LitCovid-sentences  

PMC:7128678 / 2183-32488 JSONTXT 15 Projects

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Id Subject Object Predicate Lexical cue
T20 0-15 Sentence denotes 1 Introduction
T21 16-199 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 200-355 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 356-603 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 604-750 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 751-968 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 969-1255 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 1256-1420 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 1421-1670 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 1671-1832 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 1833-2171 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 2172-2296 Sentence denotes In addition, the antiviral potential of CLQ and its derivative hydroxychloroquine (CLQ-OH) against SARS-CoV-2 were compared.
T32 2297-2441 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 2443-2467 Sentence denotes 2 Materials and methods
T34 2468-2571 Sentence denotes In-silico analyses were performed using Hyperchem and Molegro Molecular viewer as described [11,13,14].
T35 2572-3014 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 3015-3115 Sentence denotes In the next step, the ganglioside was included in a periodic box solvated with 1128 water molecules.
T37 3116-3307 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 3308-3430 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 3431-3529 Sentence denotes Initial conditions corresponded to minimized structures obtained with the Polak–Ribière algorithm.
T40 3530-3681 Sentence denotes Docked complexes were subsequently submitted to iterative cycles of molecular dynamics using the CHARMM36 force field optimized for carbohydrates [17].
T41 3682-3800 Sentence denotes Interaction energies were calculated from stable complexes using the Ligand Energy Inspector function of Molegro [13].
T42 3801-3876 Sentence denotes N-acetylneuraminic acid (Neu5Ac) was generated with the Hyperchem database.
T43 3877-3960 Sentence denotes 9-O-acetyl-N-acetylneuraminic acid (9-O-SIA) was retrieved from pdb file 6Q06 [18].
T44 3961-4026 Sentence denotes CLQ is N-(7-chloroquinolin-4-yl)-N,N-diéthyl-pentane-1,4-diamine.
T45 4027-4099 Sentence denotes Its three-dimensioanl structure was retrieved from pdb file # 4V2O [19].
T46 4100-4179 Sentence denotes CLQ-OH is (RS)-2-[{4-[(7-chloroquinolin-4-yl)amino]pentyl}(ethyl)amino]ethanol.
T47 4180-4240 Sentence denotes CLQ-OH was generated by hydroxylation of CLQ with Hyperchem.
T48 4241-4334 Sentence denotes Both CLQ and CLQ-OH were energy-minimized and merged with water molecules as described below.
T49 4336-4346 Sentence denotes 3 Results
T50 4348-4418 Sentence denotes 3.1 Structural and conformational analysis of CLQ and CLQ-OH in water
T51 4419-4487 Sentence denotes The chemical structures of CLQ and CLQ-OH are shown in Fig. 1 (a,b).
T52 4488-4589 Sentence denotes The only difference between the two molecules is the presence of a terminal hydroxyl group in CLQ-OH.
T53 4590-4695 Sentence denotes This OH group has a marked influence on the conformation and water-solubilization properties of the drug.
T54 4696-4802 Sentence denotes CLQ-OH may adopt a wide range of conformations, the most stable being the extended one shown in Fig. 1(c).
T55 4803-5022 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 5023-5137 Sentence denotes In contrast, due to an intramolecular hydrophobic effect, CLQ appeared to be more condensed than CLQ-OH Fig. 1(e).
T57 5138-5251 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 5252-5462 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 5463-5593 Sentence denotes The molecules in (c–f) are shown in either tube or sphere rendering (carbon, green; nitrogen, blue; oxygen, red; hydrogen, white).
T60 5594-5671 Sentence denotes In (c) and (e), the chlorine atom of CLQ and CLQ-OH is indicated by an arrow.
T61 5672-5838 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 5840-5896 Sentence denotes 3.2 Sialic acids as molecular targets of CLQ and CLQ-OH
T63 5897-5981 Sentence denotes Neu5Ac is the predominant sialic acid found in human glycoproteins and gangliosides.
T64 5982-6149 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 6150-6274 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 6275-6443 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 6444-6519 Sentence denotes The energy of interaction of this complex was estimated to be -47 kJ.mol−1.
T68 6520-6745 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 6746-6853 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 6854-7003 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 7004-7078 Sentence denotes The complex was further stabilized by OH-π and van der Waals interactions.
T72 7079-7227 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 7228-7272 Sentence denotes Two opposite views of the complex are shown.
T74 7273-7605 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 7606-7806 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 7807-7893 Sentence denotes Next, CLQ-OH was tested to assess whether it could, as CLQ, bind to 9-O-SIA (Fig. 3 ).
T77 7894-8085 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 8086-8222 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 8223-8481 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 8482-8664 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 8665-8709 Sentence denotes Two opposite views of the complex are shown.
T82 8710-8911 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 8913-8973 Sentence denotes 3.3 Molecular recognition of gangliosides by CLQ and CLQ-OH
T84 8974-9064 Sentence denotes In the respiratory tract, sialic acids are usually part of glycoproteins and gangliosides.
T85 9065-9210 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 9211-9326 Sentence denotes In these simulations, ganglioside GM1 was chosen as a representative example of human plasma membrane gangliosides.
T87 9327-9380 Sentence denotes A first series of simulations was performed with CLQ.
T88 9381-9500 Sentence denotes When merged with the ganglioside, CLQ had two distinct binding sites, both located in the polar saccharide part of GM1.
T89 9501-9596 Sentence denotes The first site was located at the tip of the saccharide moiety of the ganglioside Fig. 4 (a,b).
T90 9597-9656 Sentence denotes The energy of interaction was estimated to be -47 kJ.mol−1.
T91 9657-9791 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 9792-9922 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 9923-10063 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 10064-10179 Sentence denotes The second site was in a large area including both the ceramide–sugar junction and the saccharide moiety Fig. 4(c).
T95 10180-10369 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 10370-10524 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 10525-10726 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 10727-10827 Sentence denotes A similar situation was observed with CLQ-OH, which occupies the same binding site as CLQ Fig. 4(f).
T99 10828-10983 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 10984-11106 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 11107-11226 Sentence denotes Fig. 4 Molecular modelling simulations of chloroquine (CLQ) and hydroxychloroquine (CLQ-OH) binding to ganglioside GM1.
T102 11227-11677 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 11678-12193 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 12194-12301 Sentence denotes To improve clarity, CLQ-OH molecules bound to GM1 are represented in two distinct colours (blue and green).
T105 12303-12362 Sentence denotes 3.4 Structural analysis of the NTD of SARS-CoV-2 S protein
T106 12363-12538 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 12539-12618 Sentence denotes The global structure of the SARS-CoV-2 S protein [16] is shown in Fig. 5 (a–d).
T108 12619-12774 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 12775-13467 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 13468-13710 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 13711-13766 Sentence denotes The NTD contains approximately 290 amino acid residues.
T112 13767-13958 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 13959-14123 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 14124-14231 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 14232-14361 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 14362-14501 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 14502-14609 Sentence denotes However, an intriguing over-representation of aromatic and basic residues was found in the 129–158 segment:
T118 14610-14649 Sentence denotes 129-KVCEFQFCNDPFLGVYYHKNNKSWMESEFR-158.
T119 14650-14771 Sentence denotes This 30-amino acid stretch also contains Gly, Pro and/or Ser residues that are often found in ganglioside-binding motifs.
T120 14772-14891 Sentence denotes These observations supported the notion that the tip of the NTD could display a large ganglioside-attachment interface.
T121 14893-14977 Sentence denotes 3.5 Molecular interactions between gangliosides and the NTD of SARS-CoV-2 S protein
T122 14978-15153 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 15154-15298 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 15299-15422 Sentence denotes Several amino acid residues appear to be critical for this interaction, especially Phe-135, Asn-137 and Arg-158 (Table 1 ).
T125 15423-15527 Sentence denotes Overall, the complex involved 10 amino acid residues for a total energy of interaction of -100 kJ.mol−1.
T126 15528-15707 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 15708-15980 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 15981-16160 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 16161-16286 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 16287-16451 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 16452-16536 Sentence denotes The critical residues were Asp-111, Gln-134, Phe-135, Arg-158 and Ser-161 (Table 1).
T132 16537-16655 Sentence denotes Fig. 6 Molecular complex between the N-terminal domain (NTD) of SARS-CoV-2 spike protein and a single GM1 ganglioside.
T133 16656-16752 Sentence denotes The NTD is represented in ribbons superposed with a transparent surface rendering (light green).
T134 16753-16804 Sentence denotes Two symmetric views of the complex are shown (a,b).
T135 16805-16933 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 16934-17020 Sentence denotes The saccharide part of the ganglioside forms a landing surface for the tip of the NTD.
T137 17021-17136 Sentence denotes Table 1 Energy of interaction of each amino acid residue of SARS‐CoV‐2 spike protein in contact with GM1 molecules.
T138 17137-17189 Sentence denotes Amino acid residues Energy of interaction (kJ.mol‐1)
T139 17190-17218 Sentence denotes First step: one GM1 molecule
T140 17219-17232 Sentence denotes  Asp‐111 −5.6
T141 17233-17246 Sentence denotes  Lys‐113 −8.2
T142 17247-17260 Sentence denotes  Gln‐134 −8.6
T143 17261-17275 Sentence denotes  Phe‐135 −20.1
T144 17276-17289 Sentence denotes  Cys‐136 −7.0
T145 17290-17304 Sentence denotes  Asn‐137 −15.2
T146 17305-17318 Sentence denotes  Asp‐138 −6.4
T147 17319-17333 Sentence denotes  Arg‐158 −17.4
T148 17334-17347 Sentence denotes  Ser‐161 −9.7
T149 17348-17361 Sentence denotes  Ser‐162 −2.0
T150 17362-17375 Sentence denotes  Total −100.2
T151 17376-17406 Sentence denotes Second step: two GM1 molecules
T152 17407-17421 Sentence denotes  Asp‐111 −15.8
T153 17422-17436 Sentence denotes  Ser‐112 −10.7
T154 17437-17450 Sentence denotes  Lys‐113 −9.2
T155 17451-17465 Sentence denotes  Gln‐134 −11.2
T156 17466-17480 Sentence denotes  Phe‐135 −10.5
T157 17481-17494 Sentence denotes  Cys‐136 −6.2
T158 17495-17508 Sentence denotes  Asn‐137 −4.7
T159 17509-17522 Sentence denotes  Phe‐140 −5.2
T160 17523-17536 Sentence denotes  Gly‐142 −5.6
T161 17537-17550 Sentence denotes  Glu‐156 −9.0
T162 17551-17565 Sentence denotes  Phe‐157 −13.8
T163 17566-17580 Sentence denotes  Arg‐158 −19.8
T164 17581-17594 Sentence denotes  Tyr‐160 −3.2
T165 17595-17608 Sentence denotes  Ser‐161 −9.7
T166 17609-17622 Sentence denotes  Ser‐162 −2.0
T167 17623-17636 Sentence denotes  Total −136.6
T168 17637-17745 Sentence denotes Fig. 7 Molecular complex between the N-terminal domain (NTD) of SARS-CoV-2 spike protein and a dimer of GM1.
T169 17746-17803 Sentence denotes In (a), (c) and (d), the NTD is represented as in Fig. 4.
T170 17804-17869 Sentence denotes In (b), the surface of the NTD is shown without any transparency.
T171 17870-17992 Sentence denotes The amino acid residues Q-134 to S-162 belonging to the ganglioside-binding domain (GBD) are represented as green spheres.
T172 17993-18102 Sentence denotes Compared with a single GM1 molecule, the dimer of gangliosides forms a larger attractive surface for the NTD.
T173 18103-18198 Sentence denotes In the above view of (d), the anchorage of the NTD to the gangliosides is particularly obvious.
T174 18199-18417 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 18419-18557 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 18558-18843 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 18844-18953 Sentence denotes Such a dual receptor model, consistent with the topology of the SARS-CoV-2 S protein, is proposed in Fig. 8 .
T178 18954-19146 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 19147-19263 Sentence denotes Fig. 8 Dual recognition of gangliosides and angiotensin-converting enzyme-2 (ACE-2) by SARS-CoV-2 spike (S) protein.
T180 19264-19380 Sentence denotes The viral protein displays two distinct domains, the tips of which are available for distinct types of interactions.
T181 19381-19530 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 19531-19764 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 19765-20021 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 20023-20151 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 20152-20357 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 20358-20421 Sentence denotes To improve clarity, the ganglioside is not presented in Fig. 9.
T187 20422-20611 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 20612-20836 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 20837-20973 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 20974-21066 Sentence denotes On one hand, Asn-167 establishes a network of hydrogen bonds with the GalNAc residue of GM1.
T191 21067-21169 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 21170-21289 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 21290-21396 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 21397-21531 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 21532-21641 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 21642-21810 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 21811-21947 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 21948-22006 Sentence denotes Fig. 9 Mechanism of action of hydroxychloroquine (CLQ-OH).
T199 22007-22113 Sentence denotes The N-terminal domain (NTD) bound to GM1 was superposed onto GM1 in interaction with two CLQ-OH molecules.
T200 22114-22386 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 22387-22822 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 22824-22971 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 22972-23182 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 23183-23335 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 23336-23468 Sentence denotes In this region, which contains the ganglioside-binding domain identified in the present report, all amino acids are fully conserved.
T206 23469-23676 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 23677-23792 Sentence denotes In the middle of each stretch separating this typical triad, there is a N-glycosylation site (Asn-122 and Asn-149).
T208 23793-23998 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 23999-24207 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 24208-24277 Sentence denotes The amino acid residues involved in GM1 binding are indicated in red.
T211 24278-24454 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 24455-24713 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 24714-24949 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 24950-25201 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 25203-25216 Sentence denotes 4 Discussion
T216 25217-25367 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 25368-25494 Sentence denotes These viruses include major human pathogens affecting the respiratory tract, such as influenza [21] and coronaviruses [22,23].
T218 25495-25632 Sentence denotes The attachment to sialic-acid-containing cell surface structures is mediated by receptor-binding proteins that belong to the viral spike.
T219 25633-25719 Sentence denotes In the case of coronaviruses, this function is fulfilled by the S glycoprotein [9,24].
T220 25720-25861 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 25862-26053 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 26054-26209 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 26210-26380 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 26381-26612 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 26613-26753 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 26754-26881 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 26882-27007 Sentence denotes Both features have been shown to be key determinants of the recognition of sialic acids and gangliosides by proteins [20,26].
T228 27008-27259 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 27260-27415 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 27416-27576 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 27577-27665 Sentence denotes Next, it was shown that CLQ and CLQ-OH also bind to sialic-acid-containing gangliosides.
T232 27666-27758 Sentence denotes Based on these data, the drugs may also recognize the sialic acid residues of glycoproteins.
T233 27759-27804 Sentence denotes Further studies will help clarify this point.
T234 27805-27931 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 27932-28050 Sentence denotes This ganglioside-binding domain consists of a large flat interface enriched in aromatic and basic amino acid residues.
T236 28051-28195 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 28196-28349 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 28350-28496 Sentence denotes Thus, this motif displays the typical features that determine optimal binding to gangliosides (i.e. CH-π stacking and electrostatic interactions).
T239 28497-28663 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 28664-28765 Sentence denotes Two CLQ (or two CLQ-OH) molecules can bind simultaneously to the polar head group of ganglioside GM1.
T241 28766-28935 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 28936-29110 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 29111-29278 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 29279-29388 Sentence denotes It is also conserved in the bat RaTG13 isolate, which reinforces the hypothesis of bat-to-human transmission.
T245 29389-29622 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 29624-29637 Sentence denotes 5 Conclusion
T247 29638-29730 Sentence denotes Given the global health emergency, drug repurposing is obviously the option of choice [2,3].
T248 29731-29939 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 29940-30181 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 30182-30305 Sentence denotes The study data support the use of CLQ, and preferentially CLQ-OH, as initial therapy for patients infected with SARS-CoV-2.