key: cord-336793-9bbyu1qx
authors: Matsuyama, Shutoku; Kawase, Miyuki; Nao, Naganori; Shirato, Kazuya; Ujike, Makoto; Kamitani, Wataru; Shimojima, Masayuki; Fukushi, Shuetsu
title: The inhaled steroid ciclesonide blocks SARS-CoV-2 RNA replication by targeting viral replication-transcription complex in culture cells
date: 2020-08-24
journal: bioRxiv
DOI: 10.1101/2020.08.22.258459
sha: 
doc_id: 336793
cord_uid: 9bbyu1qx

We screened steroid compounds to obtain a drug expected to block host inflammatory responses and MERS-CoV replication. Ciclesonide, an inhaled corticosteroid, suppressed replication of MERS-CoV and other coronaviruses, including SARS-CoV-2, the cause of COVID-19, in cultured cells. The effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 μM. Ciclesonide inhibited formation of double membrane vesicles, which anchor the viral replication-transcription complex in cells. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in non-structural protein 3 (nsp3) or nsp4. Of note, ciclesonide still suppressed replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. Importance The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. To identify the effective antiviral agents to combat the disease is urgently needed. In the present study, we found that an inhaled corticosteroid, ciclesonide suppresses replication of coronaviruses, including beta-coronaviruses (MHV-2, MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alpha-coronavirus (HCoV-229E) in cultured cells. The inhaled ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS, and COVID-19.

In the preprint of this study (posted in BioRxiv)(14), we showed that a corticosteroid, ciclesonide, is a 62 potent blocker of SARS-CoV-2 replication. Based on the data in our preprint study, clinical trials of a 63 retrospective cohort study to treat COVID-19 patients were started in Japanese hospital in March 64 2020. The treatment regime involves inhalation of 400 μ g ciclesonide (two or three times per day) for

The 92 steroid compounds chosen from the Prestwick Chemical Library were examined to assess the 74 inhibitory effects of MERS-CoV-induced cytopathic effects. Vero cells treated with steroid 75 compounds were infected with MERS-CoV at an MOI = 0.1 and then incubated for 3 days. Four 76 steroid compounds, ciclesonide, mometasone furoate, mifepristone, and algestone acetophenide, 77 conferred a > 95% cell survival rate (Fig. 1) . Interestingly, a structural feature of these compounds is a 78 five-or six-membered monocycle attached to the steroid core. (Table   147 1). We examined replication of these mutants in the presence of ciclesonide. First, one of these 148 isolates was tested in VeroE6/TMPRSS2 cells. At 6 hpi, the amount of viral RNA derived from the 149 parental virus fell by 1000-fold in the presence of ciclesonide; by contrast, the amount of RNA 150 derived from the escape mutant increased 50-fold compared with that of the parent virus (Fig. S2 ).

There was no difference between the parental virus and the escape mutant in the presence of other suppressed replication of all escape mutants by 90% or more, suggesting that these mutants cannot 156 completely overcome ciclesonide blockade. Mutations in the ciclesonide escape mutants were 157 identified at three positions in nsp3 and at one position in nsp4 (Fig. 6b) . Of note, the amino acid 158 substitution N1543K in nsp3 was caused by a different base change (T7348G and T7348A) ( Table 1) 

Average of cell viability in the absence of virus was quantified using a WST assay (n=2, in panel b and d). 

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The inhaled corticosteroid ciclesonide blocks coronavirus RNA replication by targeting viral

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