key: cord-0775140-zp9qn3wv
authors: Yan, Haiyan; Sun, Jing; Wang, Kun; Wang, Huiqiang; Wu, Shuo; Bao, Linlin; He, Weiqing; Wang, Dong; Zhu, Airu; Zhang, Tian; Gao, Rongmei; Dong, Biao; Li, Jianrui; Yang, Lu; Zhong, Ming; Lv, Qi; Qin, Feifei; Zhuang, Zhen; Huang, Xiaofang; Yang, Xinyi; Li, Yuhuan; Che, Yongsheng; Jiang, Jiandong
title: Repurposing CFDA-approved drug carrimycin as an antiviral agent against human coronaviruses, including the currently pandemic SARS-CoV-2
date: 2021-03-11
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2021.02.024
sha: 6bc841e8a41cf797a84f53f69d21e798bee569e1
doc_id: 775140
cord_uid: zp9qn3wv

COVID-19 pandemic caused by SARS-CoV-2 infection severely threatens global health and economic development. No effective antiviral drug is currently available to treat COVID-19 and any other human coronavirus infections. We report herein that a CFDA-approved macrolide antibiotic, carrimycin, potently inhibited the cytopathic effects (CPE) and reduced the levels of viral protein and RNA in multiple cell types infected by human coronavirus 229E, OC43, and SARS-CoV-2. Time-of-addition and pseudotype virus infection studies indicated that carrimycin inhibited one or multiple post-entry replication events of human coronavirus infection. In support of this notion, metabolic labelling studies showed that carrimycin significantly inhibited the synthesis of viral RNA. Our studies thus strongly suggest that carrimycin is an antiviral agent against a broad-spectrum of human coronaviruses and its therapeutic efficacy to COVID-19 is currently under clinical investigation.

Coronaviruses (CoVs) are a large family of enveloped, positive-sense, single-stranded RNA viruses with broad host ranges 1 HCoV-HKU1, cause common cold and are speculated to be introduced into human population decades or even hundreds of years ago from unidentified animal hosts 6 . It is anticipated that emergence and re-emergence of CoV infections via cross species transmission will be a continuing challenge for human health and development of broad-spectrum antiviral agents against HCoVs are essential to cope with the current COVID-19 and future CoV epidemics.

Drug repurposing is an effective strategy for urgent treatment of emerging viral diseases 7, 8 .

In our efforts to search for the approved medicines that can suppress human CoV infections, an in-house collection of Chinese Food and Drug Administration (CFDA)-approved drugs including Chinese patent medicines, antibiotics, and antiviral agents were screened for their J o u r n a l P r e -p r o o f ability to protect the cytopathic effects (CPE) caused by HCoV-229E or HCoV-OC43 infection. We found a few macrolide antibiotics with antiviral activity against HCoV-229E and HCoV-OC43. Carrimycin, the most active one, was selected for further investigation of its antiviral activity against SARS-CoV-2 and determination of antiviral mechanism.

Human hepatocellular carcinoma cell lines Huh7 and Huh7. 5 

Carrimycin was provided by Shenyang Tonglian Group Co., Ltd. (Shenyang, China).

Clarithromycin, midecamycin, erythromycin, roxithromycin, acetylspiramycin, azithromycin, clindamycin, remdesivir (RDV), and ammonium chloride (NH 4 Cl) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Ribavirin (RBV) and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cytotoxic effects of carrimycin on different cells were assayed by cell counting kit (CCK, TransGen Biotech, Beijing, China). Briefly, cells were seeded into 96-well culture plates and J o u r n a l P r e -p r o o f were incubated overnight. Then, the medium was removed and different concentrations of carrimycin were applied in triplicate. After 2 days' incubation, the cytotoxicity of carrimycin was determined by CCK assay and then the 50% cytotoxic concentration (CC 50 ) was calculated.

The anti-coronavirus activity of carrimycin was determined by a CPE inhibition assay.

Briefly, cells were plated into 96-well culture plates and incubated for 24 h. The cells were infected with 100 times 50% tissue culture infective dose (TCID 50 ) HCoV-229E or HCoV-OC43 and the indicated concentrations of compounds were added simultaneously.

HCoV-229E infected Huh7 cells were treated for about 48 h and HCoV-OC43 infected H460 cells were treated for about 72 h. The 50% effective concentration (EC 50 ) was determined by

Reed & Muench method. The selectivity index (SI) was calculated as the ratio of CC 50 /EC 50 . 

For analysis of proteins, the cellular proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) with halt protease inhibitor single-use cocktail. Immunoblotting for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Boston, MA, USA, 1:1000) and coronavirus NP (Millipore, Billerica, MA, USA, 1:1000) was performed as described previously 9 .

J o u r n a l P r e -p r o o f

The total RNA of the infected cells was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The one-step qRT-PCR was performed with TransScript Taqman One-Step qRT-PCR SuperMix (for HCoV-OC43 detection) and TransScript II Green One-Step qRT-PCR SuperMix (for HCoV-229E detection) (TransGen Biotech) using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems) 9 . The applied primer sequences are shown in Table 1 .

Insert Table 1 2

The viral replication step(s) targeted by carrimycin was mapped by determining the effect of sequentially delayed addition of the compounds on viral NP expression, i.e., time-of-addition experiment 10 Table 1 11 . J o u r n a l P r e -p r o o f

Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). Image J software (Rawak Software Inc., Stuttgart, Germany) was used for quantitative study on Immunofluorescence data. Results are expressed as mean±standard deviation (SD). Data were analyzed by one-way ANOVA with Holm-Sidak's multiple comparisons test. P<0.05 was considered significant.

In order to identify the approved medicines that have a potential to be repurposed for the treatment of COVID-19, an in-house collection of more than 120 CFDA-approved drugs were screened for their ability to protect the CPE caused by HCoV-229E or HCoV-OC43 infection in Huh7 and H460 cells, respectively. Three macrolide antibiotics, acetylspiramycin, azithromycin, and carrimycin ( Fig. 1) , were found to inhibit the infection of both viruses with an SI higher than 5 ( Table 2 ). In particular, carrimycin demonstrated the highest antiviral potency (EC 50 value of 2.5 µg/mL) and selectivity (SI>20) against both HCoV-229E and HCoV-OC43 (Table 2) .

Insert Fig. 1 Insert Table 2 3

To ascertain the antiviral effects of carrimycin against HCoVs, we further examined the effects of carrimycin on the levels of viral nucleocapsid protein and RNA in infected cultures, with RBV as a positive control. As shown in Fig. 2 

To determine the HCoV replication step(s) targeted by carrimycin, we took the advantage of robust infection of C3A cells by HCoV-OC43 to perform a time-of-addition experiment 9 . As shown in Fig. 5A , delayed addition of carrimycin at 6 h post infection still inhibited the expression of viral NP by approximate 90%, which is at the similar extent to that observed under the condition of treatment starting at 1 h before the infection (Fig. 4B) EU for 1 h. The EU-labeled nascent RNA was extracted from cell lysates by using a Click-iT Nascent RNA Capture Kit. HCoV-OC43 specific nascent RNA was quantified by a qRT-PCR assay. Similar to RDV, carrimycin also significantly reduced the amounts of HCoV-OC43 nascent RNA synthesis (Fig. 6B) .

Insert Fig. 6 

Since the outbreak of COVID-19, there were no specific chemotherapeutic agents available to treat or prevent this disease. Currently, scientists around worldwide had focused on the repurposing of FDA approved drugs to treat COVID-19. Until now, more than 4000 clinical studies for COVID-19 were registered in the database of ClinicalTrials.gov Although some drugs, such as remdesivir, can play a certain therapeutic effect in clinical treatment, the efficacy and adverse reactions of these drugs still need to be clarified 13 .

Streptomyces spiramyceticus harboring a 4″-O-isovaleryltransferase gene (ist) from Streptomyces thermotoleran. It mainly consists of isovalerylspiramycins (ISP) I-III, with trace amounts of other 4″-acylspiramycins [14] [15] [16] . Comparing with acylspiramycin, the longer alkyl chains at positions 4″ renders carrimycin more potent antibacterial activity, especially in vivo, as a result of higher lipophilicity 17, 18 . Carrimycin was recently approved by CFDA for the treatment of acute tracheal-bronchitis caused by haemophilus influenzae, streptococcus pneumoniae, and for the treatment of acute sinusitis caused by streptococcus pneumoniae, haemophilus influenzae, streptococcus pyogenic, moraxella catarrhalis, and staphylococcus.

In this study, it was found that carrimycin exhibited broad-spectrum antiviral activity against HCoVs in multiple cells lines. As shown in Table 1 , carrimycin showed the higher antiviral potency than acetylspiramycin. It remains to be determined whether the enhanced antiviral activity of carrimycin, as compared to acylspiramycin, is the result of higher lipophilicity and membrane permeability 17, 18 .

Concerning the antiviral mechanism, the time-of-addition and pseudotyped lentiviral infection assays suggest that carrimycin efficiently inhibited the infection of multiple HCoVs by targeting one or multiple post-entry replication events. As positive strand RNA viruses, coronaviruses synthesize their RNA in the cytoplasmic replicase complexes consisting of viral nsp12-nsp7-nsp-8 core polymerase and cellular co-factors 19, 20 . As shown in Fig. 6 , similar to HCoV RNA polymerase inhibitor RDV, carrimycin significantly inhibited HCoV-OC43 RNA synthesis. Macrolide antibiotics inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit 21 

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We gratefully acknowledge Professor Ju-Tao Guo (Baruch S. Blumberg Institute, PA, USA) for helpful discussions and expert advice on the manuscript. The work was financially 

The authors declare no conflicts of interest.

HCoV