Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients. Summary Antivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.


Introduction 8
The coronavirus disease 2019 (COVID- 19) is having a catastrophic impact on human health as 9 well as on the global economy, and it will continue to affect our lives for years to come (Arthi and

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Compounds exhibiting the highest levels of CPE inhibition were initially selected. Of the 1,942 20 tested compounds, 57 were identified to significantly decrease PI incorporation (robust Z-core <

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We assessed the activity of the 57 identified hits in dose-response curve (DRC) analyses using 30 the same setting as in the screen (Supplementary Table 2). Of these, 21 showed dose-related 31 inhibition of the PI incorporation upon infection (N=2).

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Coronaviruses, including SARS-CoV-2, can use two different routes to enter their target cells.

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They either enter cells by endocytosis and release their genome into the cytosol after fusion of 34 their envelope with an endosomal membrane, or they fuse their envelope directly with the 35 4 plasma membrane. This latter entry route is triggered by the cell surface protease TMPRSS2 1 which is not expressed in Vero-81 cells (Hoffmann et al., 2020). Previously identified anti-SARS-2 CoV-2 compounds, like CQ and hydroxy-CQ, were shown to only block the endocytic entry route 3 (Hoffmann et al., 2020). An additional validation on Vero-81-TMPRSS2 cells was therefore 4 performed to discard compounds that only block the endocytic route of SARS-CoV-2 entry.

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Of the 21 molecules validated twice in Vero-81 cells, the most interesting ones were retained 6 based on a preliminary evaluation of their risk/benefit ratio in the clinic. This evaluation included 7 a comparison of the in vitro potency to plasma exposure at the approved dose. Therefore, only 8 8 out of the 21 compounds were tested in Vero-81-TMPRSS2 cells (Supplementary Table 2). Only   was not due to a cytotoxic effect of the compound. To further characterize the antiviral activity of 23 clofoctol, its effect on the production of infectious progeny virions was also quantified. As shown 24 in Fig. 2c, a dose-dependent decrease of infectious virus production was observed in these 25 experimental conditions, confirming the antiviral effect of clofoctol against SARS-CoV-2 with an 26 IC50 of 9.3 µM and 11.59 µM in Vero-81 and Vero-81-TMPRSS2 cells, respectively.

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Vero cells are the cells of choice to efficiently grow SARS-CoV-2 in culture and therefore to 28 screen large libraries of compounds for rapid identification of antivirals. However, as these cells 29 are from monkey origin, the human cell line Calu-3, derived from a lung adenocarcinoma, 30 previously shown to be permissive to SARS-CoV-2 (Hoffmann et al., 2020) was also used to 31 validate our observations. As shown in Fig. 2d, a dose-dependent decrease of viral RNA 32 production was also observed in infected Calu-3 cells treated with clofoctol, at concentrations 33 that did not exhibit a cytotoxic effect (Fig. 2b). In this cell line, clofoctol exhibited an IC50 of 7.9 34 µM.

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The life cycle of a virus can be divided into three major steps: (1) entry, (2) translation/replication 2 and (3) assembly/release. To determine at which step clofoctol inhibits SARS-CoV-2, the 3 compound was added either before infection, during virus entry, post-inoculation or throughout 4 all the steps. Remdesivir, an inhibitor of the viral polymerase (Lo et al., 2020), and CQ were 5 used as control antivirals affecting viral replication or entry, respectively. As shown in Fig. 3a,   6 remdesivir inhibited infection only when added after the entry step, whereas CQ was only 7 efficient when added at the entry step. Clofoctol inhibited SARS-CoV-2 mainly at the post-8 inoculation step, although it had also a mild effect at the entry step. These data suggest that the 9 translation/replication step is likely the major target of clofoctol. To further characterize the post-10 entry inhibitory effect of clofoctol, a time-of-addition experiment was also performed in parallel 11 with clofoctol, remdesivir and CQ. In this experiment, the compounds were added before

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Of note, clofoctol was also active against another coronavirus which is mildly pathogenic, HCoV-    UTRs-based construct in Vero-81 and Huh-7 cells, but not for the control bicistronic construct 1 (Fig. 3d), indicating that clofoctol specifically inhibits the translation of an mRNA containing the 2 UTRs of the SARS-CoV-2. This suggests that clofoctol has the potential to inhibit the translation 3 of genomic as well as sub-genomic SARS-CoV-2 RNAs.

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Because of this favorable pharmacokinetic profile in mice, we decided to test clofoctol in K18-

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In this study, we report the high-throughput screening of ~2,000 drugs, approved for human use,

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for their potential activity against SARS-CoV-2. Our data identify clofoctol as a promising       1986). Of interest, as early as 90 minutes after rectal administration, the peak concentration of 17 clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 18 measured in Vero-81 cells. In our experimental conditions, clofoctol was also detected in mice

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Together with its antiviral effects, clofoctol abrogated lung inflammation. To the best of our 28 knowledge, the anti-inflammatory effect of clofoctol has never been reported before. Together 29 with its effect on UPR pathways, clofoctol is known to interact with different targets including (i)

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Whether the UNR/KLF13 pathway triggered by clofoctol plays a role in decreasing inflammation 9 during SARS-CoV-2 infection deserves further investigation. Additional functional studies are 1 urgently needed to assess the global effect of clofoctol on COVID-19 pathology.

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In conclusion, the antiviral and anti-inflammatory properties of clofoctol, associated with its 3 safety profile and unique pharmacokinetics make a strong case for proposing clofoctol as an 4 affordable therapeutic candidate for the treatment of COVID-19 patients.

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Briefly, a mask was first determined from input image, using the intensity threshold of Hoechst 5 dye signal to create a region of interest corresponding to Hoechst-stained population. The nuclei 6 segmentation was then performed using the algorithm "Find Nuclei", as described

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The coverslips were rinsed with PBS 3 times for 5 min followed by a final water wash before

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The inflammatory score is depicted. b-c, Results are expressed as the mean ± SD (n=13 for 22 panels b and c and n=6-7 for panel d). Significant differences were determined using the Mann-23 Whitney U test (**p < 0.01; ***p < 0.001).