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Id Subject Object Predicate Lexical cue
T1 0-102 Sentence denotes Synergistic antiviral effect of hydroxychloroquine and azithromycin in combination against SARS-CoV-2:
T2 103-168 Sentence denotes What molecular dynamics studies of virus-host interactions reveal
T3 170-180 Sentence denotes Highlights
T4 181-265 Sentence denotes • Emergence of the new coronavirus, SARS-CoV-2 has led to a global pandemic disease
T5 266-346 Sentence denotes • Hydroxychloroquine/azithromycin combination therapy is currently being tested
T6 347-422 Sentence denotes • Molecular mimicry between azithromycin and ganglioside sugar is revealed
T7 423-530 Sentence denotes • Azithromycin binds to the virus spike protein of SARS-CoV-2 and hydroxychloroquine binds to gangliosides
T8 531-618 Sentence denotes • Hydroxychloroquine/azithromycin have synergistic effect against SARS-CoV-2 infection
T9 620-628 Sentence denotes Abstract
T10 629-764 Sentence denotes The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19).
T11 765-912 Sentence denotes Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results.
T12 913-999 Sentence denotes However, the molecular mechanism of action of this combination is not yet established.
T13 1000-1175 Sentence denotes Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells.
T14 1176-1344 Sentence denotes Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses.
T15 1345-1444 Sentence denotes Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein.
T16 1445-1581 Sentence denotes This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein.
T17 1582-1753 Sentence denotes CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2).
T18 1754-1891 Sentence denotes Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors.
T19 1892-2001 Sentence denotes We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane.
T20 2002-2152 Sentence denotes This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient.
T21 2153-2273 Sentence denotes This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19.
T22 2274-2409 Sentence denotes Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.
T23 2411-2426 Sentence denotes 1 Introduction
T24 2427-2587 Sentence denotes The emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19) [1].
T25 2588-2668 Sentence denotes In such a health emergency, it is critical to find a cure and stop the pandemic.
T26 2669-2893 Sentence denotes Among the potential solutions currently under investigation, a combination bitherapy consisting of the antimalarial drug, hydroxychloroquine (CLQ-OH) with the antibiotic, azithromycin (ATM) has received particular attention.
T27 2894-3085 Sentence denotes Following initial reports from China indicating a potential effect of chloroquine (CLQ) [2], a preliminary clinical trial was implemented in France on a small cohort of COVID-19 patients [3].
T28 3086-3257 Sentence denotes A significant viral load reduction was observed in the 20 patients treated with CLQ-OH [3], a CLQ derivative with increased solubility in water and decreased toxicity [4].
T29 3258-3379 Sentence denotes With the aim of preventing bacterial super-infection, six additional patients in this trial also received ATM for 5 days.
T30 3380-3444 Sentence denotes All these patients had undetectable viral load after 6 days [3].
T31 3445-3636 Sentence denotes Thus, although these promising data need clinical confirmation with more patients, CLQ-OH/ATM combination therapy already appears superior to CLQ alone as a first-line treatment for COVID-19.
T32 3637-3773 Sentence denotes Both CLQ-OH and ATM are repositioned drugs and their antiviral mechanism of action, particularly in combination, remains mostly unclear.
T33 3774-3848 Sentence denotes In vitro studies have shown that CLQ-OH inhibits SARS-CoV-2 [4], [5], [6].
T34 3849-3982 Sentence denotes Far less is known about the antiviral effects of ATM, which has been suggested to interfere with influenza virus internalization [7].
T35 3983-4100 Sentence denotes Interestingly, CLQ is also considered as an inhibitor of endocytic pathways through an elevation of endosomal pH [8].
T36 4101-4242 Sentence denotes However, several reports indicate that CLQ could also prevent virus attachment through a direct effect on host-cell surface molecules [9,10].
T37 4243-4493 Sentence denotes The first step of the replication cycle in human coronaviruses is the attachment of the virus to the host plasma membrane, which is mediated by a membrane protein receptor, i.e., angiotensin-converting enzyme-2 (ACE-2) in the case of SARS-CoV-2 [11].
T38 4494-4684 Sentence denotes Moreover, coronaviruses are also dependent upon sialylated membrane components, such as gangliosides, which act as attachment cofactors within lipid raft membrane platforms [12], [13], [14].
T39 4685-4908 Sentence denotes As ACE-2 is localized in lipid rafts [15], SARS-CoV-2 infection requires specific targeting to these plasma membrane microdomains, where multivalent interactions between the spike protein and raft components can take place.
T40 4909-5068 Sentence denotes In line with this notion, lipid raft disruption through cholesterol depletion resulted in a significant reduction of human coronavirus SARS-CoV infection [15].
T41 5069-5354 Sentence denotes The recent identification of a potential ganglioside-binding domain in the N-terminal domain (NTD) of the SARS-CoV-2 spike protein, and its potential role in membrane recognition [10], prompted this study of the molecular relationship between this domain, gangliosides, ATM and CLQ-OH.
T42 5355-5577 Sentence denotes This study involved a molecular modelling strategy that has been successfully applied to unravel the molecular mechanisms of protein binding to raft lipid components, including gangliosides [16,17] and cholesterol [18,19].
T43 5579-5603 Sentence denotes 2 Materials and Methods
T44 5605-5619 Sentence denotes 2.1 Pdb files
T45 5620-5725 Sentence denotes The SARS-CoV-2 spike protein trimer in the prefusion conformation was obtained from pdb file # 6VSB [20].
T46 5726-5826 Sentence denotes Hydroxychloroquine (CLQ-OH) is (RS)-2-[{4-[(7-chloroquinolin-4-yl)amino]pentyl}(ethyl)amino]ethanol.
T47 5827-5929 Sentence denotes CLQ-OH was generated by hydroxylation of chloroquine (CLQ) and validated as previously described [10].
T48 5930-6011 Sentence denotes CLQ was retrieved from pdb file # 4V2O (CLQ co-crystallized with saposin B) [21].
T49 6012-6292 Sentence denotes Azithromycin (ATM) is (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-2-ethyl-3,4,10-trihydroxy-13-[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one.
T50 6293-6415 Sentence denotes The 3D structure of ATM was obtained from pdb file # 5UXD (ATM co-crystallized with macrolide 2′-phosphotransferase) [22].
T51 6417-6465 Sentence denotes 2.2 Molecular modelling and visualization tools
T52 6466-6730 Sentence denotes Molecular modelling studies were performed using Hyperchem (http://www.hyper.com), Deep View/Swiss-Pdb viewer (https://spdbv.vital-it.ch) and Molegro Molecular viewer (http://molexus.io/molegro-molecular-viewer) as described previously [[16], [17], [18], [19],23].
T53 6731-6840 Sentence denotes Lennard-Jones parameters and atomic charges of ATM obtained from pdb file # 5UXD were checked with Hyperchem.
T54 6841-7013 Sentence denotes The molecular modelling protocol consisted of docking, equilibration, and subsequent 50-ns molecular dynamics (MD) simulations with CHARMM force field [16,24] in Hyperchem.
T55 7014-7226 Sentence denotes The starting point for ATM docking was done by manual positioning on the NTD and receptor-binding domain (RBD) surfaces of SARS-CoV-2 spike protein (chain A) with full consideration of the properties of the drug.
T56 7227-7334 Sentence denotes In addition, a series of 20 randomly positioned ATM molecules covering the whole NTD surface were analysed.
T57 7335-7605 Sentence denotes Energy minimization of each system was then performed with the Polak-Ribière conjugate gradient algorithm, with CHARMM force field in Hyperchem, using a maximum of 3 × 105 steps, and a root-mean-square (RMS) gradient of 0.01 kcal. Å−1.mol−1 as the convergence condition.
T58 7606-7947 Sentence denotes According to the validation ligand-binding protocol [25], the optimized docked structures were used as the initial structures for MD simulations with the following parameters: heat time 5 ps, run time 10 ps, step size 1 fs, starting temperature 0 K, simulation temperature 300 K, temperature step 30 K, bath relaxation time step size 0.1 ps.
T59 7948-8068 Sentence denotes Each complex was submitted to 50-ns long MD trajectories, replicated three times, with convergent trajectories obtained.
T60 8069-8130 Sentence denotes Frames were saved at 10 ns intervals for subsequent analysis.
T61 8131-8304 Sentence denotes The final complex obtained with chain A was reintroduced in the trimeric spike structure (pdb file # 6VSB) with Molegro Molecular viewer and with Deep View Swiss-Pdb viewer.
T62 8305-8354 Sentence denotes Similar results were obtained with both programs.
T63 8355-8468 Sentence denotes The energies of interaction were extracted from the Ligand Energy Inspector function of Molegro Molecular viewer.
T64 8469-8537 Sentence denotes Molecular volumes were calculated by the QSAR function of Hyperchem.
T65 8539-8559 Sentence denotes 2.3 Ganglioside GM1
T66 8560-9018 Sentence denotes The initial coordinates of ganglioside GM1 were obtained from CHARMM-GUI Glycolipid Modeler [24] (http://www.charmmgui.org/?doc=input/glycolipid), which uses the internal coordinate information of common glycosidic torsion angle values, orientates the ganglioside perpendicular to the membrane, and performs Langevin dynamics with a cylindrical restraint potential to keep the whole GM1 molecule cylindrical, particularly the membrane-embedded ceramide part.
T67 9019-9154 Sentence denotes In the next step, the saccharide part of the ganglioside was included in a periodic box solvated with 1128 water molecules (dimensions:
T68 9155-9199 Sentence denotes 34848 Å3 with x = 33 Å, y = 32 Å, z = 33 Å).
T69 9200-9391 Sentence denotes The system was energy-minimized 6 times switching alternatively between runs using steepest descent gradients or Polak-Ribière conjugate gradients until convergence to machine precision [16].
T70 9392-9525 Sentence denotes The dimer of ganglioside GM1 interacting with 4 CLQ-OH molecules was obtained by MD simulations of a previously described model [10].
T71 9526-9675 Sentence denotes To mimic GM1 gangliosides in a typical lipid raft membrane domain, two GM1 molecules were merged with eight cholesterol and two sphingomyelin lipids.
T72 9676-9886 Sentence denotes The whole system was optimized, merged with SARS-CoV-2 spike protein (chain A) and submitted to MD simulations with the same conditions as those used for the ATM-spike protein complex (50-ns run in triplicate).
T73 9887-10044 Sentence denotes For comparison, similar MD simulations were performed on an isolated GM1 dimer in the CHARMM-GUI membrane-compatible topology but without surrounding lipids.
T74 10046-10056 Sentence denotes 3 Results
T75 10058-10112 Sentence denotes 3.1 Molecular mimicry between ATM and ganglioside GM1
T76 10113-10164 Sentence denotes The chemical structure of ATM is shown in Fig. 1 a.
T77 10165-10303 Sentence denotes The molecule contains two sugar-like pyranyl rings, one with a nitrogen-containing group (N-pyr), the other with an acetyl group (Ac-pyr).
T78 10304-10483 Sentence denotes The remaining part of the molecule is cyclic, so that its overall conformational flexibility, although significant, is restricted to a limited spatial volume of 2082 Å3 (Fig. 1b).
T79 10484-10758 Sentence denotes Interestingly, this volume is almost the same as that of the saccharide part of ganglioside GM1 (2293 Å3, Fig. 1b), a lipid raft ganglioside that plays a critical role in the binding and endocytosis of respiratory viruses [26], including pathogenic human coronaviruses [27].
T80 10759-10978 Sentence denotes Beyond their similar spatial volume, the saccharide part of GM1 and ATM also share some analogous chemical features, including sugar rings and a solvent-accessible surface dotted with several CH and OH groups (Fig. 1b).
T81 10979-11097 Sentence denotes This molecular similarity is further illustrated in Figure S1 where ATM is superimposed on the saccharide part of GM1.
T82 11098-11247 Sentence denotes Fig. 1 Structures of azithromycin (ATM) and SARS-CoV-2 spike protein trimer. (a) ATM, with both sugar-like pyranyl groups N-pyr and Ac-pyr indicated.
T83 11248-11472 Sentence denotes The molecules are shown in chemical, tube and sphere rendering (carbon in green, nitrogen in blue, oxygen in red, hydrogen in white). (b) Molecular structure similarity between ATM and the saccharide part of ganglioside GM1.
T84 11473-11612 Sentence denotes Both structures can adopt a globular shape the surface of which is covered with a patchwork of OH (arrows 1 and 2) and CH groups (arrow 3).
T85 11613-11892 Sentence denotes The volume occupied by ATM and the saccharide part of GM1 can be estimated to be 2082 and 2293 Å3, respectively. (c) front and above views of the trimeric spike, each spike protein subunit with a distinct surface colour (cyan for chain A, yellow for chain B, purple for chain C).
T86 11893-12015 Sentence denotes Atoms belonging to the ganglioside-binding domain of each subunit are visible underneath the slightly transparent surface.
T87 12016-12083 Sentence denotes The ganglioside-binding domains, the NTD and the RBD are indicated.
T88 12084-12284 Sentence denotes Given that the SARS-CoV-2 spike protein displays a ganglioside-binding site at the tip of its NTD [10], the possibility that ATM, as a “ganglioside mimic”, could also bind to this site was considered.
T89 12285-12389 Sentence denotes The structural features of the SARS-CoV-2 spike in the prefusion conformation [20] are shown in Fig. 1c.
T90 12390-12524 Sentence denotes It consists of three interdigitated spike proteins that provide the virus its typical corona-like shape in electron microscopy images.
T91 12525-12646 Sentence denotes In each subunit, the most distant part from the viral envelope is divided into two separate domains, the NTD and the RBD.
T92 12647-12826 Sentence denotes The NTD has a flat surface available for ganglioside binding [10], and this process is independent from the ACE-2 receptor recognition, which occurs at the tip of the RBD [11,20].
T93 12827-12942 Sentence denotes When seen from above, the viral spike has a typical triangle shape, with a ganglioside-binding domain at each apex.
T94 12943-13082 Sentence denotes Thus, the spike central area is devoted to ACE-2 binding, leaving three peripheric flat surface areas available for ganglioside attachment.
T95 13083-13223 Sentence denotes Such dual ganglioside/receptor binding is commonly used by pathogenic viruses such as HIV-1 [28], [29], [30] and bacterial neurotoxins [16].
T96 13224-13415 Sentence denotes By combining the high affinity for a single protein receptor with multiple low affinity attachment sites, these pathogens have selected a very efficient pathway to gain entry into host cells.
T97 13417-13500 Sentence denotes 3.2 Characterization of an ATM binding site at the tip of SARS-CoV-2 spike protein
T98 13501-13649 Sentence denotes When ATM molecules were merged with the SARS-CoV-2 spike protein, there was a very good fit for one particular pose at the tip of protein (Fig. 2 ).
T99 13650-13801 Sentence denotes All other docking attempts on the NTD or the RBD of the spike protein were unsuccessful (Figure S2) as they did not satisfy the minimum cut-off values.
T100 13802-13874 Sentence denotes Not surprisingly, their trajectories started destabilizing before 10 ns.
T101 13875-14009 Sentence denotes In contrast, ATM #1 (coloured in yellow in Figure S2) remained bound to the spike protein throughout the simulation process (Fig. 3 ).
T102 14010-14202 Sentence denotes Interestingly, a significant movement of the drug was observed from its docked pose to a stable MD pose (dock-to-MD transition), particularly during the first 10-ns of simulations (Figure S3).
T103 14203-14261 Sentence denotes A stable complex association was then reached after 10 ns.
T104 14262-14432 Sentence denotes Three amino acid residues, referred to as the “QFN triad”, exhibited significant conformational rearrangement during the binding process: Q-134, F-135 and N-137 (Fig. 3).
T105 14433-14640 Sentence denotes The principal moves comprised a significant reorientation of the aromatic ring of F-135, from suboptimal stacking to stabilized T-shape CH-π interaction, and a concomitant retraction of the Q-134 side chain.
T106 14641-14863 Sentence denotes Fluctuations during the 10 to 50 ns period did not affect the overall geometry of the complex, which converged to a mean energy of interaction of 92.4 ± 5.8 kJ.mol−1 as determined from triplicate MD simulations (Table S1).
T107 14864-15048 Sentence denotes Schematically, the binding site is formed by two discontinuous regions of the protein, including the QFN triad with additional C-136, D-138, R-158 and S-161 residues (Fig. 4, Fig. 5 ).
T108 15049-15163 Sentence denotes These seven amino acid residues accounted for almost 90% of the whole energy of interaction (Table S1 and Fig. 5).
T109 15164-15403 Sentence denotes Fig. 2 Molecular complex between the SARS-CoV-2 spike protein trimer and ATM. (a) Detailed view of ATM bound to the NTD tip of SARS-CoV-2 spike protein chain A, shown at two distinct magnifications and orientations (left and right panels).
T110 15404-15496 Sentence denotes Note that the NTD tip displays a complementary landing surface for ATM (highlighted in red).
T111 15497-15749 Sentence denotes The protein stretch 134-138, which contains the QFN triad, is highlighted in green. (b) The trimeric structure of the SARS-CoV-2 spike is represented in surface rendition with subunit (protein chain A, B and C) in cyan, yellow and purple, respectively.
T112 15750-15831 Sentence denotes AMT (in red) is bound to the tip of the NTD domain of the A subunit (left panel).
T113 15832-15953 Sentence denotes The ribbon structure of the cyan (chain A) subunit is shown in the right panel. (c) Above views of the spike-ATM complex.
T114 15954-16049 Sentence denotes Note that the B and C subunits also display a fully accessible ATM binding site (red asterisk).
T115 16050-16194 Sentence denotes Fig. 3 Induced-fit conformational rearrangements during binding of ATM on the spike protein. (a) Docking of ATM on the spike protein (time = 0).
T116 16195-16286 Sentence denotes ATM is in yellow spheres, and the protein segment 134-137 is in balls and sticks rendition.
T117 16287-16345 Sentence denotes Three parts of the ATM molecule are marked with asterisks.
T118 16346-16500 Sentence denotes The orientation of the side chains of residues 134-137 is shown under the complex. (b) ATM bound to the spike protein after MD simulations (time = 50 ns).
T119 16501-16580 Sentence denotes Note that the complex has evolved according to a typical induced-fit mechanism.
T120 16581-16644 Sentence denotes The asterisks on ATM help visualize its conformational changes.
T121 16645-16795 Sentence denotes Reorientation of amino acid side chains is also clearly visible in the ATM-spike complex and in the isolated 134-137 fragment shown under the complex.
T122 16796-16864 Sentence denotes Fig. 4 Schematic of ATM interaction at the tip of the spike protein.
T123 16865-16909 Sentence denotes Residues lining cavity under 3.5Å are shown.
T124 16910-16970 Sentence denotes Hydrogen bonds and CH-π stacking interactions are indicated.
T125 16971-17087 Sentence denotes Note that Q-134 (Gln-134) and S-161 (Ser-161) are linked by a hydrogen bond, which stabilizes the ATM-spike complex.
T126 17088-17162 Sentence denotes Fig. 5 Energy of interaction of spike protein-ATM and spike-GM1 complexes.
T127 17163-17303 Sentence denotes Results are expressed by amino acid residue as mean ± SD of three distinct MD simulations (50 ns) with the same starting docking conditions.
T128 17304-17439 Sentence denotes In the case of GM1, the simulations are done in presence of sphingomyelin and cholesterol to mimic a lipid raft plasma membrane domain.
T129 17440-17529 Sentence denotes Each bar corresponds to a single amino acid residue, as indicated in the horizontal axis.
T130 17530-17583 Sentence denotes Detailed values and statistics are shown in Table S1.
T131 17584-17715 Sentence denotes The complex was stabilized by hydrogen bonds, CH-π and van der Waals interactions distributed over the whole ATM molecule (Fig. 4).
T132 17717-17800 Sentence denotes 3.3 SARS-CoV-2 spike protein interactions with gangliosides in a lipid raft domain
T133 17801-18041 Sentence denotes Based on recent in-silico data that led to the characterization of ganglioside-spike protein interactions [10], MD simulations were performed with GM1 gangliosides surrounded by typical raft lipids, i.e. sphingomyelin and cholesterol (Figs.
T134 18042-18052 Sentence denotes 5 and 6 ).
T135 18053-18153 Sentence denotes These new data confirmed and extended our previous results obtained with isolated gangliosides [10].
T136 18154-18329 Sentence denotes Indeed, cholesterol and sphingomyelin appeared to stabilize the typical chalice-shaped GM1 dimer, which serves as a landing platform for the SARS-CoV-2 spike protein (Fig. 6).
T137 18330-18566 Sentence denotes Moreover, the histograms of Fig. 5 show that all amino acid residues of the spike protein that are involved in ATM binding, including the QFN triad, are also essential for GM1 binding in the lipid raft environment (Table S1 and Fig. 5).
T138 18567-18734 Sentence denotes Taken together, these data indicate that the NTD of the viral glycoprotein may display a common ganglioside/ATM binding site, in agreement with our working hypothesis.
T139 18735-18933 Sentence denotes Sequence alignments revealed that this common binding site, including the QFN triad, is totally conserved among clinical isolates of SARS-CoV-2 from various geographical origins worldwide (Fig. 7 ).
T140 18934-19116 Sentence denotes It is also conserved in bat RaTG13, which further illustrates the close relationship between this bat coronavirus and the SARS-CoV-2 strains that are currently circulating the world.
T141 19117-19205 Sentence denotes This region of the NTD should therefore be considered for SARS-CoV-2 vaccine strategies.
T142 19206-19456 Sentence denotes However, the motif has a different sequence signature in other animal- and human-related SARS coronaviruses (Fig. 7), indicating a recent evolution that may be linked to the higher contagiousness of SARS-CoV-2 compared with other human coronaviruses.
T143 19457-19535 Sentence denotes Fig. 6 MD simulations of GM1-spike protein interaction in a lipid raft domain.
T144 19536-19633 Sentence denotes Two GM1 gangliosides were merged with eight cholesterol (chol) and two sphingomyelin (SM) lipids.
T145 19634-19697 Sentence denotes After initial docking, MD simulations were performed for 50 ns.
T146 19698-19742 Sentence denotes Two distinct views of the complex are shown.
T147 19743-19838 Sentence denotes Cholesterol and sphingomyelin stabilize a dimer of GM1 clamped by the NTD of the spike protein.
T148 19839-19947 Sentence denotes Fig. 7 Amino acid sequence alignments of the ganglioside/ATM binding domain of the SARS-CoV-2 spike protein.
T149 19948-20112 Sentence denotes The first group of sequences includes clinical SARS-CoV-2 isolates from various geographical origins aligned with the reference sequence (6VSB_A, fragment 111-162).
T150 20113-20152 Sentence denotes The QFN triad is highlighted in yellow.
T151 20153-20242 Sentence denotes The second group of sequences includes human and animal viruses compared with SARS-CoV-2.
T152 20243-20385 Sentence denotes Deletions are highlighted in green, amino acid changes are highlighted in blue, conserved residues of the QFN triad are highlighted in yellow.
T153 20386-20735 Sentence denotes The degree of conservation observed in each column is symbolized by an asterisk (*) when the residue is fully conserved, a colon (:) for distinct residues with strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix), and a period (.) for distinct residues with weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix).
T154 20737-20789 Sentence denotes 3.4 Synergistic antiviral effects of ATM and CLQ-OH
T155 20790-21078 Sentence denotes Overall, these molecular modelling studies are consistent with the notion that ATM might inhibit SARS-CoV-2 infection through direct binding to the virus spike and subsequent neutralization of the infection process, which requires spike protein recognition and attachment to gangliosides.
T156 21079-21130 Sentence denotes This mechanism of action is illustrated in Fig. 8 .
T157 21131-21160 Sentence denotes Comparing the models in Figs.
T158 21161-21277 Sentence denotes 8a and 8b shows that both ATM and gangliosides bind to the same site of the spike protein, centred on the QFN triad.
T159 21278-21391 Sentence denotes Thus, in the presence of ATM, the virus spike would not reach gangliosides on the host plasma membrane (Fig. 8c).
T160 21392-21526 Sentence denotes To the best of our knowledge, it is the first time that such a mechanism of action is proposed to explain the antiviral effect of ATM.
T161 21527-21835 Sentence denotes Fig. 8 CLQ-OH/ATM combination therapy at the molecular level. (a) ATM bound to the SARS-CoV-2 spike protein trimer. (b) Ganglioside dimer (two symmetrically arranged GM1 molecules in a typical chalice-like shape, just like the one observed in lipid raft simulations) bound to SARS-CoV-2 spike protein trimer.
T162 21836-21979 Sentence denotes Note that both ATM and gangliosides share the same binding region. (c) ATM prevents ganglioside binding to the SARS-CoV-2 spike protein trimer.
T163 21980-22147 Sentence denotes CLQ-OH, once bound to gangliosides (blue and orange surfaces), also prevents any interaction with the viral spike. (d) 4 CLQ-OH molecules bound to a ganglioside dimer.
T164 22148-22340 Sentence denotes Each GM1 molecule is blocked by two CLQ-OH molecules (blue and orange surfaces), which wrap around the saccharide part. (e) Detail of the 134-138 SARS-CoV-2 spike protein stretch bound to GM1.
T165 22341-22491 Sentence denotes Note that the ganglioside interacts with Q-134 and F-135, but not with D-138. (f) Detail of the 134-138 SARS-CoV-2 spike protein stretch bound to ATM.
T166 22492-22569 Sentence denotes In this case, the binding site includes D-138 in addition to Q-134 and F-135.
T167 22570-22690 Sentence denotes Note that N-137, which interacts with both ATM and GM1, is not visible in these representations as it is located behind.
T168 22691-22753 Sentence denotes This study also looked at CLQ-OH and its interaction with GM1.
T169 22754-22853 Sentence denotes We recently published a model describing a complex formed by one GM1 and two CLQ-OH molecules [10].
T170 22854-22993 Sentence denotes For comparison, the same molecular modelling approaches as for ATM (in the presence of surrounding raft lipids) were applied to this model.
T171 22994-23122 Sentence denotes A stable complex formed by a ganglioside dimer, each monomer being associated with two CLQ-OH molecules, was obtained (Fig. 8d).
T172 23123-23291 Sentence denotes The stability of this complex is reinforced by a rearrangement of CLQ-OH molecules that interact with each other as well as interacting with the saccharide part of GM1.
T173 23292-23453 Sentence denotes Consequently, the surface of the ganglioside is almost completely masked, so that the ganglioside dimer can no longer be recognized by the viral spike (Fig. 8c).
T174 23454-23635 Sentence denotes In the presence of both ATM and CLQ-OH, virus-ganglioside interactions are efficiently blocked, preventing any close contact between the virus and the plasma membrane of host cells.
T175 23636-23716 Sentence denotes The molecular details of this synergistic antiviral effect are worth mentioning.
T176 23717-23734 Sentence denotes As shown in Figs.
T177 23735-23873 Sentence denotes 5 and 8e and Table S1, the QFN triad of the virus spike protein is predicted to interact with the central region of the ganglioside dimer.
T178 23874-23993 Sentence denotes If the dimer is compared metaphorically to a butterfly, this region corresponds to the insect's head between the wings.
T179 23994-24120 Sentence denotes For its part, CLQ-OH binds to the wings (Fig. 8d), whereas ATM neutralizes the QFN triad of the virus spike protein (Fig. 8f).
T180 24121-24272 Sentence denotes Indeed, all attempts to obtain a stable raft-spike protein complex aborted when GM1 was covered by CLMQ-OH and when ATM was bound to the spike protein.
T181 24273-24529 Sentence denotes In the case of ATM, these data confirm that the QFN triad is critical for GM1 recognition and that although other residues are involved (Fig. 5 and Table S1), the whole binding process is fully controlled by the primary interaction driven by the QFN triad.
T182 24530-24675 Sentence denotes In agreement with this notion, mutating the QFN triad with alanine residues resulted in an aborted ATM binding process at the post-docking steps.
T183 24676-25040 Sentence denotes Taken together, these molecular modelling studies indicate that CLQ-OH and ATM, when bound to their respective targets, totally mask the complementary surfaces provided by the lipid raft and the virus spike (Fig. 8c): CLQ-OH binds to GM1 and covers the wing, ATM binds to the virus spike and prevents any interaction with the central area of the ganglioside dimer.
T184 25041-25140 Sentence denotes Hence, both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane.
T185 25142-25155 Sentence denotes 4 Discussion
T186 25156-25340 Sentence denotes In this study, molecular modelling approaches specifically dedicated to virus-host interactions were used to unravel the antiviral mechanism of action of ATM and CLQ-OH in combination.
T187 25341-25505 Sentence denotes The study method included a first round of molecular docking, followed by MD simulations of protein-ligand interactions to assess the robustness of each model [25].
T188 25506-25772 Sentence denotes Such computer-assisted simulations are particularly helpful for studying protein-ganglioside interactions as crystallographic approaches are usually limited to the water-soluble saccharide part of the ganglioside, neglecting the membrane embedded-ceramide part [31].
T189 25773-25970 Sentence denotes Unfortunately, the ceramide moiety of gangliosides has a marked effect on the saccharide part with which it interacts, resulting in significant restriction of its conformational possibilities [32].
T190 25971-26077 Sentence denotes Therefore, data obtained with oligosaccharides cannot be systematically transposed to intact gangliosides.
T191 26078-26226 Sentence denotes Molecular modelling approaches circumvented this difficulty and enabled study of whole gangliosides in membrane-compatible conformations [16,32,33].
T192 26227-26385 Sentence denotes The MD simulations performed in the present study were done on gangliosides surrounded by cholesterol and sphingomyelin, which mimic a lipid raft environment.
T193 26386-26533 Sentence denotes To date, there are several structural data of the SARS-CoV-2 protein in the prefusion conformation or bound to its primary receptor ACE-2 [11, 20].
T194 26534-26637 Sentence denotes However, ACE-2 is in lipid rafts and raft disruption induces a marked decrease of virus infection [15].
T195 26638-26771 Sentence denotes Thus, it is likely that the virus interacts with the raft surface through multivalent contacts involving both ACE-2 and gangliosides.
T196 26772-26907 Sentence denotes The fact that the RBD and the ganglioside-binding domain belong to distinct parts of the trimeric spike is consistent with this notion.
T197 26908-27048 Sentence denotes Such a complex network of virus-host cell membrane interactions is also consistent with previously characterized virus infection strategies.
T198 27049-27217 Sentence denotes Indeed, the HIV-1 fusion process driven by gp120 and gp41 envelope proteins involves a receptor (CD4), a coreceptor (chiefly CCR5) and glycosphingolipid cofactors [39].
T199 27218-27442 Sentence denotes Like SARS-CoV-2, the pentameric capsid protein of SV40, and polyoma viruses display three distinct binding sites for gangliosides, which serve as critical receptors for these non-enveloped viruses in lipid raft domains [40].
T200 27443-27595 Sentence denotes The MD simulations in the current study indicate that both CLQ-OH and ATM may block SARS-CoV2 binding to gangliosides via mirror competitive mechanisms.
T201 27596-27765 Sentence denotes ATM, which has some molecular similarity with GM1 sugar, can thus occupy the ganglioside-binding domain of the spike protein and neutralize virus binding to lipid rafts.
T202 27766-27889 Sentence denotes CLQ-OH covers the ganglioside surface, and thus also prevents virus-membrane interaction through a complementary mechanism.
T203 27890-28024 Sentence denotes Each of these drugs might be efficient alone to block virus attachment, ATM through virus binding, CLQ-OH through ganglioside binding.
T204 28025-28198 Sentence denotes As both drugs interfere with the same mechanism but with distinct molecular targets, they are expected to work together in synergy, as indicated by recent clinical data [3].
T205 28199-28299 Sentence denotes The posology of the combination therapy for COVID-19 is 600 mg CLQ-OH and 250 mg ATM per day [3,34].
T206 28300-28367 Sentence denotes This ratio corresponds to a molar ratio of five CLQ-OH for one ATM.
T207 28368-28475 Sentence denotes In silico calculations indicate a binding ratio of four CLQ-OH for one ATM, which is close to the posology.
T208 28476-28685 Sentence denotes Furthermore, as ATM shares structural similarity with the saccharide part of GM1, one should ask whether ATM could bind to CLQ-OH and by this way reduce the potential effectiveness of this combination therapy.
T209 28686-28845 Sentence denotes However, as ATM and CLQ-OH have been reported to synergistically decrease SARS-CoV-2 load in infected patients [3], drug cross-neutralization is very unlikely.
T210 28846-28976 Sentence denotes There was a consistent lack of evidence of any stable ATM-CLQ-OH complexes using the molecular modelling approaches in this study.
T211 28977-29158 Sentence denotes The molecular mimicry between ATM and the saccharide part of GM1 gives new perspectives on the therapeutic effect of this macrolide antibiotic and this warrants further exploration.
T212 29159-29351 Sentence denotes Moreover, the fact that ganglioside GM1 is a molecular target for CLQ-OH might explain the indication of this drug in rheumatological disorders, such as lupus and rheumatoid arthritis [35,36].
T213 29352-29444 Sentence denotes Indeed, GM1 overexpression and anti-GM1 antibodies are a hallmark of these diseases [37,38].
T214 29445-29595 Sentence denotes Thus, our data incidentally indicate that the therapeutic effect of CLQ-OH in these cases could also be related to its ganglioside-binding properties.
T215 29597-29610 Sentence denotes 5 Conclusion
T216 29611-29778 Sentence denotes In conclusion, ATM and CLQ-OH have synergistic antiviral effects on SARS-CoV-2 infection, which supports the use of this combination therapy for the COVID-19 pandemic.
T217 29779-29923 Sentence denotes In this bitherapy, one molecule (ATM) is directed against the virus, while the other (CLQ-OH) is directed against cellular attachment cofactors.
T218 29924-30122 Sentence denotes However, the two drugs are predicted to act in synergy to prevent the first step of SARS-CoV-2 infection, at the plasma membrane level, where therapeutic intervention is likely to be most efficient.
T219 30123-30313 Sentence denotes The conserved QFN triad of the SARS-CoV-2 spike protein, which is recognized by both gangliosides and ATM, should be considered as a target for neutralizing antibodies in vaccine strategies.
T220 30314-30625 Sentence denotes The molecular modelling approaches used here are based on the search for ganglioside saccharide mimicry and might be useful to identify other ATM binding sites on virus proteins, and more generally to predict the efficacy of any potential repurposed and/or innovative drug candidates before clinical evaluation.
T221 30626-30795 Sentence denotes In this respect, we suggest testing the antiviral association of ATM with short synthetic peptides specifically designed to target gangliosides without toxicity [17,33].
T222 30797-30809 Sentence denotes Declarations
T223 30810-30811 Sentence denotes  
T224 30813-30820 Sentence denotes Funding
T225 30821-30825 Sentence denotes None
T226 30827-30846 Sentence denotes Competing Interests
T227 30847-30852 Sentence denotes None.
T228 30854-30870 Sentence denotes Ethical Approval
T229 30871-30884 Sentence denotes Not required.
T230 30886-30928 Sentence denotes Appendix Supplementary materials Funding:
T231 30929-31054 Sentence denotes This research did not receive any specific grant from funding agencies in the public, commercisal, or not-for-profit sectors.
T232 31055-31185 Sentence denotes Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2020.106020.