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PMC:7128678 JSONTXT 22 Projects

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Id Subject Object Predicate Lexical cue
T1 0-142 Sentence denotes Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection
T2 144-154 Sentence denotes Highlights
T3 155-233 Sentence denotes • The COVID‐19 pandemic caused by SARS‐CoV‐2 poses a global health emergency.
T4 234-316 Sentence denotes • Promising results suggest that chloroquine could stop the spread of SARS‐CoV‐2.
T5 317-386 Sentence denotes • In-silico studies confirm the antiviral properties of chloroquine.
T6 387-440 Sentence denotes • New mechanism of action of chloroquine elucidated.
T7 441-495 Sentence denotes • Hydroxychloroquine is more potent than chloroquine.
T8 497-505 Sentence denotes Abstract
T9 506-623 Sentence denotes The recent emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is responsible for a worldwide pandemic.
T10 624-773 Sentence denotes Given the global health emergency, drug repositioning is the most reliable option to design an efficient therapy for infected patients without delay.
T11 774-960 Sentence denotes The first step of the viral replication cycle [i.e. attachment to the surface of respiratory cells, mediated by the spike (S) viral protein] offers several potential therapeutic targets.
T12 961-1108 Sentence denotes The S protein uses the angiotension-converting enzyme-2 (ACE-2) receptor for entry, but also sialic acids linked to host cell surface gangliosides.
T13 1109-1349 Sentence denotes Using a combination of structural and molecular modelling approaches, this study showed that chloroquine (CLQ), one of the drugs currently under investigation for SARS-CoV-2 treatment, binds sialic acids and gangliosides with high affinity.
T14 1350-1470 Sentence denotes A new type of ganglioside-binding domain at the tip of the N-terminal domain of the SARS-CoV-2 S protein was identified.
T15 1471-1652 Sentence denotes This domain (111–158), which is fully conserved among clinical isolates worldwide, may improve attachment of the virus to lipid rafts and facilitate contact with the ACE-2 receptor.
T16 1653-1821 Sentence denotes This study showed that, in the presence of CLQ [or its more active derivative, hydroxychloroquine (CLQ-OH)], the viral S protein is no longer able to bind gangliosides.
T17 1822-1978 Sentence denotes The identification of this new mechanism of action of CLQ and CLQ-OH supports the use of these repositioned drugs to cure patients infected with SARS-CoV-2.
T18 1979-2161 Sentence denotes The in-silico approaches used in this study might also be used to assess the efficiency of a broad range of repositioned and/or innovative drug candidates before clinical evaluation.
T19 2163-2181 Sentence denotes Graphical Abstract
T20 2183-2198 Sentence denotes 1 Introduction
T21 2199-2382 Sentence denotes The recent emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic [1], and there is an urgent need to identify active antiviral agents.
T22 2383-2538 Sentence denotes Given the global health emergency, drug repositioning is the most reliable option to design an efficient therapy for infected patients without delay [2,3].
T23 2539-2786 Sentence denotes Several drugs have already been tested, among which chloroquine (CLQ), a well-known antimalarial drug, is one of the most promising as it has shown apparent efficacy in the treatment of COVID-19-associated pneumonia in recent clinical studies [4].
T24 2787-2933 Sentence denotes However, the mechanism of action of CLQ against SARS-CoV-2 is unclear as the drug seems to exert a broad range of potential antiviral effects [5].
T25 2934-3151 Sentence denotes Therefore, although CLQ is classically considered as an inhibitor of endocytic pathways through elevation of endosomal pH [6], its detailed molecular mechanism of action as an antiviral compound remains unclear [3,5].
T26 3152-3438 Sentence denotes Interestingly, CLQ has been shown to interfere with the terminal glycosylation of angiotensin-converting enzyme-2 (ACE-2) [7], which acts as a plasma membrane receptor for both SARS-CoV [8] and SARS-CoV-2 [9], and CLQ could act at several steps of the coronavirus replication cycle [7].
T27 3439-3603 Sentence denotes These data suggest the interesting and mostly unexplored possibility that CLQ could prevent viral attachment through a direct effect on host cell surface molecules.
T28 3604-3853 Sentence denotes One important characteristic of human coronaviruses is that besides their protein membrane receptor, they also depend upon sialic-acid-containing glycoproteins and gangliosides that act as primary attachment factors along the respiratory tract [10].
T29 3854-4015 Sentence denotes The present study used a combination of structural and molecular modelling approaches [11] to investigate the potential interaction between CLQ and sialic acids.
T30 4016-4354 Sentence denotes A ganglioside-binding site in the N-terminal domain (NTD) of the spike (S) glycoprotein of SARS-CoV-2 was identified, and CLQ was shown to be a potential blocker of the S–ganglioside interaction which occurs in the first step of the viral replication cycle (i.e. attachment to the surface of respiratory cells, mediated by the S protein).
T31 4355-4479 Sentence denotes In addition, the antiviral potential of CLQ and its derivative hydroxychloroquine (CLQ-OH) against SARS-CoV-2 were compared.
T32 4480-4624 Sentence denotes Overall, this study found that CLQ and CLQ-OH may be used to fight pathogenic human coronaviruses [3], [4], [5], [6], including SARS-CoV-2 [12].
T33 4626-4650 Sentence denotes 2 Materials and methods
T34 4651-4754 Sentence denotes In-silico analyses were performed using Hyperchem and Molegro Molecular viewer as described [11,13,14].
T35 4755-5197 Sentence denotes The initial coordinates of GM1 were obtained from CHARMM-GUI Glycolipid Modeler (http://www.charmmgui.org/?doc=input/glycolipid; [15]), which uses the internal coordinate information of common glycosidic torsion angle values, orients the ganglioside perpendicular to the membrane, and performs Langevin dynamics with a cylindrical restraint potential to keep the whole GM1 molecule cylindrical, especially the membrane-embedded ceramide part.
T36 5198-5298 Sentence denotes In the next step, the ganglioside was included in a periodic box solvated with 1128 water molecules.
T37 5299-5490 Sentence denotes The system was energy-minimized six times, switching between runs using the steepest descent gradients and runs using Polak–Ribière conjugate gradients until convergence to machine precision.
T38 5491-5613 Sentence denotes The ganglioside was subsequently merged with the NTD domain of SARS-CoV-2 S protein as obtained from pdb file # 6VSB [16].
T39 5614-5712 Sentence denotes Initial conditions corresponded to minimized structures obtained with the Polak–Ribière algorithm.
T40 5713-5864 Sentence denotes Docked complexes were subsequently submitted to iterative cycles of molecular dynamics using the CHARMM36 force field optimized for carbohydrates [17].
T41 5865-5983 Sentence denotes Interaction energies were calculated from stable complexes using the Ligand Energy Inspector function of Molegro [13].
T42 5984-6059 Sentence denotes N-acetylneuraminic acid (Neu5Ac) was generated with the Hyperchem database.
T43 6060-6143 Sentence denotes 9-O-acetyl-N-acetylneuraminic acid (9-O-SIA) was retrieved from pdb file 6Q06 [18].
T44 6144-6209 Sentence denotes CLQ is N-(7-chloroquinolin-4-yl)-N,N-diéthyl-pentane-1,4-diamine.
T45 6210-6282 Sentence denotes Its three-dimensioanl structure was retrieved from pdb file # 4V2O [19].
T46 6283-6362 Sentence denotes CLQ-OH is (RS)-2-[{4-[(7-chloroquinolin-4-yl)amino]pentyl}(ethyl)amino]ethanol.
T47 6363-6423 Sentence denotes CLQ-OH was generated by hydroxylation of CLQ with Hyperchem.
T48 6424-6517 Sentence denotes Both CLQ and CLQ-OH were energy-minimized and merged with water molecules as described below.
T49 6519-6529 Sentence denotes 3 Results
T50 6531-6601 Sentence denotes 3.1 Structural and conformational analysis of CLQ and CLQ-OH in water
T51 6602-6670 Sentence denotes The chemical structures of CLQ and CLQ-OH are shown in Fig. 1 (a,b).
T52 6671-6772 Sentence denotes The only difference between the two molecules is the presence of a terminal hydroxyl group in CLQ-OH.
T53 6773-6878 Sentence denotes This OH group has a marked influence on the conformation and water-solubilization properties of the drug.
T54 6879-6985 Sentence denotes CLQ-OH may adopt a wide range of conformations, the most stable being the extended one shown in Fig. 1(c).
T55 6986-7205 Sentence denotes When immersed in a periodic box of 31.5 Å2 with 1042 water molecules, the system reached, at equilibrium, an estimated energy of interaction of -92 kJ.mol−1, accounting for 56 water molecules solvating CLQ-OH Fig. 1(d).
T56 7206-7320 Sentence denotes In contrast, due to an intramolecular hydrophobic effect, CLQ appeared to be more condensed than CLQ-OH Fig. 1(e).
T57 7321-7434 Sentence denotes At equilibrium, CLQ was surrounded by 58 water molecules with an energy of interaction of -79 kJ.mol−1 Fig. 1(f).
T58 7435-7645 Sentence denotes Fig. 1 Chemical structure of chloroquine (CLQ) and hydroxychloroquine (CLQ-OH). (a) CLQ. (b) CLQ-OH. (c) CLQ-OH extended conformer. (d) CLQ-OH in water. (e) Typical condensed conformer of CLQ. (f) CLQ in water.
T59 7646-7776 Sentence denotes The molecules in (c–f) are shown in either tube or sphere rendering (carbon, green; nitrogen, blue; oxygen, red; hydrogen, white).
T60 7777-7854 Sentence denotes In (c) and (e), the chlorine atom of CLQ and CLQ-OH is indicated by an arrow.
T61 7855-8021 Sentence denotes These water-compatible conformations of CLQ and CLQ-OH were used as initial conditions for studying the interaction of these drugs with sialic acids and gangliosides.
T62 8023-8079 Sentence denotes 3.2 Sialic acids as molecular targets of CLQ and CLQ-OH
T63 8080-8164 Sentence denotes Neu5Ac is the predominant sialic acid found in human glycoproteins and gangliosides.
T64 8165-8332 Sentence denotes When CLQ was merged with Neu5Ac, a quasi-instantaneous fit occurred between the two molecules, whose global shapes in water are geometrically complementary Fig. 2 (a).
T65 8333-8457 Sentence denotes This is particularly obvious in the views of the CLQ–Neu5Ac complex in mixed surface/balls and sticks rendition Fig. 2(a,b).
T66 8458-8626 Sentence denotes The interaction was driven by the positioning of the negative charge of the carboxylate group of Neu5Ac and one of the two cationic charges of CLQ (pKa 10.2) Fig. 2(c).
T67 8627-8702 Sentence denotes The energy of interaction of this complex was estimated to be -47 kJ.mol−1.
T68 8703-8928 Sentence denotes As coronaviruses preferentially interact with 9-O-acetyl-N-acetylneuraminic acid (9-O-SIA) [10], this study used a similar molecular modelling approach to assess whether CLQ could also interact with this specific sialic acid.
T69 8929-9036 Sentence denotes A good fit between CLQ and 9-O-SIA was obtained Fig. 2(d–f), with an energy of interaction of -45 kJ.mol−1.
T70 9037-9186 Sentence denotes In this case, the carboxylate group of the sialic acid interacted with the cationic group of the nitrogen-containing ring of CLQ (pKa 8.1) Fig. 2(d).
T71 9187-9261 Sentence denotes The complex was further stabilized by OH-π and van der Waals interactions.
T72 9262-9410 Sentence denotes Fig. 2 Molecular modelling of chloroquine (CLQ) interaction with sialic acids. (a,b) Surface representation of the CLQ–sialic acid (Neu5Ac) complex.
T73 9411-9455 Sentence denotes Two opposite views of the complex are shown.
T74 9456-9788 Sentence denotes Note the geometric complementarity between the L-shape conformer of CLQ dissolved in water (in blue) and Neu5Ac (in red). (c) Neu5Ac bound to CLQ via a combination of CH-π and electrostatic interactions with one of the cationic groups of CLQ (+). (d) Molecular modelling of CLQ bound to N-acetyl-9-O-acetylneuraminic acid (9-O-SIA).
T75 9789-9989 Sentence denotes From right to left, the dashed lines indicate a series of van der Waals, OH-π and electrostatic contacts with both cationic groups of CLQ (+). (e,f) Surface representations of the CLQ–9-O-SIA complex.
T76 9990-10076 Sentence denotes Next, CLQ-OH was tested to assess whether it could, as CLQ, bind to 9-O-SIA (Fig. 3 ).
T77 10077-10268 Sentence denotes The complex obtained with CLQ-OH was very similar to that obtained with CLQ [compare Fig. 3(a,b) with Fig. 2(e,f), although several conformational adjustments occurred during the simulations.
T78 10269-10405 Sentence denotes Interestingly, the OH group of CLQ-OH reinforced the binding of CLQ to sialic acid through establishment of a hydrogen bond Fig. 3(c,d).
T79 10406-10664 Sentence denotes Overall, this hydrogen bond compensated for the slight loss of energy caused by the conformational rearrangement, and the energy of interaction of the complex was estimated to be -46 kJ.mol−1, which is very close to the value obtained for CLQ (-45 kJ.mol−1).
T80 10665-10847 Sentence denotes Fig. 3 Molecular modelling of hydroxychloroquine (CLQ-OH) interaction with sialic acids. (a,b) Surface representation of CLQ-OH bound to N-acetyl-9-O-acetylneuraminic acid (9-O-SIA).
T81 10848-10892 Sentence denotes Two opposite views of the complex are shown.
T82 10893-11094 Sentence denotes Note the geometric complementarity between CLQ-OH (in blue) and 9-O-SIA (in red). (c,d) Molecular mechanism of CLQ-OH binding to 9-O-SIA: combination of electrostatic interactions and hydrogen bonding.
T83 11096-11156 Sentence denotes 3.3 Molecular recognition of gangliosides by CLQ and CLQ-OH
T84 11157-11247 Sentence denotes In the respiratory tract, sialic acids are usually part of glycoproteins and gangliosides.
T85 11248-11393 Sentence denotes Molecular modelling approaches were used to assess whether CLQ and CLQ-OH can recognize sialic acid units in their natural molecular environment.
T86 11394-11509 Sentence denotes In these simulations, ganglioside GM1 was chosen as a representative example of human plasma membrane gangliosides.
T87 11510-11563 Sentence denotes A first series of simulations was performed with CLQ.
T88 11564-11683 Sentence denotes When merged with the ganglioside, CLQ had two distinct binding sites, both located in the polar saccharide part of GM1.
T89 11684-11779 Sentence denotes The first site was located at the tip of the saccharide moiety of the ganglioside Fig. 4 (a,b).
T90 11780-11839 Sentence denotes The energy of interaction was estimated to be -47 kJ.mol−1.
T91 11840-11974 Sentence denotes CLQ retained the typical L-shape structure of the water-soluble conformer bound to isolated sialic acids [compare Figs 2(c) and 4(a)].
T92 11975-12105 Sentence denotes From a mechanistic point of view, the carboxylate group of the sialic acid of GM1 was oriented towards the cationic groups of CLQ.
T93 12106-12246 Sentence denotes The rings of CLQ faced the N-acetylgalactosamine (GalNAc) residue of GM1, establishing both OH-π interaction and hydrogen bonding Fig. 4(b).
T94 12247-12362 Sentence denotes The second site was in a large area including both the ceramide–sugar junction and the saccharide moiety Fig. 4(c).
T95 12363-12552 Sentence denotes The chlorine atom of CLQ was oriented towards the ceramide axis, allowing the nitrogen-containing ring of CLQ to stack on to the pyrane ring of the first sugar residue [i.e. glucose (Glc)].
T96 12553-12707 Sentence denotes The perfect geometric complementarity of the two partners Fig. 4(c,d) accounted for a particularly high energy of interaction in this case (-61 kJ.mol−1).
T97 12708-12909 Sentence denotes Interestingly, there was no overlap between the two CLQ-binding sites on GM1, so the ganglioside could accommodate two CLQ molecules Fig. 4(e), reaching a global energy of interaction of -108 kJ.mol−1.
T98 12910-13010 Sentence denotes A similar situation was observed with CLQ-OH, which occupies the same binding site as CLQ Fig. 4(f).
T99 13011-13166 Sentence denotes In this case, the energy of interaction was further increased by stabilizing contacts established between the two CLQ-OH molecules, reaching -120 kJ.mol−1.
T100 13167-13289 Sentence denotes Overall, these data showed that CLQ and CLQ-OH have a good fit for sialic acids, either isolated or bound to gangliosides.
T101 13290-13409 Sentence denotes Fig. 4 Molecular modelling simulations of chloroquine (CLQ) and hydroxychloroquine (CLQ-OH) binding to ganglioside GM1.
T102 13410-13860 Sentence denotes The surface electrostatic potential of GM1 indicates a non-polar, membrane-embedded part corresponding to ceramide (white areas), and an acidic part protruding in the extracellular space corresponding to the sialic-acid-containing saccharide part (red areas). (a) CLQ bound to the tip of the carbohydrate moiety of GM1. (b) Molecular mechanism of CLQ–ganglioside interactions. (c) Molecular dynamics simulations revealed a second site of interaction.
T103 13861-14376 Sentence denotes In this case, the aromatic cycles of CLQ are positioned at the ceramide–sugar junction, whereas the nitrogen atoms interact with the acidic part of the ganglioside (not illustrated). (d,e) Surface views of GM1 complexed with one (d) or two (e) CLQ molecules (both in blue), illustrating the geometric complementarity of GM1 and CLQ molecules. (f) One GM1 molecule can also accommodate two distinct CLQ-OH molecules simultaneously, after slight rearrangement allowing increased fit due to CLQ-OH/CLQ-OH interactions.
T104 14377-14484 Sentence denotes To improve clarity, CLQ-OH molecules bound to GM1 are represented in two distinct colours (blue and green).
T105 14486-14545 Sentence denotes 3.4 Structural analysis of the NTD of SARS-CoV-2 S protein
T106 14546-14721 Sentence denotes The next step of this study was to determine how SARS-CoV-2 could interact with plasma membrane gangliosides, and whether such interaction could be affected by CLQ and CLQ-OH.
T107 14722-14801 Sentence denotes The global structure of the SARS-CoV-2 S protein [16] is shown in Fig. 5 (a–d).
T108 14802-14957 Sentence denotes It consists of a trimer of S proteins, each harbouring two distinct domains distant from the viral envelope: the receptor-binding region (RBD) and the NTD.
T109 14958-15650 Sentence denotes Fig. 5 Structural features of the SARS-CoV-2 spike (S) protein. (a) Trimeric structure (each S protein has a distinct surface colour, ‘blue’, ‘yellow’ and ‘purple’). (b) Ribbon representation of ‘blue’ S protein in the trimer (α-helix, red; β-strand, blue; coil, grey). (c) Surface structure of the ‘blue’ S protein isolated from the trimer. (d) Ribbon structure of the ‘blue’ S protein. (e) Zoom on the N-terminal domain (NTD) of the ‘blue’ S protein. (f,g) Molecular model of a minimal NTD obtained with Hyperchem [ribbon in representation in (f), surface rendering in (g)]. (h) Highlighting of the amino acid residues of the NTD that could belong to a potential ganglioside-binding domain.
T110 15651-15893 Sentence denotes It was reasoned that if the RBD is engaged in functional interactions with the ACE-2 receptor, it would be interesting to search for potential ganglioside-binding sites on the other cell-accessible domain of the S glycoprotein (i.e. the NTD).
T111 15894-15949 Sentence denotes The NTD contains approximately 290 amino acid residues.
T112 15950-16141 Sentence denotes The tip of the NTD was of particular interest, as it displays a flat interface Fig. 5(f) ideally positioned for targeting a ganglioside-rich plasma membrane microdomain, such as a lipid raft.
T113 16142-16306 Sentence denotes The amino acid sequence of the planar interfacial surface located at the tip of the NTD was analysed for the presence of consensus ganglioside-binding domains [20].
T114 16307-16414 Sentence denotes These motifs are constituted by a triad of mandatory amino acid residues such as (K,R)-Xn-(F,Y,W)-Xn-(K,R).
T115 16415-16544 Sentence denotes The Xn intercalating segments, usually four to five residues, may contain any amino acid, but often Gly, Pro and/or Ser residues.
T116 16545-16684 Sentence denotes The strict application of this algorithm did not allow the detection of any potential ganglioside-binding domain in this region of the NTD.
T117 16685-16792 Sentence denotes However, an intriguing over-representation of aromatic and basic residues was found in the 129–158 segment:
T118 16793-16832 Sentence denotes 129-KVCEFQFCNDPFLGVYYHKNNKSWMESEFR-158.
T119 16833-16954 Sentence denotes This 30-amino acid stretch also contains Gly, Pro and/or Ser residues that are often found in ganglioside-binding motifs.
T120 16955-17074 Sentence denotes These observations supported the notion that the tip of the NTD could display a large ganglioside-attachment interface.
T121 17076-17160 Sentence denotes 3.5 Molecular interactions between gangliosides and the NTD of SARS-CoV-2 S protein
T122 17161-17336 Sentence denotes Molecular dynamic simulations of a structural motif encompassing amino acid residues 100–175 of the NTD Fig. 5(f–h) merged with ganglioside GM1 further supported this concept.
T123 17337-17481 Sentence denotes As shown in Fig. 6 , the large flat area of this structural domain fitted very well with the protruding oligosaccharide part of the ganglioside.
T124 17482-17605 Sentence denotes Several amino acid residues appear to be critical for this interaction, especially Phe-135, Asn-137 and Arg-158 (Table 1 ).
T125 17606-17710 Sentence denotes Overall, the complex involved 10 amino acid residues for a total energy of interaction of -100 kJ.mol−1.
T126 17711-17890 Sentence denotes At this stage, it was observed that approximately 50% of the interface was involved in the complex, leaving the remaining 50% available for interaction with a second GM1 molecule.
T127 17891-18163 Sentence denotes As expected, merging a second GM1 molecule with the preformed GM1–NTD complex led to a trimolecular complex consisting of two gangliosides in a typical symmetrical chalice-like structure into which the NTD could insert its interfacial ganglioside-binding domain (Fig. 7 ).
T128 18164-18343 Sentence denotes The formation of this trimolecular complex was progressive, starting with a conformational rearrangement of the first ganglioside–NTD complex triggered by the second GM1 molecule.
T129 18344-18469 Sentence denotes The energy of interaction of the new complex was consistently increased by 37%, reaching an estimated value of -137 kJ.mol−1.
T130 18470-18634 Sentence denotes At this stage, attachment of the NTD to the ganglioside-rich microdomain involved the whole interface (i.e. 15 surface-accessible residues from Asp-111 to Ser-162).
T131 18635-18719 Sentence denotes The critical residues were Asp-111, Gln-134, Phe-135, Arg-158 and Ser-161 (Table 1).
T132 18720-18838 Sentence denotes Fig. 6 Molecular complex between the N-terminal domain (NTD) of SARS-CoV-2 spike protein and a single GM1 ganglioside.
T133 18839-18935 Sentence denotes The NTD is represented in ribbons superposed with a transparent surface rendering (light green).
T134 18936-18987 Sentence denotes Two symmetric views of the complex are shown (a,b).
T135 18988-19116 Sentence denotes The amino acid residues Q-134 to D-138 located in the centre of the ganglioside-binding domain are represented as green spheres.
T136 19117-19203 Sentence denotes The saccharide part of the ganglioside forms a landing surface for the tip of the NTD.
T137 19204-19319 Sentence denotes Table 1 Energy of interaction of each amino acid residue of SARS‐CoV‐2 spike protein in contact with GM1 molecules.
T138 19320-19372 Sentence denotes Amino acid residues Energy of interaction (kJ.mol‐1)
T139 19373-19401 Sentence denotes First step: one GM1 molecule
T140 19402-19415 Sentence denotes  Asp‐111 −5.6
T141 19416-19429 Sentence denotes  Lys‐113 −8.2
T142 19430-19443 Sentence denotes  Gln‐134 −8.6
T143 19444-19458 Sentence denotes  Phe‐135 −20.1
T144 19459-19472 Sentence denotes  Cys‐136 −7.0
T145 19473-19487 Sentence denotes  Asn‐137 −15.2
T146 19488-19501 Sentence denotes  Asp‐138 −6.4
T147 19502-19516 Sentence denotes  Arg‐158 −17.4
T148 19517-19530 Sentence denotes  Ser‐161 −9.7
T149 19531-19544 Sentence denotes  Ser‐162 −2.0
T150 19545-19558 Sentence denotes  Total −100.2
T151 19559-19589 Sentence denotes Second step: two GM1 molecules
T152 19590-19604 Sentence denotes  Asp‐111 −15.8
T153 19605-19619 Sentence denotes  Ser‐112 −10.7
T154 19620-19633 Sentence denotes  Lys‐113 −9.2
T155 19634-19648 Sentence denotes  Gln‐134 −11.2
T156 19649-19663 Sentence denotes  Phe‐135 −10.5
T157 19664-19677 Sentence denotes  Cys‐136 −6.2
T158 19678-19691 Sentence denotes  Asn‐137 −4.7
T159 19692-19705 Sentence denotes  Phe‐140 −5.2
T160 19706-19719 Sentence denotes  Gly‐142 −5.6
T161 19720-19733 Sentence denotes  Glu‐156 −9.0
T162 19734-19748 Sentence denotes  Phe‐157 −13.8
T163 19749-19763 Sentence denotes  Arg‐158 −19.8
T164 19764-19777 Sentence denotes  Tyr‐160 −3.2
T165 19778-19791 Sentence denotes  Ser‐161 −9.7
T166 19792-19805 Sentence denotes  Ser‐162 −2.0
T167 19806-19819 Sentence denotes  Total −136.6
T168 19820-19928 Sentence denotes Fig. 7 Molecular complex between the N-terminal domain (NTD) of SARS-CoV-2 spike protein and a dimer of GM1.
T169 19929-19986 Sentence denotes In (a), (c) and (d), the NTD is represented as in Fig. 4.
T170 19987-20052 Sentence denotes In (b), the surface of the NTD is shown without any transparency.
T171 20053-20175 Sentence denotes The amino acid residues Q-134 to S-162 belonging to the ganglioside-binding domain (GBD) are represented as green spheres.
T172 20176-20285 Sentence denotes Compared with a single GM1 molecule, the dimer of gangliosides forms a larger attractive surface for the NTD.
T173 20286-20381 Sentence denotes In the above view of (d), the anchorage of the NTD to the gangliosides is particularly obvious.
T174 20382-20600 Sentence denotes As chloroquine also interacts with the saccharide part of GM1, its presence would clearly mask most of the landing surface available for the NTD, preventing attachment of the virus to the plasma membrane of host cells.
T175 20602-20740 Sentence denotes 3.6 Potential coordinated interactions between SARS-CoV-2 and the plasma membrane of a host cell: key role of gangliosides in lipid rafts
T176 20741-21026 Sentence denotes Taken together, these data strongly support the concept of a dual receptor/attachment model for SARS-CoV-2, with the RBD domain being involved in ACE-2 receptor recognition, and the NTD interface responsible for finding a ganglioside-rich landing area (lipid raft) at the cell surface.
T177 21027-21136 Sentence denotes Such a dual receptor model, consistent with the topology of the SARS-CoV-2 S protein, is proposed in Fig. 8 .
T178 21137-21329 Sentence denotes With this model in mind, the potential effects of CLQ and CLQ-OH were studied, both of which, according to the molecular modelling data, have a high affinity for sialic acids and gangliosides.
T179 21330-21446 Sentence denotes Fig. 8 Dual recognition of gangliosides and angiotensin-converting enzyme-2 (ACE-2) by SARS-CoV-2 spike (S) protein.
T180 21447-21563 Sentence denotes The viral protein displays two distinct domains, the tips of which are available for distinct types of interactions.
T181 21564-21713 Sentence denotes The receptor-binding domain binds to the ACE-2 receptor, and the N-terminal domain (NTD) binds to the ganglioside-rich domain of the plasma membrane.
T182 21714-21947 Sentence denotes Lipid rafts, which are membrane domains enriched in gangliosides (in yellow) and cholesterol (in blue), provide a perfect attractive interface for adequately positioning the viral S protein at the first step of the infection process.
T183 21948-22204 Sentence denotes These structural and molecular modelling studies suggest that amino acid residues 111–162 of the NTD form a functional ganglioside-binding domain, the interaction of which with lipid rafts can be efficiently prevented by chloroquine and hydroxychloroquine.
T184 22206-22334 Sentence denotes 3.7 Molecular mechanism of CLQ and CLQ-OH antiviral effect: preventing SARS-CoV-2 S protein access to cell surface gangliosides
T185 22335-22540 Sentence denotes With the aim of establishing whether CLQ and CLQ-OH could prevent the attachment of SARS-CoV-2 to plasma membrane gangliosides, the initial NTD–GM1 complex was superposed with a drug–GM1 complex (Fig. 9 ).
T186 22541-22604 Sentence denotes To improve clarity, the ganglioside is not presented in Fig. 9.
T187 22605-22794 Sentence denotes This superposition shows that the NTD and the drug (CLQ-OH in this case) share the same spatial position when bound to GM1, so GM1 cannot bind the viral protein and the drug simultaneously.
T188 22795-23019 Sentence denotes This is due to the fact that the NTD and the drugs (CLQ and CLQ-OH) bind to GM1 with a similar mechanism controlled by a dyad of functional interactions: a hydrogen bond and a geometrically perfect CH-π stacking interaction.
T189 23020-23156 Sentence denotes In the case of the NTD, the hydrogen bond involves Asn-167, whereas CH-π stacking is mediated by the aromatic ring of Phe-135 Fig. 6(b).
T190 23157-23249 Sentence denotes On one hand, Asn-167 establishes a network of hydrogen bonds with the GalNAc residue of GM1.
T191 23250-23352 Sentence denotes On the other hand, the flat aromatic ring of Phe-135 stacks on to the cycle of the Glc residue of GM1.
T192 23353-23472 Sentence denotes In the case of CLQ and CLQ-OH, it is the nitrogen-containing ring of the drug that stacks on to the Glc ring Fig. 4(c).
T193 23473-23579 Sentence denotes Note that both the Phe-135 (in red) and CLQ-OH (in green) rings are located in the same position (Fig. 9).
T194 23580-23714 Sentence denotes The other CLQ-OH molecule, which covers the tip of the sugar part of the ganglioside, interacts with the GalNAc ganglioside Fig. 4(b).
T195 23715-23824 Sentence denotes When the NTD is bound to the ganglioside, the side chain of Asn-137 is found in this exact position (Fig. 9).
T196 23825-23993 Sentence denotes Thus, once two CLQ-OH (or two CLQ) molecules are bound to a ganglioside Fig. 4(e,f), any binding of a SARS-Cov-2 S protein to the same ganglioside is totally prevented.
T197 23994-24130 Sentence denotes The energy required to overcome this steric incompatibility is estimated to be several hundred kJ.mol−1, which is far too high to occur.
T198 24131-24189 Sentence denotes Fig. 9 Mechanism of action of hydroxychloroquine (CLQ-OH).
T199 24190-24296 Sentence denotes The N-terminal domain (NTD) bound to GM1 was superposed onto GM1 in interaction with two CLQ-OH molecules.
T200 24297-24569 Sentence denotes The models only show the NTD and both CLQ-OH molecules (not GM1, to improve clarity). (a,b) The aromatic ring of F-135 (in red), which stacks onto the glucose cycle of GM1, overlaps the aromatic CLQ-OH rings (in green) which also bind to GM1 via a CH-π stacking mechanism.
T201 24570-25005 Sentence denotes The N-137 residue of the NTD interacts with the N-acetylgalactosamine residue of GM1, but this interaction cannot occur in the presence of CLQ-OH as this part of GM1 is totally masked by the drug. (c,d) Superposition of the NTD surface (in purple) with the two CLQ-OH molecules bound to GM1, illustrating the steric impossibility that prevents NTD binding to GM1 when both CLQ-OH molecules are already interacting with the ganglioside.
T202 25007-25154 Sentence denotes 3.8 Sequence alignment analysis of SARS-CoV-2 and related coronavirus: evolution of the ganglioside-binding domain at critical amino acid residues
T203 25155-25365 Sentence denotes As CLQ and CLQ-OH are potential therapies for SARS-CoV-2 infection, it is important to check whether the amino acid residues identified as critical for ganglioside binding are conserved among clinical isolates.
T204 25366-25518 Sentence denotes The alignment of the 111–162 domain of 11 clinical isolates of SARS-CoV-2 from various geographic origins (including Asia and USA) is shown in Fig. 10 .
T205 25519-25651 Sentence denotes In this region, which contains the ganglioside-binding domain identified in the present report, all amino acids are fully conserved.
T206 25652-25859 Sentence denotes Interestingly, the motif is built like a giant consensus ganglioside-binding domain: a central region displaying the critical aromatic residue (Phe-135) and a basic residue at each end (Lys-113 and Arg-158).
T207 25860-25975 Sentence denotes In the middle of each stretch separating this typical triad, there is a N-glycosylation site (Asn-122 and Asn-149).
T208 25976-26181 Sentence denotes These last regions are not directly involved in ganglioside binding, so the oligosaccharide linked to these asparagine residues could be perfectly intercalated between the sugar head group of gangliosides.
T209 26182-26390 Sentence denotes Fig. 10 Amino acid sequence alignments of the ganglioside-binding domain (GBD) of the SARS-CoV-2 spike protein. (a) Clinical SARS-CoV-2 isolates aligned with the reference sequence (6VSB_A, fragment 111–162).
T210 26391-26460 Sentence denotes The amino acid residues involved in GM1 binding are indicated in red.
T211 26461-26637 Sentence denotes Two asparagine residues acting as glycosylation sites are highlighted in yellow. (b) Alignments of human and animal viruses compared with SARS-CoV-2 (6VSB_A, fragment 111–162).
T212 26638-26896 Sentence denotes Deletions are highlighted in green, amino acid changes in residues involved in ganglioside binding are highlighted in blue, conserved residues of the GBD are highlighted in red, and asparagine residues acting as glycosylation sites are highlighted in yellow.
T213 26897-27132 Sentence denotes It was also noted that the ganglioside-binding domain of the NTD is fully conserved in bat RaTG13, which indicates a close relationship between the bat coronavirus and the human isolates that are currently circulating around the world.
T214 27133-27384 Sentence denotes However, the motif is slightly different in other bat- and human-related coronaviruses (Fig. 10), suggesting a recent evolution which could explain, at least in part, why SARS-CoV-2 is more contagious than previously characterized human coronaviruses.
T215 27386-27399 Sentence denotes 4 Discussion
T216 27400-27550 Sentence denotes Sialic acids linked to glycoproteins and gangliosides are used by a broad range of viruses as receptors and/or attachment factors for cell entry [10].
T217 27551-27677 Sentence denotes These viruses include major human pathogens affecting the respiratory tract, such as influenza [21] and coronaviruses [22,23].
T218 27678-27815 Sentence denotes The attachment to sialic-acid-containing cell surface structures is mediated by receptor-binding proteins that belong to the viral spike.
T219 27816-27902 Sentence denotes In the case of coronaviruses, this function is fulfilled by the S glycoprotein [9,24].
T220 27903-28044 Sentence denotes SARS-CoV and SARS-CoV-2 interact with the ACE-2 protein, which has been identified as a key determinant of the contagiousness of viruses [8].
T221 28045-28236 Sentence denotes However, considering the increased transmissibility of SARS-CoV-2 compared with SARS-CoV, binding to ACE-2 alone might not be enough to ensure robust infection of the upper respiratory tract.
T222 28237-28392 Sentence denotes Thus, it is likely that SARS-CoV-2 might also bind to other cell surface attachment factors, such as sialic-acid-containing glycoproteins and gangliosides.
T223 28393-28563 Sentence denotes Consistent with this notion, it has been shown that depletion of cell surface sialic acids by neuraminidase treatment inhibited MERS-CoV entry of human airway cells [25].
T224 28564-28795 Sentence denotes These data, which provide direct evidence that sialic acids play a critical role in human coronavirus attachment, broaden the therapeutic options to block the replication of SARS-CoV-2 that is responsible for the COVID-19 pandemic.
T225 28796-28936 Sentence denotes Few drugs have shown consistent antiviral efficiency in vitro together with reported efficiency in patients infected with SARS-CoV-2 [3,12].
T226 28937-29064 Sentence denotes Of these, CLQ is of interest as its chemical structure is based on a combination of cationic nitrogen atoms and aromatic rings.
T227 29065-29190 Sentence denotes Both features have been shown to be key determinants of the recognition of sialic acids and gangliosides by proteins [20,26].
T228 29191-29442 Sentence denotes Modelling approaches have been used successfully to decipher various molecular mechanisms of protein–sugar interactions accounting for the interaction of virus [27], bacteria [28], membrane [13] and amyloid proteins [20] with cell surface glycolipids.
T229 29443-29598 Sentence denotes This in-silico strategy was applied to unravel the molecular mechanisms underlying the antiviral mechanisms of CLQ and CLQ-OH against SARS-CoV-2 infection.
T230 29599-29759 Sentence denotes First, it was shown that CLQ and CLQ-OH bind readily to sialic acids with high affinity, including the typical 9-O-SIA subtype recognized by coronaviruses [23].
T231 29760-29848 Sentence denotes Next, it was shown that CLQ and CLQ-OH also bind to sialic-acid-containing gangliosides.
T232 29849-29941 Sentence denotes Based on these data, the drugs may also recognize the sialic acid residues of glycoproteins.
T233 29942-29987 Sentence denotes Further studies will help clarify this point.
T234 29988-30114 Sentence denotes This molecular modelling study has identified a new type of ganglioside-binding domain in the NTD of the SARS-CoV-2 S protein.
T235 30115-30233 Sentence denotes This ganglioside-binding domain consists of a large flat interface enriched in aromatic and basic amino acid residues.
T236 30234-30378 Sentence denotes It covers a stretch of 52 amino acid residues (111–162), which is longer than all linear ganglioside-binding domains characterized to date [29].
T237 30379-30532 Sentence denotes However, the new SARS-CoV-2 motif is organized into three distinct regions, including a central aromatic domain and two terminal basic domains (Fig. 10).
T238 30533-30679 Sentence denotes Thus, this motif displays the typical features that determine optimal binding to gangliosides (i.e. CH-π stacking and electrostatic interactions).
T239 30680-30846 Sentence denotes A major outcome of this study is the demonstration that CLQ and CLQ-OH display molecular groups that fully mimic the way in which the S protein binds to gangliosides.
T240 30847-30948 Sentence denotes Two CLQ (or two CLQ-OH) molecules can bind simultaneously to the polar head group of ganglioside GM1.
T241 30949-31118 Sentence denotes Interestingly, these simulations indicated that CLQ-OH is more potent than CLQ, in line with the reported increased antiviral activity of CLQ-OH against SARS-CoV-2 [30].
T242 31119-31293 Sentence denotes Once bound to GM1, the drugs prevent any access to the Glc and GalNAc units of the ganglioside, which are the critical binding residues for Phe-135 and Asn-137, respectively.
T243 31294-31461 Sentence denotes This amino acid dyad, as well as all the other residues that mediate ganglioside binding by the SARS-CoV-2 spike, is fully conserved among clinical isolates worldwide.
T244 31462-31571 Sentence denotes It is also conserved in the bat RaTG13 isolate, which reinforces the hypothesis of bat-to-human transmission.
T245 31572-31805 Sentence denotes From an epidemiological point of view, it can be hypothesized that the evolution of this motif has conferred an enhanced attachment capacity of human coronaviruses to the respiratory tract through improved S–ganglioside interactions.
T246 31807-31820 Sentence denotes 5 Conclusion
T247 31821-31913 Sentence denotes Given the global health emergency, drug repurposing is obviously the option of choice [2,3].
T248 31914-32122 Sentence denotes However, a considerable amount of time could be saved by in-silico testing to determine the capability of any potential anti-SARS-CoV-2 to disrupt the interaction of the S protein with the host cell membrane.
T249 32123-32364 Sentence denotes Applied to both RBD–ACE-2 and NTD–ganglioside interactions, this molecular modelling method will help select those drugs that are likely to interfere with the initial attachment of virus particles to the respiratory tract surface epithelium.
T250 32365-32488 Sentence denotes The study data support the use of CLQ, and preferentially CLQ-OH, as initial therapy for patients infected with SARS-CoV-2.
T251 32490-32498 Sentence denotes Funding:
T252 32499-32504 Sentence denotes None.
T253 32505-32525 Sentence denotes Competing interests:
T254 32526-32540 Sentence denotes None declared.
T255 32541-32558 Sentence denotes Ethical approval:
T256 32559-32572 Sentence denotes Not required.