bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.258459; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The inhaled steroid ciclesonide blocks SARS-CoV-2 RNA replication by targeting viral 2 replication-transcription complex in culture cells 3 4 Shutoku Matsuyamaa#, Miyuki Kawasea, Naganori Naoa, Kazuya Shiratoa, Makoto Ujikeb, Wataru 5 Kamitanic, Masayuki Shimojimad, and Shuetsu Fukushid 6 7 aDepartment of Virology III, National Institute of Infectious Diseases, Tokyo, Japan 8 bFaculty of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo, Japan 9 cDepartment of Infectious Diseases and Host Defense, Gunma University Graduate School of 10 Medicine, Gunma, Japan 11 dDepartment of Virology I, National Institute of Infectious Diseases, Tokyo, Japan. 12 13 Running Head: Ciclesonide blocks SARS-CoV-2 replication 14 15 #Address correspondence to Shutoku Matsuyama, [email protected] 16 17 Word count: Abstract 149, Text 3,016 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.258459; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 Abstract 19 We screened steroid compounds to obtain a drug expected to block host inflammatory responses and 20 MERS-CoV replication. Ciclesonide, an inhaled corticosteroid, suppressed replication of MERS-CoV 21 and other coronaviruses, including SARS-CoV-2, the cause of COVID-19, in cultured cells. The 22 effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial 23 tracheal epithelial cells was 0.55 μM. Ciclesonide inhibited formation of double membrane vesicles, 24 which anchor the viral replication-transcription complex in cells. Eight consecutive passages of 43 25 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single 26 amino acid substitutions in non-structural protein 3 (nsp3) or nsp4. Of note, ciclesonide still 27 suppressed replication of all these mutants by 90% or more, suggesting that these mutants cannot 28 completely overcome ciclesonide blockade. These observations indicate that the suppressive effect of 29 ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the 30 treatment of COVID-19 patients. 31 32 Importance 33 The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. To identify the effective antiviral 34 agents to combat the disease is urgently needed. In the present study, we found that an inhaled 35 corticosteroid, ciclesonide suppresses replication of coronaviruses, including beta-coronaviruses 36 (MHV-2, MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alpha-coronavirus (HCoV-229E) in 37 cultured cells. The inhaled ciclesonide is safe; indeed, it can be administered to infants at high 38 concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective 39 against many members of the coronavirus family. It could be prescribed for the treatment of MERS, 40 and COVID-19. 41 42 Introduction 43 The COVID-19 outbreak began in December 2019 in Wuhan, China(1). The causative virus, severe 44 acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread rapidly worldwide and was declared bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.258459; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 a global health emergency by the World Health Organization. Thus, effective antiviral agents to 46 combat the disease are urgently needed. Several drugs are effective against SARS-CoV-2 in cultured 47 cells(2–4). Of these, Remdesivir has undergone clinical trials in COVID-19 patients, with both 48 positive and negative results (5, 6). Lopinavir/ritonavir and chloroquine/hydroxychloroquine are of no 49 benefit (7, 8). 50 The virus can have inflammatory effects; therefore, steroids are used to treat severe inflammation, 51 with beneficial effects in some cases. For example, high-dose steroids reduce symptoms in those with 52 influenza encephalopathy(9). It would be highly beneficial if a virus-specific inhibitor was identified 53 among the many steroid compounds that have been well characterized. However, systemic treatment 54 with steroids is contraindicated in cases of severe pneumonia caused by Middle East respiratory 55 syndrome coronavirus (MERS-CoV) or severe acute respiratory syndrome coronavirus (SARS-CoV); 56 this is because steroids suppress innate and adaptive immune responses(10, 11), resulting in increased 57 viral replication. In fact, for SARS (2003) and MERS (2013), systemic treatment with cortisone or 58 prednisolone is associated with increased mortality(12, 13). Therefore, if steroid compounds are to be 59 used to treat patients suffering from COVID-19, their tendency to increase virus replication must be 60 abrogated. Here, we reconsidered the use of steroids for treatment of pneumonia caused by 61 coronavirus. 62 In the preprint of this study (posted in BioRxiv)(14), we showed that a corticosteroid, ciclesonide, is a 63 potent blocker of SARS-CoV-2 replication. Based on the data in our preprint study, clinical trials of a 64 retrospective cohort study to treat COVID-19 patients were started in Japanese hospital in March 65 2020. The treatment regime involves inhalation of 400 μg ciclesonide (two or three times per day) for 66 2 weeks. Three cases of COVID-19 pneumonia treated successfully with ciclesonide have been 67 reported(15), as have several case reports(16–18). None of these studies reported significant side 68 effects. The aim of the present study is to outline the scientific rationale for conducting these clinical 69 trials. 70 71 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.258459; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 Results 73 Antiviral effect of steroid compounds on MERS-CoV 74 The 92 steroid compounds chosen from the Prestwick Chemical Library were examined to assess the 75 inhibitory effects of MERS-CoV-induced cytopathic effects. Vero cells treated with steroid 76 compounds were infected with MERS-CoV at an MOI = 0.1 and then incubated for 3 days. Four 77 steroid compounds, ciclesonide, mometasone furoate, mifepristone, and algestone acetophenide, 78 conferred a > 95% cell survival rate (Fig. 1). Interestingly, a structural feature of these compounds is a 79 five- or six-membered monocycle attached to the steroid core. 80 81 Next, we assessed the ability of eight steroid compounds (denoted by arrows in Fig. 1) to suppress 82 both growth of MERS-CoV and virus-mediated cytotoxicity in Vero cells over a range of drug 83 concentrations (0.1–100 µM). Ciclesonide exhibited low cytotoxicity and potent suppression of viral 84 growth (Fig. 2a). Algestone acetophenide, mometasone, and mifepristone also suppressed viral 85 growth; however, at 10 μM, the percentage viability of cells treated with algestone acetophenide and 86 mometasone was lower than that of cells treated with ciclesonide, and the ability of mifepristone and 87 mometasone to suppress viral growth was lower than that of ciclesonide. Cortisone and prednisolone, 88 which are commonly used for systemic steroid therapy, dexamethasone, which has strong 89 immunosuppressant effects, and fluticasone, a common inhaled steroid drug, did not suppress viral 90 growth (Fig. 2a). A time-of-addition assay to compare the viral inhibition efficacies of the steroids 91 with those of E64d, a cathepsin-dependent virus entry inhibitor, and lopinavir, a viral 3CL protease 92 inhibitor, previously reported for SARS-CoV(19, 20), demonstrated that ciclesonide functions at the 93 post-virus entry stage (Supplemental Fig. S1). 94 95 The antiviral effects of mometasone and ciclesonide against various viral species were tested by 96 quantifying propagated virus in the culture medium of infected cells. Ciclesonide and mometasone 97 suppressed replication of MHV-2, MERS-CoV, SARS-CoV, HCoV-229E, and SARS-CoV-2 (all of 98 which have a positive strand RNA genome), but did not affect replication of respiratory syncytial bioRxiv preprint doi: https://doi.org/10.1101/2020.08.22.258459; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 99 virus (RSV) or influenza virus (which have a negative strand RNA genome) (Fig. 2b). In addition, 100 ciclesonide slightly, but significantly, inhibited replication of rubella virus (which has a positive strand 101 RNA genome) (Fig. 2b). 102 103 Target of ciclesonide during MERS-CoV replication 104 In an attempt to identify a druggable target for viral replication, we performed 11 consecutive 105 passages of MERS-CoV in the presence of 40 μM ciclesonide or 40 μM mometasone. A mutant virus 106 displaying resistance to ciclesonide (but no virus displaying resistance to mometasone) was generated. 107 Viral replication in the presence of ciclesonide was confirmed by measuring the virus titer in the 108 culture medium of infected Vero cells at 24 h post-infection (hpi) and the amount of viral RNA in 109 infected cells at 6 hpi (Fig. 3a and 3b). Next-generation sequencing revealed that an amino acid 110 substitution at A25V (C19647T in the reference sequence NC_019843.3) in non-structural protein 15 111 (NSP15), a coronavirus endoribonuclease (21–23), was predicted to cause resistance to ciclesonide. 112 Subsequently, a recombinant virus carrying the A25V amino acid substitution in nsp15 (Re-Nsp15- 113 A25V) was generated from the parental MERS-CoV/EMC strain (Re-EMC/MERS) using a bacterial 114 artificial chromosome (BAC) reverse genetics system(24).