key: cord-0844747-12th7nja
authors: Fantini, Jacques; Chahinian, Henri; Yahi, Nouara
title: nSynergistic antiviral effect of hydroxychloroquine and azithromycin in combination against SARS-CoV-2: what molecular dynamics studies of virus-host interactions reveal
date: 2020-05-13
journal: Int J Antimicrob Agents
DOI: 10.1016/j.ijantimicag.2020.106020
sha: 8fbd2ab26d6789477da19a74381732d110b0caf5
doc_id: 844747
cord_uid: 12th7nja

The emergence of SARS-coronavirus-2 (SARS-CoV-2) is responsible for a global pandemic disease referred to as coronavirus disease 19 (Covid-19). Hydroxychloroquine/azithromycin combination therapy is currently tested for curing Covid-19, with promising results. However, the molecular mechanism of action of this combination is not established yet. Using molecular dynamics (MD) simulations we show that both drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. We reveal unexpected molecular similarity between azithromycin and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, azithromycin interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by azithromycin and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. We also show that hydroxychloroquine molecules can saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor ACE-2. Taken together, these data show that azithromycin is directed against the virus, whereas hydroxychloroquine is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where the most efficient therapeutic intervention probably stands. This molecular mechanism may explain the beneficial effects of hydroxychloroquine/azithromycin combination therapy in patients with Covid-19. Incidentally, our data suggest that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.

pandemic disease referred to as coronavirus disease . 23 Hydroxychloroquine/azithromycin combination therapy is currently tested for curing 24 Covid-19, with promising results. However, the molecular mechanism of action of this 25 combination is not established yet. Using molecular dynamics (MD) simulations we show 26 that both drugs act in synergy to prevent any close contact between the virus and the 27 plasma membrane of host cells. We reveal unexpected molecular similarity between 28 azithromycin and the sugar moiety of GM1, a lipid raft ganglioside acting as a host 29 attachment cofactor for respiratory viruses. Due to this mimicry, azithromycin interacts 30 with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site 31 shared by azithromycin and GM1 displays a conserved amino acid triad Q-134/F-135/N- 32 137 located at the tip of the spike protein. We also show that hydroxychloroquine 33 molecules can saturate virus attachment sites on gangliosides in the vicinity of the 34 primary coronavirus receptor ACE-2. Taken together, these data show that azithromycin 35 is directed against the virus, whereas hydroxychloroquine is directed against cellular 36 attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS- 37 CoV-2 attachment to the host cell membrane. This is consistent with a synergistic 38 antiviral mechanism at the plasma membrane level, where the most efficient therapeutic 39 intervention probably stands. This molecular mechanism may explain the beneficial 40 effects of hydroxychloroquine/azithromycin combination therapy in patients with Covid- 41 19. Incidentally, our data suggest that the conserved Q-134/F-135/N-137 triad could be 1. Introduction 53 The emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is 54 responsible for a global pandemic disease referred to as coronavirus disease 19 (Covid- The very first step of human coronaviruses replication cycle is the attachment to the 77 plasma membrane of target cells, which is mediated by a membrane protein receptor, i.e. 78 angiotensin converting enzyme-2 (ACE-2) in the case of SARS-CoV-2 [11]. Moreover, 79 coronaviruses are also dependent upon sialylated membrane components such as 80 gangliosides that act as attachment cofactors within lipid raft membrane platforms [12- 81 14] . As ACE-2 is localized in lipid rafts [15] , SARS-CoV-2 infection requires specific 82 targeting to these plasma membrane microdomains, where multivalent interactions 83 between the spike protein and raft components can take place. In line with notion, lipid 84 raft disruption through cholesterol depletion resulted in a significant reduction of human 85 coronavirus SARS-CoV infection [15] . The recent identification of a potential 86 ganglioside-binding domain in the N-terminal domain (NTD) of the SARS-CoV-2 spike 87 protein, and its potential role in membrane recognition [10], prompted us to study the 88 molecular relationship between this domain, gangliosides, ATM and CLQ-OH. To this 89 end, we used our molecular modeling strategy that has been successfully applied for 90 unraveling the molecular mechanisms of protein binding to raft lipid components 91 including gangliosides [16, 17] and cholesterol [18, 19] . The SARS-CoV-2 spike protein trimer in the prefusion conformation was obtained from 95 pdb file # 6VSB [20] . Hydroxychloroquine (CLQ-OH) is (RS)-2-[{4-[(7-chloroquinolin-96 4-yl)amino]pentyl}(ethyl)amino]ethanol. CLQ-OH was generated by hydroxylation of 97 chloroquine (CLQ) and validated as previously described [10] . CLQ was retrieved from 98 pdb file # 4V2O (CLQ co-crystallized with saposin B) [21] . Azithromycin (ATM) is (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-100 (dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-2-ethyl-3,4,10-trihydroxy- [(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy-3,5,6,8,10,12,14-102 heptamethyl-1-oxa-6-azacyclopentadecan-15-one. The 3D structure of ATM was 103 obtained from pdb file # 5UXD (ATM co-crystallized with macrolide 2'-104 phosphotransferase) [22] . (http://molexus.io/molegro-molecular-viewer) as described previously [16] [17] [18] [19] 23] . (Table S1 ). Schematically, the binding site is formed by two discontinuous regions of the 200 protein, including the QFN triad with additional C-136, D-138, R-158 and S-161 residues 201 (Figures 4 and 5) . These seven amino acid residues accounted for almost 90% of the 202 whole energy of interaction (Table S1 and Figure 5 ).

The complex was stabilized by hydrogen bonds, CH- and van der Waals interactions 204 distributed over the whole ATM molecule (Figure 4) . Table S1 , the QFN triad 249 of the virus spike protein is predicted to interact with the central region of the ganglioside 250 dimer. If we metaphorically compare the dimer to a butterfly, this region corresponds to 251 the insect's head between the wings. For its part, CLQ-OH binds to the wings ( Figure   252 8d), whereas ATM neutralizes the QFN triad of the virus spike protein (Figure 8f) . 253 Indeed, all attempts to obtain a stable raft-spike protein complex aborted when GM1 was 254 covered by CLMQ-OH and when ATM was bound to the spike protein. In the particular 255 case of ATM, these data confirm that the QFN triad is critical for GM1 recognition and 256 that although other residues are involved ( Figure 5 and Table S1), the whole binding 257 process is fully controlled by the primary interaction driven by the QFN triad. In 

In this study, we used molecular modelling approaches specifically dedicated to virus-268 host interactions to unravel the antiviral mechanism of action of ATM and CLQ-OH in 269 combination. Our method included a first round of molecular docking, followed by MD 270 simulations of protein-ligand interactions to assess the robustness of each model [25] . water-soluble saccharide part of the ganglioside, neglecting their membrane embedded 274 ceramide part [31] . Unfortunately, the ceramide moiety of gangliosides has a marked 275 effect on the saccharide part with which it interacts, resulting in significant restriction of 276 its conformational possibilities [32] . Therefore, data obtained with oligosaccharides 

In any case, the molecular mimicry between ATM and the saccharide part of GM1 gives 314 new perspectives on the therapeutic effect of this macrolide antibiotic which warrants 315 further exploration. Moreover, the fact that ganglioside GM1 is a molecular target for 316 CLQ-OH might explain the indication of this drug in rheumatologic disorders such as 317 lupus and rheumatoid arthritis [35, 36] . Indeed, GM1 overexpression and anti-GM1 318 antibodies are a hallmark of these diseases [37, 38] . Thus, our data incidentally suggest 319 that the therapeutic effect of CLQ-OH in these cases could also be related to its 320 ganglioside-binding properties. virus proteins, and more generally to predict the efficacy of any potential repurposed 333 and/or innovative drug candidates before clinical evaluation. In this respect, we suggest 334 testing the antiviral association of ATM with short synthetic peptides specifically 335 designed to target gangliosides without toxicity [17, 33] . Table S1 . 

Identification of sialic acid-binding 402 function for the Middle East respiratory syndrome coronavirus spike glycoprotein

Structures of MERS-CoV spike glycoprotein in complex with sialoside 407 attachment receptors

Importance of cholesterol-rich membrane microdomains in the 412 interaction of the S protein of SARS-coronavirus with the cellular receptor angiotensin-413 converting enzyme 2

Gangliosides interact with synaptotagmin to form the 418 high-affinity receptor complex for botulinum neurotoxin B

Deciphering the glycolipid code of Alzheimer's and Parkinson's 422 amyloid proteins allowed the creation of a universal ganglioside-binding peptide

A mirror code for 426 protein-cholesterol interactions in the two leaflets of biological membranes

The fusogenic tilted peptide (67-78) of α-synuclein is 430 a cholesterol binding domain

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

The lysosomal protein saposin B binds chloroquine

The evolution of substrate discrimination in macrolide antibiotic resistance 443 enzymes

Hybrid in silico/in vitro approaches for the identification of 446 functional cholesterol-binding domains in membrane proteins

Molecular Dynamics Simulation for All

Host Lipid Rafts Play a Major Role in Binding

Endocytosis of Influenza A Virus

Lipid rafts are involved in SARS-CoV entry into Vero E6 462 cells

Clues 466 to Innovative Therapeutic Strategies for Brain Disorders

Fantini 470 J. Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with 471 monolayers of galactosylceramide and ganglioside GM3

Lipid rafts: structure, function and role in 474

Alzheimer's and prion diseases

Structure of dual receptor binding to 478 botulinum neurotoxin B

Cholesterol accelerates the binding of Alzheimer's β-481 amyloid peptide to ganglioside GM1 through a universal hydrogen-bond-dependent sterol 482 tuning of glycolipid conformation

Common molecular mechanism of amyloid pore formation by Alzheimer's β-amyloid 487 peptide and α-synuclein

Chloroquine and 490 hydroxychloroquine as available weapons to fight COVID-19

Hydroxychloroquine in systemic lupus erythematosus (SLE)

Mechanisms of action of hydroxychloroquine and 497 chloroquine: implications for rheumatology

Anti-glycosphingolipid 501 autoantibodies in rheumatologic disorders

Jury 505 EC. Normalizing glycosphingolipids restores function in CD4+ T cells from lupus 506 patients

Human erythrocyte 509 glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1 510 (HIV-1) entry: evidence for CD4-induced interactions between HIV-1 gp120 and 511 reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3)

Structural basis of GM1 ganglioside 514 recognition by simian virus 40