key: cord-0930991-pvfk33i8
authors: Terrier, Olivier; Dilly, Sébastien; Pizzorno, Andrés; Henri, Julien; Berenbaum, Francis; Lina, Bruno; Fève, Bruno; Adnet, Frédéric; Sabbah, Michèle; Rosa-Calatrava, Manuel; Maréchal, Vincent; Schwok, Anny Slama
title: Broad-spectrum antiviral activity of naproxen: from Influenza A to SARS-CoV-2 Coronavirus
date: 2020-05-01
journal: bioRxiv
DOI: 10.1101/2020.04.30.069922
sha: d4cd6672737a9767c0482dd79650d743f2bb376e
doc_id: 930991
cord_uid: pvfk33i8

There is an urgent need for specific antiviral drugs directed against SARS-CoV-2 both to prevent the most severe forms of COVID-19 and to reduce viral excretion and subsequent virus dissemination; in the present pandemic context, drug repurposing is a priority. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus in order to inhibit its association with viral RNA could be a strategy to impeding viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, belonging to the NSAID family, previously demonstrated against Influenza A virus, were evaluated against SARS-CoV-2. Naproxen binding to the nucleoprotein of SARS-CoV2 was shown by molecular modeling. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2 induced-damage. The benefit of naproxen addition to the standard of care is tested in an on-going clinical study.

The current pandemic of novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Wuhan, Hubei province, China, in December 2019 1, 2 There is an urgent need for specific and effective antiviral drugs directed against pandemic SARS-CoV-2 to prevent the most severe forms of COVID-19. In that context, drug repurposing is a priority. Ongoing advances in our knowledge of this new virus and its pathogenesis are revealing an exacerbated inflammatory response in severe COVID-19 cases, with a similar cytokine storm observed in severe cases of H5N1 Influenza virus infection and 1918 Influenza A pandemics 3 . In that regard, the symptoms of respiratory distress caused by COVID-19 could be reduced by drugs combining anti-inflammatory and antiviral effects 4 .

Several current therapeutic approaches involve drug repositioning, in light of what has been achieved for other viruses, particularly influenza viruses 5 . Our previous work showed that naproxen, an approved antiinflammatory drug, is an inhibitor of both cyclooxygenase (COX) and of Influenza A virus nucleoprotein 6 .

Although NSAIDs were the subject of a precautionary measure by the French Ministry of Health in mid-March 2020 regarding the use of these drugs in the event of COVID-19 infection, there is currently no evidence of any particular toxicity of this family of drugs [7] [8] . Moreover, the ability of naproxen to decrease viral load and inflammation through the inhibition of cyclooxygenase activity could be beneficial for limiting the cytokine storm that may happen in severe forms of COVID19. Importantly, naproxen has the advantage of being a generic drug, readily available and often used for other indications than COVID-19 by fragile populations.

Naproxen binding to the Influenza A virus nucleoprotein blocked viral RNA association with the nucleoprotein and impeded its self-association 6, 9 ; consequently, naproxen strongly reduced viral transcription/ replication in infected cells and protected mice against an infection with Influenza A virus 6, 10 . Viral nucleoproteins are unique to the virus and no equivalent of these proteins are found in the host cell, which makes them attractive targets for potential antivirals [12] [13] . Moreover, the viral nucleoprotein is an important diagnostic marker of infection with SARS-COV and /or Influenza A [14] [15] . SARS-CoV-1 virions contains multiple copies of the N protein (ca 1000 copies) located inside the particle, thus N is one of the most abundant structural proteins in CoVs 16 . Through its interaction with the coronavirus membrane (M) protein, the N protein drives virus assembly and budding. N binds to the long viral RNA genome to form a virion core comprising a ribonucleoprotein (RNP) complex that assumes a long helical structure. The RNP is important for replication and transcription in which the N protein also plays a critical role. Interactions between N and the non-structural protein-3 are also important for replication. In addition to its role in different aspects of the viral cycle, the N protein not only highjacks cellular processes, including the progression of the host cell cycle and apoptosis, but also modulates the immune response, by for example inhibiting the interferon response 17 .

In this study, we combined structure-based modeling of naproxen binding to the nucleoprotein of SARS-CoV-2 with experimental approaches to explore and validate whether naproxen harbors antiviral activity against SARS-CoV-2 pandemic virus, as previously implemented for Influenza A virus 9-10, 18 .

Structure-based modeling of naproxen binding to the nucleoprotein of SARS-CoV-2: The nucleoproteins N of enveloped, positive-sense, single-stranded viruses Coronavirus (CoV) share with negative-sense singlestranded viruses such as Influenza A virus the ability to bind to-and protect genomic viral RNA without sequence specificity and to form self-associated oligomers [12] [13] [18] [19] . Despite the limited sequence similarity between them, the N-terminal domains of three coronaviruses SARS-Cov-1, MERS-CoV, SARS-CoV-2 and Influenza A virus all presented a wide, positively-charged groove in which the viral negatively-charged RNA binds, Figure 1 Figure 1I ). For instance, SARS-CoV-2 nucleoprotein amino-terminal domain counts 14 aromatic residues among a total of 124 modeled in 6VYO.pdb. The consecutive, 5-aromatics pentapeptide 108 WYFYY 112 is located at the center of the electropositive groove ( Figure 1I , red arrow). This contrast with the nucleoprotein of Influenza A virus which has a single aromatic residue (Y148) within its RNA binding groove in which naproxen ( Figure 1J ) was shown to bind, making electrostatic (blue arrow) and hydrophobic interactions (red arrow) with conserved residues of the RNA binding groove and C-terminal domain (Sup. Figure 1E , F. Naproxen stabilized this "non -natural" interface as found in the crystal structure for the designed ligands by Hou et al 13 . Figures 1L, O and M, P and Sup. Figure 1H , E indeed show that naproxen can also bind at this interface in both MERS-CoV and SARS-Cov-2, respectively, but an additional binding site in SARS-CoV-2 monomer was also identified (Sup. Figure 1F ). Supplementary Figure   1A -D shows that naproxen binds at the interface between hydrophobic and hydrophilic parts of N proteins, to account for the hydrophobic interactions of its aromatic core with usually aromatic residues ( -stacking) and methyl group of the methoxy group with hydrophobic apolar residues and polar and/or electrostatic interactions via its carboxylate moiety. Accordingly, the following order of free binding energies of 

(HAE): Based on the docking analysis described above, we then evaluated the potential anti SARS-CoV-2 activity of naproxen in vitro. As shown in Figure 2A Developed from biopsies of nasal or bronchial cells differentiated in the air/liquid interphase, these models reproduce with high fidelity most of the main structural, functional and innate immune features of the human respiratory epithelium that play a central role in the early stages of infection and constitute robust surrogates to study airway disease mechanisms and for drug discovery 23 . Post-infection treatment of nasal HAE with 90 or 300 µM naproxen did not show an antiviral effect at 48 hpi compared to the mock-treated control (Fig. 2C, upper panel) .

Conversely, significant reductions in intracellular SARS-CoV-2 viral titers were observed for the two treatment conditions in bronchial HAE (73 and 82% reduction vs mock-treated control, respectively). This reduction in viral titers correlated with naproxen inducing a protective effect of the bronchial epithelium integrity, as shown by Trans-epithelial electrical resistance (TEER), considered as a surrogate of epithelium integrity. TEER values in both naproxen groups were comparable to those of the uninfected and untreated control (Fig. 2C , lower panel) and significantly higher than those of the untreated control (Fig. 2C , lower panel). The antiviral effects of naproxen in reconstituted human bronchial epithelium are consistent with the IC50 value determined in Vero E6 cells. However, the lack of antiviral effect of naproxen on nasal epithelium is puzzling. The viral load is usually lower in the nasal cavity as compared to the lungs 16, 24 . The nasal epithelium is an important portal for initial infection, and may serve as a key reservoir for viral spread across the respiratory mucosa and an important locus mediating viral transmission. The number of host cells as pneumocytes and alveolar macrophages permissive to viral entry is higher in the lungs than the mucous cells in the nasal cavity. Moreover, recent interactome analysis support the hypothesis that SARS-CoV-2 preferentially hijacks host proteins available in lung tissue, and N targets stress granule protein G3BP1, an essential antiviral protein which is known to induce innate immune response 7 .

Interestingly, we previously determined that the peak of viral replication was reached earlier in bronchial (48-72 hpi) than in nasal HAE, in which a progressive increase in infectious viral titers was observed until at least 96 hpi 23 . Using this model, this differential antiviral effect between airway sites was observed for naproxen in this work and for remdesivir in a recent report that indicates an antiviral effect mainly observed in the lower respiratory tract of non-Human primates 23 . It is interesting to note that naproxen is found very protective against viral-induced damages at 48 h post-infection in the reconstituted bronchial epithelium ( Figure 2C and Figure 4 in 23 ) . Therefore, we speculate that the lack of naproxen effect in nasal epithelium as opposed to its antiviral effect in pulmonary epithelium could be associated with differential expression or activity of host factor(s), and/or with slower replication kinetics in the nasal vs pulmonary epithelia. 

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The ligand crystallized in this structure is depicted in green for comparison. Panel M shows naproxen binding to Influenza A N that involved electrostatic interactions of the carboxylate with R361, a cation - interaction with R355 and hydrophobic interaction with Y148 and interactions of the last C-terminal F (not shown) with the methyl of the methoxy group.Supplementary Figure 1