key: cord-0840948-ex2zhpaw
authors: Persoons, Leentje; Vanderlinden, Evelien; Vangeel, Laura; Wang, Xinyu; Thuc Do, Nguyen Dan; Caroline Foo, Shi-Yan; Leyssen, Pieter; Neyts, Johan; Jochmans, Dirk; Schols, Dominique; De Jonghe, Steven
title: Broad spectrum anti-coronavirus activity of a series of anti-malaria quinoline analogues
date: 2021-07-01
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2021.105127
sha: f7ded011feb81ed24623ab9ca23959aed35c6741
doc_id: 840948
cord_uid: ex2zhpaw

In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral activity against a panel of alpha- and beta-coronaviruses, including the severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were the most potent with antiviral EC(50) values in the range of 0.12–12 μM. Chloroquine displayed the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with amodiaquine, ferroquine and mefloquine, although this was associated with substantial cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked coronaviruses replication at higher concentrations, while piperaquine completely lacked antiviral and cytotoxic effect. A time-of-addition experiment in HCoV-229E-infected HEL cells revealed that chloroquine interferes with viral entry at a post-attachment stage. Using confocal microscopy, no viral RNA synthesis could be detected upon treatment of SARS-CoV-2-infected cells with chloroquine. The inhibition of SARS-CoV-2 replication by chloroquine and hydroxychloroquine coincided with an inhibitory effect on the autophagy pathway as visualized by a dose-dependent increase in LC3-positive puncta. The latter effect was less pronounced or even absent with the other quinolines. In summary, we showed that several quinoline analogues, including chloroquine, hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus activity in vitro.

In this study, a series of 10 quinoline analogues was evaluated for their in vitro antiviral 24 activity against a panel of alpha-and beta-coronaviruses, including the severe acute 25 respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2), as well as the 26 human coronaviruses (HCoV) 229E and OC43. Chloroquine and hydroxychloroquine were 27 the most potent with antiviral EC 50 values in the range of 0.12-12 µM. Chloroquine displayed 28 the most favorable selectivity index (i.e. ratio cytotoxic versus antiviral concentration), being 29 165 for HCoV-OC43 in HEL cells. Potent anti-coronavirus activity was also observed with 30 amodiaquine, ferroquine and mefloquine, although this was associated with substantial 31 cytotoxicity for mefloquine. Primaquine, quinidine, quinine and tafenoquine only blocked 32 coronaviruses replication at higher concentrations, while piperaquine completely lacked 33 antiviral and cytotoxic effect. In summary, we showed that several quinoline analogues, including chloroquine, 42 hydroxychloroquine, amodiaquine, ferroquine and mefloquine, exhibit broad anti-coronavirus 43 activity in vitro. 44 J o u r n a l P r e -p r o o f simplex virus type 1 (Boonyasuppayakorn et al., 2014; Delvecchio et al., 2016; Dowall et al., 77 2015; Khan et al., 2010; Ooi et al., 2006; Savarino et al., 2001; Singh et al., 1996) . In 2004, 78 Keyaerts et al. reported that chloroquine effectively inhibits SARS-CoV-1 in Vero E6 cells. 79

Also, MERS-CoV, HCoV-229E and HCoV-OC43 were blocked by chloroquine in vitro (de 80 Wilde et al., 2014; Keyaerts et al., 2009 ). In addition, chloroquine was highly effective against 81

HCoV-OC43 infection in newborn mice. However, the antiviral effectiveness of chloroquine 82 against SARS-CoV-2 was cell-type dependent, since it inhibited virus replication in Vero E6 83 cells, but not in Calu3 and Caco2 cells (Ellinger et al., 2021; Hoffmann et al., 2020) . 84

Hydroxychloroquine showed consistent anti-SARS-CoV-2 activity in Vero E6 and variable 85 activity in Caco2 cells, but lacked antiviral activity in Calu3 cells (Clementi et al., 2020; 86 Ellinger et al., 2021; Hoffmann et al., 2020) . In vivo studies in preclinical animal models were 87 disappointing since hydroxychloroquine conferred no protection against SARS-CoV-2 in 88 macaques and a hamster model (Kaptein et al., 2020; Maisonnasse et al., 2020) . Also, the 89 overall conclusion of numerous clinical studies is that chloroquine treatment of SARS-CoV-2 90 infected patients is not beneficial (Horby et al., 2020) . Recently, other anti-malarial quinoline 91 analogues were reported to inhibit SARS-CoV-2 in Vero E6 cells (Gendrot et al., 2020) . 92

Mefloquine was also shown to be effective against SARS-CoV-2 in Caco2 cells and a 93 hamster model (Ellinger et al., 2021; Jan et al., 2021) . 94 95 However, a systematic side-by-side comparison of the antiviral activity of quinoline 96 analogues against several human alpha-and betacoronaviruses in different cell lines has not 97 been reported. In this study, the FDA-approved antimalarial drugs chloroquine, 98 hydroxychloroquine, amodiaquine, ferroquine, mefloquine, quinidine, quinine, piperaquine, antiviral activity was expressed as the concentration producing 50% antiviral effect (EC 50 ). In 115 parallel, mock-infected cells were treated with serial dilutions of the compounds to evaluate 116 their cellular toxicity, which was expressed as the 50% cytotoxic concentration (CC 50 ). 117

The majority of the quinoline analogues did show antiviral activity against one or more 118 coronaviruses tested. Only piperaquine completely lacked antiviral activity and cytotoxicity at 119 the highest concentration tested (i.e. 100 µM). Among the quinolines tested, chloroquine and 120 hydroxychloroquine were the most potent analogues with EC 50 values in the range of 0.12 -121 12 µM against the various coronaviruses (Fig. 1A) . Especially, the activity of chloroquine 122 against HCoV-OC43 in HEL cells is noteworthy with an EC 50 value of 0.12 µM and a 123 selectivity index (i.e. ratio of the cytotoxic concentration versus antiviral concentration) of 124 165. However, overall, the profile of hydroxychloroquine looked somewhat more attractive 125 because of its lower cytotoxicity in the various cell lines, giving rise to more favorable 126 selectivity indexes. Although mefloquine showed potent antiviral activity against the different 127 coronaviruses, this was accompanied with substantial cytotoxicity in the HEL, Huh7 and Vero 128 E6 cells. This is in agreement with previous findings, reporting cellular toxicity of mefloquine 129 At 11 h p.i., viral RNA was quantified by one-step qRT-PCR (see Supplementary Information  160 for details). In the absence of compound, the viral RNA copy number increased by 20-fold at 161 11 h p.i. (Fig 1C) . HHA, which interferes with virus entry at a post-attachment step, needs to 162 be added prior to the virus (van der Meer et al., 2007) , and quickly loses its inhibitory effect 163 when added post infection. GS-441524, the parent nucleoside of the ProTide remdesivir, 164 blocks viral RNA synthesis (Yin et al., 2020) , and thus can be administered up to 3 h p.i. 165

Chloroquine completely blocked viral RNA synthesis when added at 1 h p.i., but gradually 166 showed a diminished antiviral efficacy, at 3 h p.i. or later on. A similar time-dependency was 167 reported for chloroquine in SARS-CoV-1 infected Vero E6 cells, while in HCoV-OC43-168 infected HRT-18G cells chloroquine lost its antiviral activity when added at 2 h p.i. (Keyaerts 169 et al., 2009; Keyaerts et al., 2004) . Altogether, these data suggest that chloroquine interferes 170

with the viral entry process at a post attachment stage. 171

172 Regarding its precise mechanism of action, several hypotheses have been proposed. 173

Chloroquine has been reported to interfere with the terminal glycosylation of ACE2, the 174 cellular receptor used by SARS-CoV-1 and SARS-CoV-2, resulting in a reduced binding of 175 the viral spike protein to its receptor (Vincent et al., 2005) . However, this hypothesis does not In SARS-CoV-2-infected cells, viral RNA synthesis was completely blocked in the presence 195 of chloroquine at 5 µM (Fig 2) . The higher concentration of chloroquine (i.e. 50 µM) required 196 to fully inhibit HCoV-229E replication corresponds with its 4-fold higher antiviral EC 50 for 197

HCoV-229E compared to SARS-CoV-2 in Huh7 cells (Fig 1A) . Chloroquine treatment also 198 J o u r n a l P r e -p r o o f resulted in a dose-dependent increase of LC3-positive puncta in infected and uninfected 199 cells. High content imaging showed that chloroquine and hydroxychloroquine both produced 200 a 6-fold increase of the intensity of cytoplasmic LC3 puncta, while for the other quinoline 201 analogues only a moderate or no effect on LC3 staining was observed (Fig 1D) . This finding 202 confirms the inhibitory effect of both chloroquine and hydroxychloroquine on the autophagy 203 pathway and is in agreement with the fact that chloroquine and hydroxychloroquine are 204 structurally very similar. In contrast, the other 8 analogues, although also based on a 205 quinoline scaffold and having a basic side chain, are structurally more different and therefore 206 it is not unexpected that they behave differently in various assays. These other quinoline 207 analogues exert their antiviral effect probably via another mechanism of action that might be 208 clinically more relevant. identifies TMEM106B as a proviral host factor for SARS-CoV-2. Nat Genet. 227

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and toxicity of quinoline derivatives against alpha-and beta-coronaviridae assessed in HEL, 343Huh7 or Vero E6 cells. Antiviral activity is expressed as the 50% antiviral effective 344 concentration (EC 50 ), and cellular toxicity as the 50% cytotoxic concentration (CC 50 ). Data 345shown are the mean ± SD of at least two independent tests performed in duplo; nd: not done.