-8 assay. Consistent with previous studies, simeprevir exhibited moderate cytotoxicity with a 50% cytotoxic concentration (CC 50 ) of 32 .71 ± 0.94 M (Fig. 1A) Given the promising antiviral potency of simeprevir in vitro, we further assessed its performance in vivo in a hACE2 transgenic mouse model. As shown in Fig. 1D , rapid weight loss of mice was observed in all three treatment groups, a typical sign of SARS-CoV-2 infection in transgenic mice [15] . High copies of viral RNA were observed in multiple organs of virus-infected mock-treated mice, including the lung, brain, liver, kidney and intestine (Fig. 1E) , indicating active viral replication and distribution in these organs. The highest viral burden was observed in the lungs of infected mice (Fig. 1E) . In contrast to findings reported in in vitro antiviral activity studies, no significant difference in viral load was observed between the simeprevir-treated and buffer-treated groups ( Fig. 1E) , indicating that simeprevir, at a dose much higher than that recommended in the treatment of HCV (150 mg/day, ~2.5 mg/kg) [16] , has little or no effects on the replication of SARS-CoV-2 in vivo. Similarly, the remdesivir-treatment group showed no significant reduction in virus titres compared with the control group (Fig. 1E ). Simeprevir is a strong HCV NS3/4A protease inhibitor and has been suggested as a potent inhibitor of SARS-CoV-2 M pro protease. While HCV and SARS-CoV-2 proteases share some structural similarities, they are fundamentally different thus limiting the 13 potency of simeprevir against SARS-CoV-2 [14] . Additionally, cytochrome P450 3A4 (CYP3A4) enzyme, which is involved in the biotransformation of simeprevir, is preferentially found in the liver, which may result in an uneven distribution of the active metabolite into relevant tissues such as the lungs [17] . Moreover, hepatic absorption of simeprevir is mediated by organic anion-transporting polypeptide 1B3 (OATP1B3), which is not expressed in our current mouse model. Factors such as uneven drug distribution from preferential metabolism in various organs and the complexity of pharmacokinetics, which do not affect standard cell culture protocols, may explain the discrepancies between the in vitro and in vivo performance of simeprevir. Poor pharmacokinetics of remdesivir in mouse models have been reported, which may explain its low efficacy in this study. The presence of high levels of serum esterase vastly affects the plasma stability of remdesivir in mice [18] . In vitro studies have demonstrated that concomitant use of remdesivir and simeprevir results in a synergistic antiviral response against SARS-CoV-2 [12, 13] . We cannot rule out with certainty synergy between the two drugs in vivo solely based on our study findings. Further in vivo antiviral evaluation of this combination in a model that can adequately metabolise remdesivir should be considered. Similar cases of prospective drugs inhibiting SARS-CoV-2 replication in vitro but failing to reflect the same in vivo have been reported for other compounds, including chloroquine and hydroxychloroquine [19] . However, we acknowledge that factors such as dosage and route of administration may influence the bioavailability of a drug and thereby significantly impact its efficacy. In the present study, we tested the in vivo 14 antiviral potency of simeprevir at 10 mg/kg, a dose we considered to be within the approved therapeutic margins for the treatment of HCV. However, a major challenge in drug repurposing is the requirement of higher doses than those that effectively treat the original indication. Therefore, a limitation of this study is that simeprevir at the current dosage under investigation may have been too low to provide sufficient exposure in relevant tissues. Additionally, pharmacokinetics and drug exposure profiles of intraperitoneally administered simeprevir were not extensively studied to rule out poor metabolism as a reason for failed efficacy. Further investigation of the reasons for the lack of in vitro to in vivo translation of antiviral efficacy of simeprevir against SARS-CoV-2 should be considered. Future studies may explore the in vivo antiviral efficacy of simeprevir at higher doses or as a combination therapy with other drugs such as remdesivir that have been reported to augment its antiviral effects. In summary, we report here that simeprevir shows inhibitory effects against SARS-CoV-2 in cell culture but shows little inhibitory activity in transgenic mice. Although the mechanisms of failed protection by simeprevir in vivo require further study, the current data suggest that simeprevir may not be an effective antiviral candidate for SARS-CoV-2 at a dosage of 10 mg/kg. 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