key: cord-0922823-6470qlu1
authors: Lo, Ho Sing; Hui, Kenrie Pui Yan; Lai, Hei-Ming; Khan, Khadija Shahed; Kaur, Simranjeet; Huang, Junzhe; Li, Zhongqi; Chan, Anthony K. N.; Cheung, Hayley Hei-Yin; Ng, Ka-Chun; Wang Ho, John Chi; Chen, Yu Wai; Ma, Bowen; Cheung, Peter Man-Hin; Shin, Donghyuk; Wang, Kaidao; Lee, Meng-Hsuan; Selisko, Barbara; Eydoux, Cecilia; Guillemot, Jean-Claude; Canard, Bruno; Wu, Kuen-Phon; Liang, Po-Huang; Dikic, Ivan; Zuo, Zhong; Chan, Francis K. L.; Hui, David S. C.; Mok, Vincent C. T.; Wong, Kam-Bo; Ko, Ho; Aik, Wei Shen; Chan, Michael Chi Wai; Ng, Wai-Lung
title: Simeprevir potently suppresses SARS-CoV-2 replication and synergizes with remdesivir
date: 2020-09-03
journal: bioRxiv
DOI: 10.1101/2020.05.26.116020
sha: 8ac5480dc1ce2937fb95fbc7f880be5cdc16bf93
doc_id: 922823
cord_uid: 6470qlu1

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence Summary Discovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

The recent outbreak of infection by the novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to almost all countries and claimed more than 760,000 lives worldwide (WHO situation report 209, August 16, 2020) . Alarming features of COVID-19 include a high risk of clustered outbreak both in community and nosocomial settings, and up to one-fifth severe/critically ill proportion of symptomatic inpatients reported [1] [2] [3] [4] . Furthermore, a significant proportion of infected individuals are asymptomatic, substantially delaying their diagnoses, hence facilitating the widespread dissemination of COVID-19 5 . With a dire need for effective therapeutics that can reduce both clinical severity and viral shedding, numerous antiviral candidates have been under clinical trials or in compassionate use for the treatment of SARS-CoV-2 infection 6 .

Several antivirals under study are hypothesized or proven to target the key mediator of a specific step in the SARS-CoV-2 viral replication cycle. For instance, lopinavir/ritonavir (LPV/r) and danoprevir have been proposed to inhibit the SARS-CoV-2 main protease (M pro , also called 3CL Pro ) needed for the maturation of multiple viral proteins; chloroquine (CQ) / hydroxychloroquine (HCQ) [alone or combined with azithromycin (AZ)] may abrogate viral replication by inhibiting endosomal acidification crucial for viral entry 7, 8 ; nucleoside analogues such as remdesivir, ribavirin, favipiravir and EIDD-2801 likely inhibit the SARS-CoV-2 nsp12 RNA-dependent RNA polymerase (RdRp) and/or induce lethal mutations during viral RNA replication [9] [10] [11] . Unfortunately, on the clinical aspect, LPV/r failed to demonstrate clinical benefits in well-powered randomized controlled trials (RCTs), while HCQ and/or AZ also failed to demonstrate benefits in observational studies [12] [13] [14] . Meanwhile, LPV/r, CQ/HCQ and AZ may even increase the incidence of adverse events [14] [15] [16] . Although remdesivir is widely considered as one of the most promising candidates, latest RCTs only revealed marginal shortening of disease duration in patients treated 17 . Therefore, further efforts are required to search for more potent, readily repurposable therapeutic agents for SARS-CoV-2 infection, either as sole therapy or in combination with other drugs to enhance their efficacy.

Ideally, the candidate drugs need to be readily available as intravenous and/or oral formulation(s), possess favourable pharmacokinetics properties as anti-infectives, and do not cause adverse events during the treatment of SARS-CoV-2 infection (e.g. non-specific immunosuppression, arrhythmia or respiratory side effects). Two complementary approaches have been adopted to identify novel drugs or compounds that can suppress SARS-CoV-2 replication. One approach relies on in vitro profiling of the antiviral efficacy of up to thousands of compounds in early clinical development, or drugs already approved by the U.S. Food and Drug Administration (FDA) [18] [19] [20] [21] [22] . On the other hand, as the crystal structure of the M pro 23,24 , papain-like protease (PL pro ) 25 and the cryo-EM structure of the nsp12-nsp7-nsp8 RdRp complex 11, 26 of the SARS-CoV-2 virus became available, the structure-based development of their specific inhibitors becomes feasible. Structure-aided screening will enable the discovery of novel compounds as highly potent inhibitors 27 as well as the repurposing of readily available drugs as anti-CoV agents for fast-track clinical trials.

Here we report our results regarding the discovery of FDA-approved drugs potentially active against the SARS-CoV-2. In vitro experiments led to the identification of simeprevir, a hepatitis C virus (HCV) NS3A/4 protease inhibitor 28 , as a potent inhibitor of SARS-CoV-2 replication (Fig. 1A) . Importantly, simeprevir acts synergistically with remdesivir, whereby the effective dose of remdesivir could be lowered by multiple-fold by simeprevir at physiologically feasible concentrations. Interestingly, biochemical and molecular characterizations revealed that simeprevir inhibits both the M pro protease and RdRp polymerase activities. This unexpected anti-SARS-CoV-2 mechanism of simeprevir provides hints on novel antiviral strategies.

A prioritized screening identifies simeprevir as a potent suppressor of SARS-CoV-2 replication in a cellular infection model Given our goal of identifying immediately usable and practical drugs against SARS-CoV-2, we prioritized a list of repurposing drug candidates for in vitro testing based on joint considerations on safety, pharmacokinetics, drug formulation availability, and feasibility of rapidly conducting clinical trials (Table S1) . We focused on FDAapproved antivirals (including simeprevir, saquinavir, daclatasvir, ribavirin, sofosbuvir and zidovudine), and drugs whose primary indication was not antiviral but had reported antiviral activity (including bromocriptine and atovaquone). Remdesivir was also tested for comparison of efficacy and as a positive control. (Fig. 1B) . More detailed dose-response characterization found that simeprevir has a potency comparable to remdesivir (Fig. 1C, D) . The half-maximal effective concentration (EC50) of simeprevir was determined to be 4.08 μ M, while the 50% cytotoxicity concentration (CC50) was 19.33 μ M (Fig. 1C, D) . In a physiologically relevant human lung epithelial cell model, ACE2-expressing A549 cells (A549-ACE2) infected with SARS-CoV-2, we also observed the strong antiviral effect of simeprevir 29 (Supplementary Fig. 1) . The cytotoxicity data are also in line with the reported in vitro and in vivo safety pharmacological profiling using human cell lines, genotoxicity assays, and animal models 28,30 . These data suggest that a desirable therapeutic window exists for the suppression of SARS-CoV-2 replication with simeprevir. 

While simeprevir is a potential candidate for clinical use alone, we hypothesized that it may also have a synergistic effect with remdesivir, thereby mitigating its reported adverse effects, improving its efficacy, and broadening its applicability 17 . Indeed, combining simeprevir and remdesivir at various concentrations apparently provided much greater suppression of SARS-CoV-2 replication than remdesivir alone, while they did not synergize to increase cytotoxicity ( Fig. 2A) . Importantly, such effects were not merely additive, as the excess over Bliss score suggested synergism at 3. 

The desirable anti-SARS-CoV-2 effect of simeprevir prompted us to determine its mechanism of action. Given that simeprevir is an HCV NS3/4A protease inhibitor, we first investigated its inhibitory activity against SARS-CoV-2 M pro and PL pro using biochemical assays 31,32 (Fig. 3) . We found inhibition of M pro by simeprevir with halfmaximal inhibitory concentration (IC 50 ) of 9.6 ± 2.3 μ M (Fig. 3A) , two times higher than the EC 50 determined from our cell-based assay. The substrate cleavage was further verified with SDS-PAGE (Supplementary Fig. 3 ). Docking simeprevir against the apo vir ug 3 of re ly its e ng an er po protein crystal structure of SARS-CoV-2 M pro suggested a putative binding mode with a score of -9.9 kcal mol -1 (Supplementary Fig. 4 ). This binding mode is consistent with a recent docking study using a homology model of SARS-CoV-2 M pro 33 . On the other hand, no inhibition of PL pro activity was observed at physiologically feasible concentrations of simeprevir, with either ISG15 or ubiquitin as substrate (Fig. 3B,  Supplementary Fig. 5, 6 ).

We speculated that the weak inhibition of M pro protease activity by simeprevir could not fully account for its antiviral effect towards SARS-CoV-2. To identify additional target(s), we next docked simeprevir alongside several nucleoside analogues (remdesivir, ribavirin, and favipiravir) against the motif F active site of the cryo-EM structure of the SARS-CoV-2 nsp12 RdRp (Supplementary Fig. 7A) . Interestingly, the docking results revealed that simeprevir had a higher binding score than the nucleoside analogues ( Supplementary Fig. 7B ). To test this experimentally, we established and performed RdRp primer extension assays using recombinant nsp12, nsp7, and nsp8 of SARS-CoV 34 . Intriguingly, simeprevir showed low micromolar-range inhibition towards SARS-CoV RdRp as validated by both a gel-based assay (Supplementary Fig. 8 ) and a Picogreen fluorescence-based assay, with an IC 50 value of 5.5 ± 0.2 μ M (Fig. 3C) . Collectively, the assay data suggested that simeprevir inhibits the enzymatic activities of both M pro and RdRp but not PL pro . 

While inhibition of viral targets seems to be a primary mechanism of action of simeprevir, the weak inhibitory effects observed in biochemical assays as well as its cytotoxicity suggest the possibility of additional host-mediated antiviral response. To further elucidate the antiviral mechanism of simeprevir, we next performed RNA sequencing on Vero E6 cells to reveal the transcriptomic changes upon drug treatment (Fig. 4A) . In line with the literature, SARS-CoV-2 infection induced type I interferon and chemokine response (Supplementary Fig. 9 ) 35, 36 . In mock-infected cells, simeprevir treatment (at 1.1 μ M or 3.3 μ M) did not induce any significant changes of differentially expressed genes (DEGs); while in SARS-CoV-2-infected cells, a small number of DEGs was observed (Fig. 4B) . Gene set enrichment analyses (GSEA) of infected cells using Reactome gene sets revealed significant positive enrichment of 93 gene sets in the simeprevir-treated samples, including histone lysine/arginine methylation, histone demethylation, and cell cycle control (Fig. 4C, Supplementary table 2) . On the other hand, gene sets with the gene ontology (GO) terms "defense response to virus" and "response to type I interferon" were negatively enriched, suggesting overall downregulation of these gene sets (Fig. 4D) . In the latter set, green monkey orthologs of crucial human innate immune-related genes (e.g. IFIT1-3, USP18) and interferonstimulated genes (e.g. ISG15) were downregulated with simeprevir treatment in a dosedependent manner (Fig. 4E) . Similarly, the downregulation of some of these genes as well as the proinflammatory cytokines IL-6 and interferon IFNL1 were also observed with remdesivir treatment (Fig. 4F) . 

The novel coronavirus SARS-CoV-2 has gone from an emerging infection to a global pandemic with its high transmissibility. As human activities are becoming more aggressive and damaging to nature, future coronavirus pandemics are bound to happen. It is therefore essential to reduce the casualties by effective pharmacological based on available pharmacokinetic data 37, 38 .

In addition, we discovered that simeprevir can synergize with remdesivir in inhibiting SARS-CoV-2 replication in a cellular model, potentially allowing lower doses of both drugs to be used to treat COVID-19. In a global pandemic with patients having diverse clinical characteristics, providing additional therapeutic options to remdesivir will be important to treat those who are intolerant or not responding to the drug 17 , which can easily amount to tens of thousands of patients. As there is only one confirmed and approved therapy for COVID-19, a potentially repurposable drug can be rapidly tested in animal models before clinical trials to prepare for supply shortages or when remdesivirresistant mutations arise. Combination treatment, such as simeprevir-remdesivir, may also help to reduce the dose required to alleviate side effects.

We note, however, there are also several limitations of simeprevir and the proposed simeprevir-remdesivir combination. Simeprevir requires dose adjustments in patients with Child-Pugh Class B or C cirrhosis, as well as in patients with East Asian ancestry 37 . In addition, simeprevir has been taken off the market since 2018 due to the emergence of next-generation HCV protease inhibitors, hence its supply may not be ramped up easily. Noteworthily, simeprevir is metabolized by the CYP3A4 enzyme with saturable kinetics 37 while remdesivir itself is not only a substrate of CYP3A4 but also a CYP3A4 inhibitor. Whether such theoretical pharmacokinetic interaction will exacerbate liver toxicity or provide additional pharmacokinetic synergy (in addition to pharmacodynamic synergy) in vivo remains to be tested.

Mechanistically, we found that simeprevir suppresses SARS-CoV-2 replication by targeting at least two viral proteins -it weakly inhibits M pro at ~10 μ M and unexpectedly inhibits RdRp at ~5 μ M. The potency towards M pro is consistent with the IC 50 of ~13.7 μ M as determined in a parallel study 39 . Our gel-based assay (Supplementary Fig. 9 ) suggested that simeprevir interferes with RNA-binding of RdRp because less probe was extended but to full length. This is also supported by the in silico docking results, in which simeprevir is docked to a highly conserved RNA binding site showing no amino acid polymorphism between SARS-CoV and SARS-CoV-2 (Supplementary Fig. 8A ). This putative binding mode hints that simeprevir might block the RNA binding site while remdesivir might target the nucleoside entry site, potentially resulting in a synergistic effect. Importantly, the high similarity in sequence (96% identity) and structure between SARS-CoV-2 and SARS-CoV also suggest that simeprevir might act as a broadspectrum antiviral in the Coronaviridae family. Furthermore, the discrepancy between RdRp and M pro inhibitory potency versus in vitro inhibitory potency of SARS-CoV-2 replication suggested additional mechanism(s) of action of simeprevir. Our RNA-seq and GSEA analysis revealed several molecular pathways that warrant future investigation. A possible direction is immune modulation via epigenetic regulations, which could mediate viral infection (via SWI/SNF chromatin remodeling complex and histone H3.3 complex) 40 , interferoninduced antiviral response (via H3K79 methylation) 41 , and host immune evasion (via alteration of DNA methylome) 42 . Whether the downregulation of type I IFN-related genes stems directly from simeprevir's action or a reduction in viral load remains an open question. Collectively, simeprevir targets two viral proteins but may also act on the host proteins to suppress SARS-CoV-2 replication.

Given that simeprevir is originally a non-nucleoside antiviral targeting HCV protease, its inhibition towards RdRp is largely unexpected and represents a novel mechanism of action. Simeprevir thus holds promise to be a lead compound for the future development of dual inhibitors of M pro and RdRp. It should be noted that the potencies of M pro and RdRp inhibition may not entirely account for the strong suppression of SARS-CoV-2 viral replication. Therefore, further investigation of the mechanism of action of simeprevir may uncover new druggable targets for inhibiting SARS-CoV-2 replication.

Bromocriptine mesylate (BD118791), saquinavir (BD150839), bictegravir (BD767657), atovaquone (BD114807) and asunaprevir (BD626409) were purchased from BLD Pharmatech (Shanghai, China). Entecavir (HY-13623), zidovudine (HY-17413), sofosbuvir (HY-15005), daclatasvir (HY-10466), simeprevir (HY-10241), remdesivir (HY-104077) and remdesivir triphosphate sodium (HY-126303A) were purchased from MedChemExpress (Monmouth Junction, NJ). Drug stocks were made with DMSO.

SARS-CoV-2 virus (BetaCoV/Hong Kong/VM20001061/2020, SCoV2) was isolated from the nasopharyngeal aspirate and throat swab of a COVID-19 patient in Hong Kong using Vero E6 cells (ATCC CRL-1586). Vero E6 or A549-ACE2 cells were infected with SCoV2 at a multiplicity of infection (MOI) of 0.05 or 0.5, respectively, in the presence of varying concentrations and/or combinations of the test drugs. DMSO as the vehicle was used as a negative control. Antiviral activities were evaluated by quantification of SARS-CoV-2 ORF1b copy number in the culture supernatant by using quantitative real-time RT-PCR (qPCR) at 48 h post-infection with specific primers targeting the SARS-CoV-2 ORF1b 43 .

In vitro cytotoxicity of the tested drugs was evaluated using thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich)-based cell viability assays. Vero E6 cells were seeded onto 48-well plates and treated with indicated concentrations of simeprevir and/or remdesivir for 48 h. Treated cells were incubated with DMEM supplemented with 0.15 mg/mL MTT for 2 h, and formazan crystal products were dissolved with DMSO. Cell viability was quantified with colorimetric absorbance at 590 nm.

Three-dimensional representations of chemical structures were extracted from the ZINC15 database (http://zinc15.docking.org) 44 , with the application of three selection filters --Protomers, Anodyne, and Ref. ZINC15 subset DrugBank FDA (http://zinc15.docking.org./catalogs/dbfda/) were downloaded as the mol2 file format. The molecular structures were then converted to the pdbqt format (the input file format for AutoDock Vina) using MGLTools2-1.1 RC1 (sub-program "prepare_ligand") (http://adfr.scripps.edu/versions/1.1/downloads.html). AutoDock Vina v1.1.2 was employed to perform docking experiments 45 . Docking of simeprevir on SARS-CoV-2 M pro was performed with the target structure based on an apo protein crystal structure (PDB ID: 6YB7); the A:B dimer was generated by crystallographic symmetry. Docking was run with the substrate-binding residues set to be flexible. Docking of simeprevir and other active triphosphate forms of nucleotide analogues was performed against the nsp12 portion of the SARS-CoV-2 nsp12-nsp7-nsp8 complex cryo-EM structure (PDB ID: 6M71).

The sequence of SARS-CoV-2 M pro was obtained from GenBank (accession number: YP_009725301), codon-optimized, and ordered from GenScript. A C-terminal hexahistidine-maltose binding protein (His 6 -MBP) tag with two in-between Factor Xa digestion sites were inserted. Expression and purification of SARS-CoV-2 M pro was then performed as described for SARS-CoV M pro 31 . The protein substrate, where the cleavage sequence "TSAVLQSGFRKM" of M pro was inserted between a cyan fluorescent protein and a yellow fluorescent protein, was expressed and purified as described 31 .

The inhibition assay was based on fluorescence resonance energy transfer (FRET) using a fluorescent protein-based substrate previously developed for SARS-CoV M pro 31, 46 . 0.1 μ M of purified SARS-CoV-2 M pro was pre-incubated with 0 -250 μ M simeprevir in 20 mM HEPES pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT for 30 min before the reaction was initiated by addition of 10 μ M protein substrate 32 . Protease activity was followed at 25 °C by FRET with excitation and emission wavelengths of 430 nm and 530 nm, respectively, using a multi-plate reader as previously described 31, 46 . Reduction of fluorescence at 530 nm was fitted to a single exponential decay to obtain the observed rate constant (k obs ). Relative activity of M pro was defined as the ratio of k obs with inhibitors to that without. The relative IC 50 value of simeprevir was determined by fitting the relative activity at different inhibitor concentration to a four-parameter logistics equation.

The fusion protein nsp7-nsp8 (nsp7L8) was generated by inserting a GSGSGS linker sequence between the nsp7 and nsp8 coding sequences 34 . The nsp7L8, nsp8 and nsp12 were produced and purified independently as described 47 . The complex, referred to as the replication/transcription complex (RTC), was reconstituted with a 1:3:3 ratio of nsp12:nsp7L8:nsp8.

The assay was performed as previously described 48 . The compound concentration leading to a 50% inhibition of RTC-mediated RNA synthesis was determined as previously described. Briefly, poly(A) template and the SARS-CoV RTC was incubated 5 min at room temperature and then added to increasing concentration of compound. Reaction was started by adding UTP and incubated 20 min at 30°C. Reaction assays were stopped by the addition of 20 µl EDTA 100 mM. Positive and negative controls consisted of a reaction mix with 5% DMSO (final concentration) or EDTA (100 mM) instead of compounds, respectively. Picogreen® fluorescent reagent was diluted to 1/800 final in TE buffer according to the data manufacturer and aliquots were distributed into each well of the plate. The plate was incubated for 5 min in the dark at room temperature and the fluorescence signal was then read at 480 nm (excitation) and 530 nm (emission) using a TecanSafire2 microplate reader. IC 50 was determined using the following equation: % of active enzyme = 100/(1+(I) 2 /IC 50 ), where I is the concentration of inhibitor and 100% of activity is the fluorescence intensity without inhibitor. IC 50 was determined from curve-fitting using the GraphPad Prism 8.

Enzyme mix (10 µM nsp12, 30 µM nsp7L8, 30 µM nsp8) in complex buffer (25 mM HEPES pH 7.5, 150 NaCl, 5 mM TCEP, 5 mM MgCl 2 ) was incubated for 10 min on ice and then diluted with reaction buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl 2 ) to 2 µM nsp12 (6 µM nsp7L8 and nsp8) to a final volume of 10 µl. The resulting enzyme complex was mixed with the 10 µl of 0.8 µM primer/ template (P/T) carrying a Cy5 fluorescent label at the 5' end (P:Cy5-GUC AUU CUC C, T: UAG CUU CUU AGG AGA AUG AC) in reaction buffer, and incubated at 30°C for 10 min. Inhibitor was added in 2 µl to the elongation complex and reactions were immediately started with 18 µl of NTP mix in the reaction buffer. Final concentrations in the reactions were 0.5 µM nsp12 (1.5 µM nsp7L8 and nsp8), 0.2 µM P/T, 50 µM NTPs and the given concentrations of inhibitors. Samples of 8 µl were taken at given time points and mixed with 40 µl of formamide containing 10 mM EDTA. Ten-µl samples were analyzed by denaturing PAGE (20 % acrylamide, 7 M urea, TBE buffer); and product profiles visualized by a fluorescence imager (Amersham Typhoon). Quantification of product bands and analysis were performed using ImageQuant and Excel.

The purification and assay of PL pro activity was adapted from as previously described 25 . Briefly, a ISG15-C-term protein tagged with rhodamine was used as a substrate for the enzymatic assay. Because of the solubility of Simeprevir, we used an optimized reaction buffer (5% DMSO, 15% PEG300, 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM DTT). 5 uL of solution containing 0 -2 mM of Simeprevir and 2 µM of ISG15c-rhodamine were aliquoted into a 384-well plate. Reaction was initiated by addition of 5 µL of 40 nM PL pro to the well. Initial velocities of rhodamine release (36 -240 seconds) were normalized against DMSO control. Reactions were conducted for 12 minutes with monitoring of fluorescence intensity at 485/520 nm using a microplate reader (PHERAstar FSX, BMG Labtech). The same experiment was repeated with ubiquitin-rhodamine substrate.

All proteins used for ubiquitination and PL pro biochemical assays were expressed in E. coli RIL cells with 0.6 mM IPTG over-induction at 16 ˚C. Rsp5 WW3-HECT with 2 mutations (Q808M, E809L) at the C-terminus was expressed using GST-fusion affinity tag followed by TEV protease digestion and purification by size exclusion chromatography. His-tagged PL pro , UBA1, UBCH7, ubiquitin K63R with extra cysteine at the N-terminus were also expressed and purified similarly, except hexahistidine tag used. Ubiquitin was further cross-linked with fluorescein-5-maleimide (Anaspec, Fremont, CA, US) and the poly-ubiquitination sample was generated following previous protocol 49 . Deubiquitination assays using PL pro (SARS-CoV-2) were carried out at 37 ˚C for 10 minutes using 1-100 µM Simeprevir and 1 µM PL pro . Final DMSO concentration of each reaction is 2%. The reaction was quenched by SDS sample buffer and analyzed by 4-20% SDS-gel (GenScript, Piscataway, NJ, US). Fluorescent ubiquitin signals were imaged using Thermo iBright exposed for 750 ms.

Approximately 4 x 10 5 Vero E6 cells were seeded onto each well of 12-well plates, in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L glutamine, 25 mM HEPES and 1% penicillin/streptomycin. Infections of SARS-CoV-2 were performed at a MOI of 1 for 24 hours, followed by drug treatment or 2% DMSO in triplicates. Uninfected cells were also treated with the same concentrations of drug or DMSO in triplicates. After 24 hours of drug treatment, total RNA from infected cells and uninfected cells were extracted using Qiagen RNeasy mini kit (Qiagen) following the manufacturer's instructions. Then, we performed pair-end sequencing on a NovaSeq 6000 PE150 platform and generated 20 million reads per sample at Novogene Bioinformatics Institute (Novogene, Beijing, China).

The raw reads quality was checked by the FastQC (0.11.7) and aligned to ChlSab1.1 (Chlorocebus sabaeus) reference genome by the STAR (2.5.0a) with default parameters. The count matrix was generated by the featureCounts (as a component of Subread package 2.0.1) program. Differentially expressed genes (DEGs) were calculated by DESeq2 package (1.26.0) under R environment (3.6.1) and characterized for each sample (|L2FC| > 1, p-adjusted-value < 0.05). Gene set enrichment analysis (GSEA) was performed as previously described using normalized counts with orthology gene converting to human gene by biomaRt package (2.42.1) 50 . Bubble plots and heatmaps were generated using ggplot package and heatmap() function in R respectively.

For in vitro experiments, four-parameter dose response curve fitting was performed with constraints: Top = 1, IC50 > 0 using GraphPad Prism 8.

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We thank Dr. Martin Chan for helpful discussions during the early