key: cord-0750426-y1v8wzxq
authors: Yuan, Shuofeng; Yin, Xin; Meng, XiangZhi; Chan, Jasper; Ye, Zi-Wei; Riva, Laura; Pache, Lars; Chan, Chris Chun-Yiu; Lai, Pok-Man; Chan, Chris; Poon, Vincent; Matsunaga, Naoko; Pu, Yuan; Yuen, Chun-Kit; Cao, Jianli; Liang, Ronghui; Tang, Kaiming; Sheng, Li; Du, Yushen; Xu, Wan; Sze, Kong-Hung; Zhang, Jinxia; Chu, Hin; Kok, Kin-Hang; To, Kelvin; Jin, Dong-Yan; Sun, Ren; Chanda, Sumit; Yuen, Kwok-Yung
title: Clofazimine is a broad-spectrum coronavirus inhibitor that antagonizes SARS-CoV-2 replication in primary human cell culture and hamsters
date: 2020-10-07
journal: Res Sq
DOI: 10.21203/rs.3.rs-86169/v1
sha: 34e6f10060cca5243fca223de6d768cbc059f54c
doc_id: 750426
cord_uid: y1v8wzxq

COVID-19 pandemic is the third zoonotic coronavirus (CoV) outbreak of the century after severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) since 2012. Treatment options for CoVs are largely lacking. Here, we show that clofazimine, an anti-leprosy drug with a favorable safety and pharmacokinetics profile, possesses pan-coronaviral inhibitory activity, and can antagonize SARS-CoV-2 replication in multiple in vitro systems, including the human embryonic stem cell-derived cardiomyocytes and ex vivo lung cultures. The FDA-approved molecule was found to inhibit multiple steps of viral replication, suggesting multiple underlying antiviral mechanisms. In a hamster model of SARS-CoV-2 pathogenesis, prophylactic or therapeutic administration of clofazimine significantly reduced viral load in the lung and fecal viral shedding, and also prevented cytokine storm associated with viral infection. Additionally, clofazimine exhibited synergy when administered with remdesivir. Since clofazimine is orally bioavailable and has a comparatively low manufacturing cost, it is an attractive clinical candidate for outpatient treatment and remdesivir-based combinatorial therapy for hospitalized COVID-19 patients, particularly in developing countries. Taken together, our data provide evidence that clofazimine may have a role in the control of the current pandemic SARS-CoV-2, endemic MERS-CoV in the Middle East, and, possibly most importantly, emerging CoVs of the future.

The current pandemic of novel Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a global public health crisis. SARS-CoV-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~ 6.4% worldwide 1 . As of September 29, 2020, over 33 million cases had been reported in 235 countries, areas or territories with more than 1 million deaths, whereas a sizable portion of infected but non-symptomatic people with potential of transmissibility was also reported 2 .

The genetically diverse coronavirus (CoV) family, currently composed of four genera (α, β, γ, and δ), infects birds, bats and a variety of mammals 3 . Within a decade, the world's human population has undergone three major CoV outbreaks. SARS-CoV-1 emerged in Guangdong, China in 2002 and, with the aid of commercial air travel, spread rapidly and globally, causing more than 8,000 cases with 10% Currently, there are no widely available speci c antiviral therapies for CoV in humans. Remdesivir exhibited pan-coronavirus inhibitory potential 6 , and was recently granted emergency use authorization by the FDA for the treatment of COVID-19 based on the signi cant reduced time to recovery 7 . However, the therapy is far from optimal, particularly for severe COVID-19 patients, and can only be administered intravenously to hospitalized patients 8, 9 . Thus development of additional therapeutic options is urgent, as well as the establishment of combinatorial regimens, such as the triple antiviral combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin, which has been shown to be bene cial in a clinical trial 10 .

In efforts to accelerate the development of novel therapies for COVID-19, we previously pro led a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules 11 . In this study, we focused on the antiviral mechanisms of action and in vivo e cacy of clofazimine, an FDA-approved molecule discovered as an anti-tuberculosis drug in 1957 and later used for treatment of leprosy 12 . Treating tuberculosis, clofazimine exhibits a minimum inhibitory concentration of 0.016 µg/ml (equivalent to 33.80 nM). The effective concentration of clofazimine against SARS-CoV-2 (half maximal effective concentration 310 nM) is clinically achievable with standard dosage in patients (peak serum concentration 861 nM) 13 . Here, we report the capability of clofazimine to confer protection against SARS-CoV-2 infection in primary human cell and animal models. Most importantly, clofazimine is affordable by COVID-19 patients in developing countries which may substantially relieve the critical care pressure caused by continuing pandemic 14 .

Clofazimine has been found to be well tolerated in humans, showing a desirable safety pro le at doses of 200mg/day in human 13 , a C max of > 861nM, and a selectivity index (CC 50 /EC 50 ) around 30~50 against SARS-CoV-2 infection 15 ( Figure 1a ). These data suggest that therapeutic dosing of clofazimine may be achievable in patients at concentrations likely to have in vivo antiviral activity. Using SARS-CoV-2 infection as a model, we further characterized the antiviral activity of clofazimine in human embryonic stem cell-derived cardiomyocytes that robustly support SARS-CoV-2 replication 16 . Strikingly, and in a dose-dependent manner, clofazimine treatment reduced viral titers in the cell lysate by >3-log10 at a concentration of 10µM when compared with the DMSO control ( Figure 1b ). Next, we assessed the antiviral activity of clofazimine in an ex vivo lung culture system. Donor lung tissue was infected with SARS-CoV-2 for 24 h with drug treatment starting at 2 hours post-inoculation (hpi). Our results revealed that clofazimine potently antagonized viral replication in tissues that re ect the primary site of SARS-CoV-2 replication (Figure 1c ). To explore whether clofazimine confers protection against another epidemic CoV, we performed a plaque reduction assay for MERS-CoV. Clofazimine reduced MERS-CoV replication in VeroE6 cells with an EC 50 of 1.48±0.17 µM (Figure 1d ). Immuno uorescence staining for MERS-CoV NP illustrated dramatic suppression of virus infection upon clofazimine treatment (upper panel, Figure 1e ), which is supported by the ow cytometry analysis that the percentage of MERS-CoVinfected cells after clofazimine treatment decreased from 44.6% (DMSO) to 23.0% (clofazimine) at 24 hpi in Huh7 cells (lower panel, Figure 1e ). Overall, clofazimine exhibited potent broad spectrum anti-CoV, and antagonized SARS-CoV-2 replication in human primary cell and ex vivo lung models.

To understand the impact of clofazimine on the virus life cycle, antiviral activity was rst evaluated by a time-of-drug addition assay in a single infectious cycle. Treatment with clofazimine during inoculation strongly inhibited SARS-CoV-2 infection, indicating that clofazimine exerts inhibitory effect on viral entry. Intriguingly, clofazimine also blocked SARS-CoV-2 infection at a post-entry step as evidenced by the observed reduction of viral infection when clofazimine was added at 5 hpi (Figure 2a ). To further evaluate the impact of clofazimine on viral entry, we employed vesicular stomatitis virus (VSV)-based SARS-CoV-2 Spike (S) pseudotyped virions. Clofazimine treatment dramatically reduced the infectivity of both SARS-CoV-1 S and SARS-CoV-2 S pseudotyped virions in VeroE6 cells. Interestingly, clofazimine did not impact MERS-CoV S pseudotyped virus particles (Figure 2b) , and this lack of entry inhibition may contribute to a lower potency observed for MERS-CoV. To con rm whether clofazimine also inhibits post-entry steps of viral replication, we evaluated the impact of clofazimine on viral RNA production by electroporating in vitro transcribed viral RNA into VeroE6 cells, which bypasses clofazimine-mediated inhibition on the entry process, and directly measures RNA synthesis ( Figure 2c ). As expected, remdesivir could effectively reduce the synthesis of negative-stranded RNA in a dose-dependent manner ( Figure 2d ). Intriguingly, viral RNA levels were also reduced by 1~1.5 logs in the cells treated with clofazimine at concentrations above 5 µM (Figure 2e ). However, no signi cant effect was observed on electroporated GFP mRNA translation ( Figure 2f) . Collectively, these results demonstrated that clofazimine inhibit multiple steps in SARS-CoV-2 replication by interfering with spike-mediated entry as well as viral RNA replication.

To explore what is the impact of clofazimine on the transcriptional response of host cells, we employed RNA-Seq to pro le the transcriptomic-wide changes during clofazimine treatment. We found that in human colorectal Caco-2 cells, clofazimine exhibited comparable anti-SARS-CoV-2 potency as that of remdesivir (Figure 3a) , which was chosen for the downstream analysis. Transcriptional analysis was performed in Caco-2 cells which were either infected with SARS-CoV-2, treated with clofazimine (10 µM) or both. Principal Component Analysis (PCA) on RNA-Seq results suggested that at 3 hpi, clofazimine treatment (3hpi. CFZ) caused overall transcriptome shift towards mock-infection group when compared with the vehicle control group (3 hpi) (Figure 3b ), which is consistent with our data indicating that the drug inhibits viral infection at early time point post infection (Figure 2 ). At 6 hpi, there were 607 and 448 genes up-and down-regulated by SARS-CoV-2 infection, respectively (FDR<0.05, fold change>2 or <0.5 compared with mock). The RNA level of more than 90% of these genes was reverted by clofazimine treatment, indicating that clofazimine treatment abrogated transcriptomic changes caused by SARS-CoV-2 infection. This is consistent with the PCA plot that treatment with clofazimine at 6 hpi (6 hpi. CFZ) caused a dramatic shift towards mock (Figure 3b , Extended Data Figure 1a) . Interestingly, clofazimine treatment in the absence of infection (6h. CFZ) up-regulated genes that were enriched into innate immunity-related pathways, including MAPK, interleukin and TNF responses (Figure 3c and 3d, Extended Data Figure 1b) . Particularly, transcription factors critical for immediate-early cellular response, including AP-1, SMAD, MAFF families, were upregulated by clofazimine (Figure 3c ). When clofazimine was applied onto infected cells, most of these innate immune pathways were further enriched in upregulated genes (6hpi. CFZ, Figure 3d , Extended Data Figure 1b and 1c) . These results suggest that clofazimine rewires the transcriptional landscape to prime the innate immunity-related pathways. While de cient early stage innate immune responses have been attributed to poor disease outcome, additional studies are required to determine if this enhanced antiviral response contributes to the in vitro and in vivo e cacy of the drug 17-19 .

Clofazimine is useful for the treatment of disease due to multidrug resistant Mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases 13 .Previous pharmacokinetics studies revealed that clofazimine absorption varies from 45 to 62% following oral administration in leprosy patients. Coadministration of a 200mg dose of clofazimine with food resulted in a C max of 0.41 mg/L (equivalent to 861 nM) with a T max of 8h. Administered in a fasting state, however, the corresponding C max of clofazimine was 30% lower while the time to C max was 12h 20 . Intriguingly, clofazimine exerts antiin ammatory properties due to the suppression of macrophage activity, which may further mitigate the cytokine storm of SARS-CoV-2 infection in addition to its direct antiviral effects 21 . To determine the in vivo antiviral e cacy of clofazimine, we employed a golden Syrian hamster model that serves as a suitable tool to study antiviral effects and disease pathogenesis 22 . Since administration of clofazimine with a high fat meal provides better bioavailability 13 , we delivered the drug through oral route utilizing corn oil as vehicle. 25 mg/kg/day of clofazimine given on 3 consecutive days exhibited no signi cant observable toxicity to the animals. Remdesivir was included as a positive control drug and dosed at 15 mg/kg/day based on its effective dosage in SARS-CoV-infected mice 6 .

Clofazimine has a relatively long duration of action with the mean elimination half-life approximately 25 days, thus we performed prophylactic treatment of hamsters with clofazimine before intranasally challenged with 10 5 PFU of SARS-CoV-2 ( Figure 4a ). Expectedly, the DMSO-treated control hamsters developed the clinical signs of lethargy, hunched back posture, and rapid breathing starting from 2 dpi, whereas the hamsters treated with clofazimine did not develop any clinical signs. At 2 dpi when the viral loads and histopathological changes were expected to be worse, clofazimine decreased virus plaque forming units in lung tissues by ~1 to 1.5 logs (Figure 4b) . Consistently, suppression of SARS-CoV-2 viral load in hamster lungs was con rmed in the clofazimine-treated hamsters (Figure 4c ). To explore if the presence of clofazimine in the gastrointestinal tract, after intragastric administration, would prevent SARS-CoV-2 shedding, animal feces were collected at 2 dpi for viral RNA detection. Signi cantly less (p=0.0353) viral copies were detectable in clofazimine-treated group when compared with the DMSO group, indicating its potential to diminish fecal shedding of SARS-CoV-2 ( Figure 4d ). Increased proin ammatory cytokines and chemokines is associated with disease severity of COVID-19 patients. To ascertain if the therapeutic effect of clofazimine alleviates virus-induced cytokine dysregulation, we determined the expression levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and C-C chemokine receptor type 4 (CCR4), which are prognostic markers for severe COVID-19 23 . As shown in Figure 4e , mRNA expression of IL-6 (p=0.0001), TNF-α (p=0.0006), and CCR4 (p=0.0029) were remarkably reduced in the hamsters treated with clofazimine. Previous reports have shown that clofazimine can inhibit lymphocyte function 24 . To explore if this is the case in our animal model, hamster sera were collected at 14dpi for measurement of anti-NP antibody using an ELISA-based enzyme immunoassay. Apparently, similarly high levels of antibody responses were triggered in DMSO and clofazimine groups, indicating insigni cant suppression of humoral immune response of B lymphocyte by clofazimine ( Figure 4i ). Taken together, prophylactic administration of clofazimine conferred protection against SARS-CoV-2 challenge by reducing the virus replication and the associated in ammatory dysregulation.

To recapitulate the scenario that most COVID-19 patients will receive treatment after diagnosis or disease onset, it was of interest to determine whether therapeutic treatment of clofazimine, with the rst dosing given 24 h after virus exposure, would also ameliorate SARS-CoV-2 disease. SARS-CoV-2 infected hamsters were given 3 doses in total before being scari ed at 4 dpi for lung viral yield detection. Generally, both therapeutic clofazimine and remdesivir suppressed virus lung titers when compared with the DMSO control (Figure 4f and 4g). The diminished clinical signs were also associated with substantially decreased IL-6 protein amount in the clofazimine (p=0.0119) and remdesivir-treated (p=0.0074) hamster sera (Figure 4h ), as increased serum IL-6 level has been correlated with respiratory failure and adverse clinical outcome 25 .

As for the severity of lung damage, histological examination of hematoxylin and eosin (H&E) stained lung tissues was performed. Signi cant amelioration of lung damage was observed after clofazimine treatment (Figure 4j ). For prophylactic administration, lung tissues from the DMSO group showed severe bronchiolar cell death with massive cell debris lling the lumen, alveolar wall thickened with alveolar exudation; whereas prophylaxis with clofazimine showed no apparent pathological changes. With therapeutic administration, DMSO-treated lung sections showed large areas of lung consolidation with alveolar in ltration and exudation, while clofazimine treated lungs exhibited a mild degree of alveolar wall thickening and capillary congestion. Generally, prophylactic administration conferred more dramatic improvements of lung pathology when compared with therapeutic administration, which might be attributed to the relatively long T max of clofazimine. Nevertheless, both prophylactic and therapeutic treatment with clofazimine reduced SARS-CoV-2 disease in vivo.

Since the emergency use authorization by the US FDA, remdesivir is considered the standard of care for the treatment of COVID-19. To understand the impact of combinatorial treatments of remdesivir and clofazimine on SARS-CoV-2 replication, we conducted a matrixed dose response analysis. We found that co-application of clofazimine and remdesivir impacts SARS-CoV-2 replication in a manner that extends beyond the additive combinatorial activity predicted by the Bliss independence model (maximal Bliss Synergy Score of 44.28; Figure 5a , Extended Data Figure 2) , and indicates these two drugs harbor a synergistic antiviral relationship. Clofazimine can be safely dosed at 200 mg/day for the treatment of leprosy, which results in average serum concentrations of 1.79 µM, although the bioavailable fraction of the molecule will be a function of plasma protein binding. The addition of 1.25 µM clofazimine in an in vitro cellular assay with a 10% concentration of FBS resulted in a nearly 20-fold decrease in concentrations of remdesivir required to inhibit viral replication by 90% (Figure 5b) . Importantly, the combination of drugs did not elicit additional cellular cytotoxicity (Figure 5c ).

Clofazimine was rst used to treat leprosy in 1969, and gained FDA approval in 1996 26 . It is an orally bioavailable drug that is included in the WHO Model List of Essential Medicines. It is generally welltolerated, with adverse events that include skin discoloration, ichthyosis, and gastrointestinal intolerance 27 . Besides treating leprosy, clofazimine is an intriguing medication that has implications for multi-drugresistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). Showing good safety evidence, clofazimine is a part of WHO group C in terms of the treatment guidelines for MDR-TB 28 . This is supported by clinical trials in China, Bangladesh, and Brazil where patients were receiving clofazimine for 18~21 months at a dose of 100 mg/day 29, 30 .

We observed that clofazimine shows pleotropic antiviral activities against SARS-CoV-2, including inhibition of spike-dependent entry. While it has been reported that clofazimine is internalized through endocytosis, further investigation is required to elucidate if the drug directly impinges on endosomal function to inhibit viral entry 31 , and why the leprosy drug selectively blocks SARS-CoV, but not MERS-CoV, entry into cells. Importantly, this drug is a lipophilic rhimophenazine dye which inhibits mycobacteria through intercalation into bacterial DNA, likely inhibiting DNA replication and proliferation 32 . While we observe that clofazimine inhibits the RNA replication of SARS-CoV-2, additional studies are also required to determine if the drug similarly inhibits CoV RNA unwinding or template function.

In SARS-CoV-2 infection, a delayed innate immune response may result in uncontrollable cytokine storm 19, 33 . Clofazimine's effect on rewiring the transcriptional landscape of the cell towards an antiviral status may be important in a disease setting, and understanding the contribution of this activity toward in vivo disease amelioration can provide insight towards its potential to improve viral control through enhancement of innate immune activities. Paradoxically, clofazimine has been reported to possess antiin ammatory activity through the inhibition of macrophage function and T lymphocyte activation and proliferation 34 . Further elucidation of how clofazimine treatment may balance regulation of innate and adaptive immune responses to improve disease outcome will be important to understand its potential clinical e cacy.

In this study, a prophylaxis regimen with three daily doses substantially protected animal from SARS-CoV-2 infection (Figure 4) . In contrast to orally bioavailable clofazimine, remdesivir is currently given through intravenous administration, which makes it di cult to provide on an outpatient or prophylactic basis. Moreover, remdesivir requires a complex synthesis process to manufacture, resulting in a high treatment cost (US$520 per vial, or US$3,120 per treatment course) and availability for only several million patients over the next two years 35 . In view of the potentially-long epidemic dynamics and pressures on critical care capacity over the next 5 years, as well as the potential resurgence of SARS-CoV-2 in the future, clofazimine, which only costs US$1.43/100mg tablets, can be considered as one of the potential countermeasures for global control of the COVID-19 pandemic 14 , especially in developing countries. Additionally, co-administration with clofazimine could signi cantly reduce costs for remdesivir-based treatment of COVID-19, and extend worldwide supplies of remdesivir, and a combinatorial approach can also help mitigate the emergence of drug-resistant viral strains.

Clofazimine, in conjunction with interferon, is currently being evaluated in clinical trials for the treatment of COVID-19 (Trial Number: NCT04465695). The ex vivo and in vivo e cacy of clofazimine suggests that clinical evaluation of the drug as monotherapy in outpatient setting for treatment of early stage disease, or in combination with remdesivir in hospitalized patients, is critical for establishing its potential as a rapidly scalable treatment option for COVID-19.

Human hepatoma Huh7 (JCRB, 0403) cells and monkey VeroE6 cells (ATCC, CRL-592 1586) were maintained in DMEM culture medium supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin and 50 µg/ml streptomycin. Ventricular cardiomyocyte were differentiated from the human embryonic stem cell HES2 (ESI) maintained in mTeSR1 medium (STEMCELL Technologies) 36 . Brie y, HES2 cells were dissociated with Accutase (Invitrogen) into single cells suspensions on day 0. Cells were seeded on low-attachment culture vessels (Corning) and cultured in mTeSR1 medium supplemented with 40 µg/ml Matrigel, 1 ng/ml BMP4 (Invitrogen) and 10µM Rho kinase inhibitor (ROCK) (R&D) under hypoxic environment with 5% O 2 . From day 1 to 3, cells were cultured in StemPro34 SFM (Invitrogen) with 50 µg/ml ascorbic acid (AA) (Sigma), 2 mM Gluta-MAX (Invitrogen), 10 ng/ml BMP4, and 10 ng/ml human recombinant activin-A (Invitrogen). From day 4 to day 7, 5 µM Wnt inhibitor IWR-1(Tocris) was added. Antiviral evaluation in human ex vivo lung tissues Human lung tissues for ex vivo studies were obtained from patients undergoing surgical operations at Queen Mary Hospital, Hong Kong as previously described 38 . The donors gave written consent as approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364). The freshly obtained lung tissues were processed into small rectangular pieces and were rinsed with advanced DMEM/F12 medium (Gibco) supplemented with 2 mM of HEPES (Gibco), 1×GlutaMAX (Gibco), 100 U/ml penicillin, and 100 μg/mL streptomycin. The specimens were infected with SARS-CoV-2 HKU-001a with an inoculum of 1×10 6 PFU/ml at 500 μL per well. After two hours, the inoculum was removed, and the specimens were washed 3 times with PBS. The infected human lung tissues were then cultured in 1 ml of advanced DMEM/F12 medium with 2 mM HEPES (Gibco), 1×GlutaMAX (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, 20 μg/mL vancomycin, 20 μg/mL cipro oxacin, 50 μg/mL amikacin, and 50 μg/mL nystatin. Supernatants were collected at 24 hours post inoculation (hpi) for plaque assays.

Male and female Syrian hamster, aged 6-10 weeks old, were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum as we previously described 22 . All experimental protocols were approved by the Animal Ethics Committee in the University of Hong Kong (CULATR) and

were performed according to the standard operating procedures of the biosafety level 3 animal facilities (Reference code: CULATR 5370-20). Experimentally, each hamster was intranasally inoculated with 10 5 PFU of SARS-CoV-2 in 100 μL PBS under intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) anesthesia. Prophylactic treatment used oral administration of clofazimine given on -3, -2 and -1dpi (25 mg/kg/day), followed by virus challenge at 0dpi, while therapeutic post-exposure and oral administration of clofazimine were performed on 1, 2, and 3 dpi (25 mg/kg/day) with the rst dosage given at 24 hpi. Clofazimine was delivered using corn oil (Sigma-Aldrich, C8267) as vehicle. Remdesivir (15 mg/kg/day, MedChemExpress) was used as a positive control through intraperitoneal injection. One percent DMSO in corn oil was used as a placebo control through oral route. Animals were sacri ced at 2 dpi and 4 dpi for virological and histopathological analyses. Viral yield in the lung tissue homogenates and/or feces were detected by plaque assay and/or qRT-PCR methods. Cytokine and chemokine pro le of the hamster lungs were detected by 2 -ΔΔCT method using probe-based one step qRT-PCR (Qiagen). ELISA kit was utilized to determine the Interleukin 6 (IL-6) amount in the hamster sera on 4 dpi according to the manufacture's recommendations (ELISAGenie, HMFI0001). Tissue pathology of infected animals was examined by H&E staining in accordance to the established protocol 39 . On 14 dpi, enzyme immunoassay (EIA) was utilized to determine the antibody titer of hamster sera against SARS-CoV-2 NP antigen 40 . Brie y, 96-well immune-plates (Nunc) were coated with 100 μL/well (0.1 μg/well) of SARS-CoV-2 NP in 0.05 M NaHCO 3 (pH 9.6) overnight at 4°C. After blocking, 100 μL of heat-inactivated serum samples were serial-diluted before adding to the wells and incubated at 37°C for 1 h. The attached antibodies were detected using horseradish-peroxidase-conjugated rabbit anti-hamster IgG antibody (Invitrogen, A18895). The reaction was developed by adding diluted 3,3',5,5'-tetramethylbenzidine single solution (Invitrogen) and stopped with 0.3 N H 2 SO 4 . The optical density (OD) was read at 450/620 nm using a microplate reader.

Fastq les from RNA-seq were quality examined by FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were processed by cutadapt to remove reads with low quality and to trim adapters. Trimmed reads were aligned to hg38 reference genome using Tophat2 41 . Reads assigned to each gene were counted by featureCounts 42 with refseq gene sets as references. Genes without at least 1 read mapped on average in each sample were considered undetectable and were ltered out. Read counts were normalized by Trimmed Mean of Mvalues (TMM) method and differential expression was calculated using R package edgeR and Genewise Negative Binomial Generalized Linear Models with Quasi-likelihood Tests (glmQLFit) method was used for statistical tests. Cut-offs imposed for differential expression analysis was set as False Discovery Rate (FDR) of 0.05 and fold change >2 or <0. 5 Pseudotyping of VSV and Pseudotype-based inhibition assay Vesicular Stomatitis Virus (VSV) pseudotyped with spike proteins of MERS-CoV, SARS-CoV-1, and SARS-CoV-2 were generated as previously reported with some modi cations 45 . Brie y, BHK-21/WI-2 cells (Kerafast, MA) overexpressing the spike proteins were inoculated with VSV-G pseudotyped ΔG-luciferase VSV (Kerafast, MA). After 2 h inoculation at 37°C, the inoculum was removed and cells were refed with DMEM supplemented with 5% FBS and VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC). Pseudotyped particles were collected at 24 h post-inoculation, then centrifuged at 1,320 × g to remove cell debris and stored at −80°C until use.

To determine the effect of the compounds on viral entry, VeroE6 cells were treated with clofazimine at a concentration of 2.5 µM for 1 h prior to inoculation with respective pseudotyped VSV. After 2 h inoculation in the presence of the compounds, the inoculum was removed and cells were refed with fresh medium for further culture. The activity of re y luciferase was measured using bright-Glo™ luciferase assay (Promega) for quantitative determination at 16 h post-transduction.

The full-length SARS-CoV-2 viral RNA transcripts were in vitro synthesized from an infectious clone of SARS-CoV-2 (kindly provided by Pei-Yong Shi, UTMB ) according to a recently published protocol 46 . 10 µg of total RNA transcripts and 5 μg SARS-CoV-2 NP gene transcript were mixed with VeroE6 cells stably expressing SARS-CoV-2 NP protein and then added into a 0.2 cm cuvette for nucleofection with the 4D-NucleofectorTM Core Unit (Lonza) using pulse code V-001. Immediately after electroporation, 1000 µL of pre-warmed media was added to the cuvette and cells were subsequently aliquoted into 384-well plates. Two hours post-seeding, compounds at different concentrations were added into each well. At 12 hours post-electroporation, intracellular and viral RNA was puri ed from the treated cells with TurboCapture 384 mRNA Kit (Qiagen) in accordance with the manufacturer's instructions. The puri ed RNA was subjected to rst-strand cDNA synthesis using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Inc) with the following primer (TagRdRp-F: 5'-CGGTCATGGTGGCGAATAACCCTGTGGGTTTTACACTTAA-3'). Real-time PCR analysis was performed using TaqPath 1-step RT-qPCR Master Mix (Applied Biosystems, Inc). The following primers and probe were used for negative-stranded RNA detection: Tag-F: 5'-CGGTCATGGTGGCGAATAACCCTGT-3', ORF1ab-R: 5'-ACGATTGTGC ATCAGCTGA-3', ORF1ab-P:

Time-of-addition assay Time-of-drug-addition assay was performed to investigate which stage of SARS-CoV-2 life cycle clofazimine interfered with as previously described 47 . Brie y, VeroE6 cells were seeded in 96-well plates (4×10 4 cells/well). The cells were infected by SARS-CoV-2 USA-WA1/2020 at an MOI of 1.5 and then incubated for additional 1 h. The viral inoculum was then removed and the cells were washed twice with PBS. At 1 hpi (i.e., post entry), clofazimine at a concentration of 5 µM was added to the infected cells at time-points indicated, followed by the incubation at 37 °C in 5% CO2 until 10 hpi (i.e. one virus life cycle).

Cells were xed at 10 hpi for quanti cation of the percentage of infected cells using an immuno uorescence assay targeting SARS-CoV-2 NP.

Complete sequences of SARS-CoV-2 HKU-001a and SARS-CoV-2 USA-WA1/2020 are available through GenBank (accession numbers MT230904 (HKU-001a), MT246667 and MN908947 (USA-WA1/2020)). The raw RNA-Seq data reported in Figure 3 have been deposited in GEO. Other supporting raw data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Foundation Limited, Tse Kam Ming Laurence, and Norman & Cecilia Yip Foundation. This work was also supported by the grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972, as well as generous philanthropic donations from Dinah Ruch and Susan & James Blair. This research was also partly funded by CRIP (Center for Research for In uenza Pathogenesis), a NIAID supported Center of Excellence for In uenza Research and Surveillance (CEIRS, contract # HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by an administrative supplement to NIAID grant U19AI142733, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)). The funding sources had no role in the study design, data collection, analysis, interpretation, or writing of the report. 63/010630, entitled Methods and Compositions for Antiviral Treatment relates to aspects of this work and was led on 15 April 2020. The corresponding authors had full access to all the data in the study and had nal responsibility for the decision to submit for publication. The other authors declare no competing interests.

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Metabolism of clofazimine in leprosy patients

Receptor Antagonist Production To Induce a Systemic Anti-In ammatory State

Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility

Interleukin-6 as a potential biomarker of COVID-19 progression

Clofazimine: current status and future prospects

Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19

Safety and availability of clofazimine in the treatment of multidrug and extensively drug-resistant tuberculosis: analysis of published guidance and meta-analysis of cohort studies

Evaluation of Clinical Drug Interaction Potential of Clofazimine Using Static and Dynamic Modeling Approaches

World Health Organization treatment guidelines for drug-resistant tuberculosis, 2016 update

Clofazimine for the treatment of multidrug-resistant tuberculosis: prospective, multicenter, randomized controlled study in China

Effectiveness and safety of clofazimine in multidrug-resistant tuberculosis: a nationwide report from Brazil

Molecular imaging of intracellular drug-membrane aggregate formation

The mode of action of clofazimine DNA binding studies. International journal of leprosy and other mycobacterial diseases : o cial organ of the International Leprosy Association

Longitudinal analyses reveal immunological mis ring in severe COVID-19

Clofazimine Inhibits Human Kv1.3 Potassium Channel by Perturbing Calcium Oscillation in T Lymphocytes

A simple, cost-effective but highly e cient system for deriving ventricular cardiomyocytes from human pluripotent stem cells

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target

Comparative replication and immune activation pro les of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19

SARS-CoV-2 infects and damages the mature and immature olfactory sensory neurons of hamsters

Temporal pro les of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions

featureCounts: an e cient general purpose program for assigning sequence reads to genomic features

clusterPro ler: an R package for comparing biological themes among gene clusters

Cytoscape: a software environment for integrated models of biomolecular interaction networks

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

An Infectious cDNA Clone of SARS-CoV-2

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing

SynergyFinder 2.0: visual analytics of multi-drug combination synergies

The authors acknowledge the assistance of the University of Hong Kong Li Ka Shing Faculty of Medicine Centre for PanorOmic Sciences. This study was partly supported by funding to University of