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Id Subject Object Predicate Lexical cue
T1 0-86 Sentence denotes Dual inhibition of SARS-CoV-2 spike and main protease through a repurposed drug, rutin
T2 87-89 Sentence denotes A.
T3 90-103 Sentence denotes Kumari et al.
T4 104-150 Sentence denotes Journal of Biomolecular Structure and Dynamics
T5 152-160 Sentence denotes Abstract
T6 161-169 Sentence denotes Abstract
T7 170-332 Sentence denotes The global health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to alarming numbers of fatalities across the world.
T8 333-465 Sentence denotes So far the researchers worldwide have not been able to discover a breakthrough in the form of a potent drug or an effective vaccine.
T9 466-539 Sentence denotes Therefore, it is imperative to discover drugs to curb the ongoing menace.
T10 540-665 Sentence denotes In silico approaches using FDA approved drugs can expedite the drug discovery process by providing leads that can be pursued.
T11 666-879 Sentence denotes In this report, two drug targets, namely the spike protein and main protease, belonging to structural and non-structural class of proteins respectively, were utilized to carry out drug repurposing based screening.
T12 880-1133 Sentence denotes The exposed nature of the spike protein on the viral surface along with its instrumental role in host infection and the involvement of main protease in processing of polyproteins along with no human homologue make these proteins attractive drug targets.
T13 1134-1228 Sentence denotes Interestingly, the screening identified a common high efficiency binding molecule named rutin.
T14 1229-1357 Sentence denotes Further, molecular dynamics simulations in explicit solvent affirmed the stable and sturdy binding of rutin with these proteins.
T15 1358-1659 Sentence denotes The decreased Rg value (4 nm for spike-rutin and 2.23 nm for main protease-rutin) and stagnant SASA analysis (485 nm/S2/N in spike-rutin and 152 nm/S2/N in main protease-rutin) for protein surface and its orientation in the exposed and buried regions suggests a strong binding interaction of the drug.
T16 1660-1804 Sentence denotes Further, cluster analysis and secondary structure analysis of complex trajectories validated the conformational changes due to binding of rutin.
T17 1806-1824 Sentence denotes Graphical Abstract
T18 1825-1853 Sentence denotes Communicated by Ramaswamy H.
T19 1854-1859 Sentence denotes Sarma
T20 1861-1863 Sentence denotes 1.
T21 1865-1877 Sentence denotes Introduction
T22 1878-1970 Sentence denotes Pandemics involving pathogenic human coronaviruses have wreaked havoc on the human populace.
T23 1971-2238 Sentence denotes During the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic in 2002–2003, a high fatality rate of 10% was observed in the approximately 8,000 individuals infected (Marra et al., 2003; Peiris et al., 2003; Rota et al., 2003; SARS Working Group, 2003).
T24 2239-2431 Sentence denotes In 2012, the middle east respiratory syndrome coronavirus (MERS-CoV) had a much higher fatality rate of 36% and infected more than 1,700 individuals (de Groot et al., 2013; Zaki et al., 2012).
T25 2432-2642 Sentence denotes However, the recent SARS-CoV outbreak of 2019, also referred to as SARS-CoV-2 or COVID-19 (Chen et al., 2020), began in Wuhan in China and spread rapidly due to close human-to-human proximity (Li et al., 2020).
T26 2643-2745 Sentence denotes The SARS-CoV-2 has caused significant devastation globally due to the sheer number of people infected.
T27 2746-3051 Sentence denotes According to statistics published by the World Health Organization (WHO), there were 4,334,451 known cases of Covid-19 worldwide, out of which 19,737,794 cases alone are reported from America, while Europe attributes 9,664,042 cases, causing death casualties of over 1,157,509 globally as of Oct 27, 2020.
T28 3052-3481 Sentence denotes Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 (exhibiting 80% and 50% homology, respectively) (Kim et al., 2020; Zhu et al., 2020) belongs to the genera beta coronavirus (family Coronaviridae in the order Nidovirales) (Enjuanes et al., 2006; Perlman & Netland, 2009), which is known to infect mammals (Li, 2016) and has manifested an illustrious capability of cross-species transmission, including humans (Menachery et al., 2017).
T29 3482-3668 Sentence denotes The SARS-CoV-2 is an enveloped virus characterized by a positive-sense, single-stranded RNA genome of almost 30 kb, surrounded by a helical capsid comprised of nucleo capsid protein (N).
T30 3669-3936 Sentence denotes The viral envelope is associated with three primary structural proteins viz. membrane proteins (M) and envelope proteins (E), which perform virus assembly; and spike proteins (S), which facilitate the viral attachment and thus, the virus entrance into the host cells.
T31 3937-4094 Sentence denotes The large protrusions formed by spikes on the surface of the virus give it the appearance of having crowns, which, in Latin, translates to corona (Li, 2016).
T32 4095-4220 Sentence denotes These shorter, sgRNA-encoded structural proteins and several accessory proteins are known to be conserved (Kim et al., 2020).
T33 4221-4319 Sentence denotes The large ectodomain of the spike consists of receptor binding S1 and membrane fusion S2 subunits.
T34 4320-4386 Sentence denotes For many CoVs, the S1 and S2 domains remain non-covalently linked.
T35 4387-4468 Sentence denotes In β coronaviruses, the cleavage between the S1 and S2 regions is not obligatory.
T36 4469-4680 Sentence denotes However, the host proteases have been observed to cause cleavage within the fusion domain (S2), which leads to irreversible conformational changes, activating the protein for membrane fusion (Zhou et al., 2020).
T37 4681-4818 Sentence denotes The S2 subunit contains two regions with 4, 3 hydrophobic heptad repeats (HR), HR1 and HR2, which are conserved in sequence and position.
T38 4819-4928 Sentence denotes The HR2 is located adjacent to the trans-membrane domain, while the HR1 occurs ∼170 residues upstream of HR2.
T39 4929-5015 Sentence denotes The HR region has been observed to be a common motif in several viral fusion proteins.
T40 5016-5307 Sentence denotes Structurally, the HR1 domain exists as a homo-trimeric coil, packing the HR2 domain (in an anti-parallel manner) in its hydrophobic grooves, thereby bringing the N-terminal fusion peptide closer to the trans-membrane anchor, further facilitating fusion due to proximity (Bosch et al., 2003).
T41 5308-5596 Sentence denotes Various host receptors viz. angiotensin-converting enzyme 2 (ACE2), amino peptidase N (APN), dipeptidyl peptidase 4 (DPP4), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and sugar recognize S1 domain of the spike protein to render virus entry into cells (Li, 2016).
T42 5597-6070 Sentence denotes Several reports have been published recently discovering inhibitors against the target via a computational methodologies, which involve the usage of FDA approved compounds and natural compounds (Wei et al., 2020), spice molecules (Rout et al., 2020), small-molecule compounds of ZINC Drug Database (Kadioglu, 2020; Wu et al., 2020) along with traditional Chinese medicine and natural products and derivatives (Wu et al., 2020) and medicinal compounds (Salman et al., 2020).
T43 6071-6392 Sentence denotes Apart from structural proteins which play a crucial role in the lifecycle of the virus, remarkable number of functional proteins categorized as non-structural proteins (Nsp’s), involved in viral replication, transcription, translation and protein modifications, and host infection are equally important (Wu et al., 2020).
T44 6393-6626 Sentence denotes Among many such proteins, a well characterized distinguished drug target for SARS-Cov-2 is the 33.8 kDa, 3 C-like main protease, also called as the main protease or Nsp5 (3CLpro or Mpro or Nsp5) (Wu et al., 2020; Zhang et al., 2020).
T45 6627-6784 Sentence denotes Together with the papain like proteases (Plpro), the enzyme is responsible for the processing of polyproteins produced by the viral RNA (Zhang et al., 2020).
T46 6785-6867 Sentence denotes The Mpro along with Plpro cleaves the 790 kDa polyprotein1ab to generate 15 Nsp’s.
T47 6868-6965 Sentence denotes The Mpro processes the polyprotein1ab at 11 sites to produce mature Nsp4-Nsp16 (Wu et al., 2020).
T48 6966-7055 Sentence denotes Recently, the crystal structure of Mpro has also been reported at a resolution of 1.75 Å.
T49 7056-7439 Sentence denotes The Mpro structure consists of three domains, domains I and II comprising of six-stranded anti-parallel β barrels spanning from residues 10 to 99 and 100 to 182, respectively forming the substrate-binding site between them and the domain III which is responsible for modulating the Mpro dimerization is a cluster of five helices ranging from residues 198 to 303 (Zhang et al., 2020).
T50 7440-7678 Sentence denotes Due to the availability of the crystal structure and the absence of any human homologue, the Mpro is a lucrative drug target to work upon for the discovery of novel antiviral agents (Jin et al., 2020; Wu et al., 2020; Zhang et al., 2020).
T51 7679-8332 Sentence denotes Recently, numerous studies have emerged against this target, discovering novel inhibitors by utilizing computational approaches which include the use of natural molecules (Aanouz et al., 2020; Das et al., 2020), commercially available drugs and zinc library (Das et al., 2020; Elmezayen, 2020; Ton et al., 2020), antiviral compounds (Khan, 2020; Kumar et al., 2020; Muralidharan, 2020), peptide molecules (Pant, 2020), generative chemistry approaches for drug design (Alex, 2020), combinatorial strategy of using anti-virals, natural products, anti-fungals, anti-protozoals and anti-nematodes (Das et al., 2020), and spice molecules (Rout et al., 2020).
T52 8333-8642 Sentence denotes Many pharmacological agents such as antiviral agent’s viz. remdesvir, ribavirin, favipiravir, chloroquine, hydroxychloroquine, oseltamivir and umifenovir; immunomodulatory agents including toclizumab, inteferons; adjunctive agents like azithromycin, corticosteroids have been repurposed for treating COVID-19.
T53 8643-8762 Sentence denotes However, so far no therapy has been (Lam et al., 2020) recognized as an effective treatment against the deadly disease.
T54 8763-8925 Sentence denotes It has, therefore, become the need of the hour to adopt computational approaches for drug discovery and repurpose other existing marketed drugs for its treatment.
T55 8926-9039 Sentence denotes Drug repurposing via high-throughput screening of various databases is a cost-effective and time-saving approach.
T56 9040-9209 Sentence denotes The present report pertains to the above-described drug discovery effort using a repurposing screening strategy to target the SARS-CoV-2 spike protein and main protease.
T57 9210-9483 Sentence denotes Together, these proteins play a key role in binding to host cell receptors and causing fusion to render viral entry, determining the host range and tissue tropism, eliciting an immune response by the host, viral replication, transcription and protein processing (Li, 2016).
T58 9485-9487 Sentence denotes 2.
T59 9489-9509 Sentence denotes Material and methods
T60 9511-9515 Sentence denotes 2.1.
T61 9517-9561 Sentence denotes Preparation of protein and ligand structures
T62 9562-9740 Sentence denotes The spike protein and main protease regulatory enzyme of SARS-CoV-2 have been revealed to play a vital role in COVID-19 infection in the host (Ke et al., 2020; Xue et al., 2008).
T63 9741-9880 Sentence denotes In order to delineate the drug treatment, the deduced three dimensional structures of target SAR-CoV-2 spike protein in open state (PDB ID:
T64 9881-9913 Sentence denotes 6VYB) and main protease (PDB ID:
T65 9914-9985 Sentence denotes 6LU7) were retrieved from RCSB Protein Data Bank (Burley et al., 2019).
T66 9986-10110 Sentence denotes The crystal structures of target proteins were imported to Schrödinger Maestro (Moore, 2015) for molecular docking analysis.
T67 10111-10394 Sentence denotes Pre-processing and preparation of target proteins were performed using Schrödinger Protein Preparation Wizard (Schrödinger, 2011) by adding the missing hydrogen atoms, correcting the bond orders, capping the protein termini, creating disulfide bonds and removing the water molecules.
T68 10395-10531 Sentence denotes Also, the H-bonds were optimized and further the target proteins were equilibrated using OPLS 2005 force field (Robertson et al., 2015).
T69 10532-10697 Sentence denotes The library of ligands was downloaded from DrugBank (Wishart et al., 2018) and the coordinate files were prepared and optimized using LigPrep module (Release, 2017).
T70 10698-10847 Sentence denotes The energy minimization of ligand structures was carried using OPLS 2005 force field and ionization states were generated at pH 7.0 ± 2.0 using Epik.
T71 10848-10990 Sentence denotes This generated an output file with various stereoisomers and tautomeric conformers of each ligand producing chemical and structural diversity.
T72 10992-10996 Sentence denotes 2.2.
T73 10998-11061 Sentence denotes Determination of binding pocket and generation of receptor grid
T74 11062-11331 Sentence denotes The active sites enclosed in the binding pocket of spike protein and main protease were predicted using Computed Atlas of Surface Topography of proteins (CASTp) webserver (Tian et al., 2018) which calculates geometric and topological measurements of protein structures.
T75 11332-11467 Sentence denotes It measures the volume and surface area of the binding pocket and also predicts the atoms involved in formation of this binding cavity.
T76 11468-11676 Sentence denotes Although the active sites of spike protein and the main protease are known but still research is undergoing in which it is predicted that new amino acids might be involved in the inhibition of virus activity.
T77 11677-11774 Sentence denotes In order to undertake all the possibilities, binding pocket analysis was performed through CastP.
T78 11775-11929 Sentence denotes Further for the ease of molecular docking, a receptor grid was generated around the active sites of target proteins using Receptor Grid generation module.
T79 11930-12029 Sentence denotes The receptor grid file was created with a grid box around the centroid of selected active residues.
T80 12031-12035 Sentence denotes 2.3.
T81 12037-12086 Sentence denotes Protein ligand interactions and molecular docking
T82 12087-12246 Sentence denotes Protein ligand docking was conducted using GLIDE module of Schrödinger (Release, 2018) which performs all the docking calculations using OPLS 2005 force field.
T83 12247-12386 Sentence denotes A total of 2652 FDA approved drugs were screened against spike protein and main protease of SAR-CoV-2 through molecular docking approaches.
T84 12387-12748 Sentence denotes The virtual screening of compounds against the target protein was conducted using two docking methodologies, initially through a rapid screening of large set of compounds using high throughput virtual screening (HTVS) method followed by further screening a subset of top scoring HTVS compounds with a more precise and accurate extra precise (XP) docking method.
T85 12749-12872 Sentence denotes Protein ligand complexes were ranked using GlideScore function to predict the binding efficacy of ligands with the protein.
T86 12873-13052 Sentence denotes The docked complex with the lowest docking score was selected for further molecular dynamic simulation analysis to elucidate the inhibition mechanism against SARS-CoV-2 infection.
T87 13054-13058 Sentence denotes 2.4.
T88 13060-13134 Sentence denotes Molecular dynamics trajectory analysis of protein and their docked complex
T89 13135-13296 Sentence denotes The SARS-CoV-2 spike protein and main protease were used separately for molecular dynamics (MD) simulations alone and in complex with the repurposed drug, rutin.
T90 13297-13510 Sentence denotes All the four MD simulations were executed through GROMACS v5.0 under the force field GROMOS96 54a7 having water model SPC216 along with the time step of 1 fs for 100 ns (Abraham et al., 2015; Darden et al., 1993).
T91 13511-13683 Sentence denotes Varied sizes of the simulation box were created for each MD simulation event, which were further loaded with about respective amount of water molecules using the SPC model.
T92 13684-13974 Sentence denotes The total charge on spike protein, main protease and the two in complex with rutin were neutralized by adding –6, –4, –6, and –4 charges, respectively, and were incorporated into the simulation system by compensating the water molecules in the arbitrary locations inside the simulation box.
T93 13975-14077 Sentence denotes The NPT ensembles, along with periodic boundary conditions, were utilized to carry out MD simulations.
T94 14078-14154 Sentence denotes A cut-off of about 12 Å was used in order to manage the Vander Waals forces.
T95 14155-14302 Sentence denotes The Particle Mesh Ewald model manifesting a cut-off of 14 Å was further utilized to calculate the electrostatic interactions (Darden et al., 1993).
T96 14303-14432 Sentence denotes The spike protein, main protease and the two in complex with rutin were solvated through a slab of about 10 Å in every direction.
T97 14433-14487 Sentence denotes The neighbor list was updated to a frequency of 10 ps.
T98 14488-14568 Sentence denotes The MD simulations were achieved for each system employing the four major steps.
T99 14569-14758 Sentence denotes The first step deals with the energy minimization of the entire system utilizing the integrator of steepest descent in continuation with second integrator of conjugate gradients algorithms.
T100 14759-14989 Sentence denotes The second step involves the minimization and molecular dynamics of NVT and NPT ensembles for 500 ps and 1000 ps, respectively allowing the solvents and ions to evolve by keeping the same starting configuration for the structures.
T101 14990-15191 Sentence denotes In the third step the systems were heated using a lower temperature coupling (τ = 0.1 ps) along with pressure coupling (τ = 0.5 ps) to attain equilibrium at 300 K and 1 atm of temperature and pressure.
T102 15192-15320 Sentence denotes In the equilibration phase, the thermostat and barostat were evaluated through the Berendsen algorithm (Berendsen et al., 1984).
T103 15321-15432 Sentence denotes The hydrogen-containing bond lengths were constrained with the help of the LINCS algorithm (Hess et al., 1997).
T104 15433-15664 Sentence denotes Finally, the last step also called as the production step was carried out, where the MD simulation for 100 ns at 300 K temperature with 2 fs of time step were performed for both systems, and the last final structures were achieved.
T105 15665-15763 Sentence denotes The Maxwell Boltzmann distribution was utilized in order to reassign the velocities at every step.
T106 15764-15926 Sentence denotes The Nose Hoover thermostat and Parrinello Rahman barostat were the respective thermostat and barostat for the final MD or production run (Berendsen et al., 1984).
T107 15927-16013 Sentence denotes Various analyses were performed with the help of inbuilt analysis commands of GROMACS.
T108 16014-16257 Sentence denotes The root mean square deviation (RMSD) is a magnitude of the dimensional disparity among the two stagnant structures, and RMSD calculation is achieved depending upon the native structure and each consecutive trajectory frames in the simulation.
T109 16258-16466 Sentence denotes In addition, root mean square fluctuation (RMSF) profile measures the affability of every protein residue depending on the fluctuation about an average location within all MD simulations (Knapp et al., 2011).
T110 16467-16585 Sentence denotes Therefore, RMSD and RMSF of each simulation system were determined to examine the stability and residual fluctuations.
T111 16586-16716 Sentence denotes Further, the radius of gyration (Rg) analysis was performed to evaluate the compactness of both the simulation systems separately.
T112 16717-17006 Sentence denotes Also, the hydrogen bond analysis was performed to check the neighboring interactions with both simulation systems separately, including the hydrophobic interactions with the help of the LigPlot tool for both spike protein and main protease complexes with rutin before and after simulation.
T113 17007-17152 Sentence denotes Additionally, the solvent accessibility surface area (SASA) was also computed to examine the solvent attributable areas of all simulation system.
T114 17153-17399 Sentence denotes The cluster analysis having a cut-off value of 0.25 and 0.2 nm for spike protein and main protease respectively, depending upon the RMSD profile were utilized to demonstrate the conformations found utmost intermittently throughout the trajectory.
T115 17400-17537 Sentence denotes Here, all the structures having RMSD values of below 0.25 nm for all components within a cluster are incorporated to the initial cluster.
T116 17538-17671 Sentence denotes It is rare that a molecule having a higher value for RMSD than 0.25 nm from other cluster supposedly would be treated as a structure.
T117 17672-17769 Sentence denotes The secondary structure analysis was also performed using the DSSP program (Martin et al., 2005).
T118 17770-17959 Sentence denotes The visualization of protein nature during the entire simulation was accomplished by using Visual Molecular Dynamics (VMD) (Humphrey et al., 1996) and UCSF Chimera (Pettersen et al., 2004).
T119 17961-17963 Sentence denotes 3.
T120 17965-17987 Sentence denotes Results and discussion
T121 17988-18144 Sentence denotes SARS-CoV-2 is an enveloped non-segmented positively stranded RNA infectious virus that mainly affects enteric and respiratory systems (Graham et al., 2013).
T122 18145-18284 Sentence denotes Its infection and amelioration in the host are majorly governed by several structural proteins (Joshi et al., 2020; Prajapat et al., 2020).
T123 18285-18561 Sentence denotes Amidst numerous structural proteins, spike protein and main protease of SARS-CoV-2 are stated to show essential impacts on viral replication through proteolytic machinery and involvement in transcription, translation, and amplification of viral proteins (Paules et al., 2020).
T124 18562-18724 Sentence denotes Spike protein plays a critical role in binding to host cell receptor and is thought to represent a key determinant of the host range restriction (de Wilde, 2017).
T125 18725-19001 Sentence denotes Main protease on the other hand exclusively cleaves polypeptide sequences after the glutamine residue and to the best of our knowledge is known as an ideal drug target due to the absence of human host cell protease showing this substrate specificity (Ullrich & Nitsche, 2020).
T126 19002-19153 Sentence denotes The 3 D structures of spike protein and main protease were preprocessed, optimized and minimized to obtain the refined structures for drug repurposing.
T127 19155-19159 Sentence denotes 3.1.
T128 19161-19184 Sentence denotes Binding pocket analysis
T129 19185-19268 Sentence denotes The binding pocket volume and surface area were determined through CASTp webserver.
T130 19269-19353 Sentence denotes Binding pocket was forecasted at the surface as well as in the interior of proteins.
T131 19354-19501 Sentence denotes Binding pocket volume of spike protein and main protease was 22,908 and 319 (SA), respectively which signifies an optimum space for ligand binding.
T132 19502-20006 Sentence denotes The binding residues as predicted in the binding cavity for spike protein were Y38-S45, V47-L54, P85-D88,T108,K195-Y200,K202-Y204,P225-D228,I233-R237,Q271-T274,C291-A292,E298,C301-K304,Y313-N317,R319-V327,D364-S366,Y369-N370,Y380-T393,P412,D427-T430,F515-H519,A522,P527-T531,N540-T549,Q564-D568,A570-T573,V576-R577,P579,I587,P589-F592,G594,M740-Y741,G744,F855-N856,Q954,Q957-A958,N960-D979,R983,V991,D994-R995,T998-R1000,Q1002-S1003,T1006-Y1007,Q1010, and R1014 around which the receptor grid was formed.
T133 20007-20175 Sentence denotes The receptor grid was also created around the catalytic region (T24-L27, H41, C44-S46, M49, P52, Y54, F140-C145, H163-P168, H172, D187-T190, and Q192) of main protease.
T134 20177-20181 Sentence denotes 3.2.
T135 20183-20251 Sentence denotes Probable drugs against spike protein and main protease of SARS-CoV-2
T136 20252-20409 Sentence denotes The molecular docking studies were performed to determine the ligand interactions and their binding affinity with SARS-CoV-2 spike protein and main protease.
T137 20410-20503 Sentence denotes The prepared ligands structures were docked against the binding sites of the target proteins.
T138 20504-20662 Sentence denotes The HTVS docking approach filtered out a large number of compounds on the basis of their binding interactions within the binding sites of the target proteins.
T139 20663-20761 Sentence denotes The compounds displaying a HTVS glide score of more than −6.0 kJ/mol were employed for XP docking.
T140 20762-20938 Sentence denotes The docking simulations evaluated a high Glide score for the compounds with best binding affinity and their interactions within the binding pocket of the binding site residues.
T141 20939-21078 Sentence denotes The docking analysis revealed a common compound, rutin (DrugBank ID: DB01698) which binds strongly to both the major targets of SARS-CoV-2.
T142 21079-21260 Sentence denotes Our study here presents rutin exhibiting high binding efficiency against spike protein and main protease with an XP Glide score of −8.367 kcal/mol and −11.553 kcal/mol respectively.
T143 21261-21442 Sentence denotes Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) belongs to the flavonol class of compound, generously present in plants including buckwheat, apple, passion flower and tea.
T144 21443-21684 Sentence denotes It is well known to exhibit many bioactivities, such as anti-viral, anti-bacterial, ant-larvicidal, cytoprotective, anticarcinogenic, antioxidant, vasoprotective, cardioprotective and neuroprotective activities (Saluja & Ganeshpurkar, 2016).
T145 21685-21909 Sentence denotes The list of best ten candidates derived from the docking calculations with their corresponding docking score against spike protein and main protease are shown in supporting information (supporting material Tables S2 and S3).
T146 21910-22046 Sentence denotes To determine the interaction pattern between rutin and target proteins, their docked complexes were visualized using Ligplot (Figure 1).
T147 22047-22257 Sentence denotes The interaction pattern between rutin and spike protein revealed the formation of five hydrogen bonds and four hydrophobic interactions at the binding site, thereby contributing to the stability of the complex.
T148 22258-22383 Sentence denotes The residues involved in the formation of hydrogen bonds between rutin and spike protein were F970, N969, H49, Q52, and T274.
T149 22384-22472 Sentence denotes While the hydrophobic contact between the two involved residues T51, S50, S967 and S968.
T150 22473-22732 Sentence denotes The interactions between rutin and main protease was stabilized by residues E166, T190, Y54, D187, T26, N142 forming hydrogen bond and various hydrophobic contacts with residues P168, L167, H41, G143, T25, L27, M49, M165, H164, R188 and Q189 (Tables 1 and 2).
T151 22733-22909 Sentence denotes A similar study exploiting a blind molecular docking approach utilizing the Swiss Dock server also highlights main protease inhibition by rutin where by common amino acids viz.
T152 22910-22979 Sentence denotes E166, T190, N142, H41 are found to be interacting with the inhibitor.
T153 22980-23098 Sentence denotes In our study however, the inhibitor displayed slightly better binding efficiency than their report (Das et al., 2020).
T154 23099-23262 Sentence denotes From the contact analyses of both proteins, it can be attributed that rutin has high affinity and wide molecular contacts for both spike protein and main protease.
T155 23263-23462 Sentence denotes Also, it was further scrutinized by molecular dynamics simulations to attain insights towards the inhibitory aspects and efficacy to combat spike protein and main protease drug targets of SARS-CoV-2.
T156 23463-23472 Sentence denotes Figure 1.
T157 23474-23567 Sentence denotes Protein-ligand interactions diagram of spike protein and main protease in complex with rutin.
T158 23568-23769 Sentence denotes 2D Ligplot representation showing hydrogen bond and hydrophobic interactions, and 3D structure representation showing rutin in the binding pocket of spike protein (A and C) and main protease (B and D).
T159 23770-23778 Sentence denotes Table 1.
T160 23780-23890 Sentence denotes Intermolecular hydrogen bonds and hydrophobic residues showing close contact between spike protein with Rutin.
T161 23891-23912 Sentence denotes Spike protein + rutin
T162 23913-23992 Sentence denotes Interacting residue H-bond distance (Å) H-bond (D-H--A) Hydrophobic residues
T163 23993-24068 Sentence denotes His 49.A NE2–Rutin.het O15 3.28 HNE2-H--O15 Ser50, Thr51, Ser967, Ser968
T164 24069-24117 Sentence denotes GLN 52.A OE1–Rutin.het O14 2.89 HOE1-H--O14  
T165 24118-24167 Sentence denotes THR 274.A OG1–Rutin.het O14 3.30 HOG1-H--O14  
T166 24168-24217 Sentence denotes ASN 969.A ND2–Rutin.het O10 3.17 HND2-H--O10  
T167 24218-24261 Sentence denotes PHE 970.A N–Rutin.het O9 3.10 HN-H--O9  
T168 24262-24270 Sentence denotes Table 2.
T169 24272-24382 Sentence denotes Intermolecular hydrogen bonds and hydrophobic residues showing close contact between main protease with Rutin.
T170 24383-24404 Sentence denotes Main protease + rutin
T171 24405-24484 Sentence denotes Interacting residue H-bond distance (Å) H-bond (D-H--A) Hydrophobic residues
T172 24485-24571 Sentence denotes THR 26.A O–Rutin.het O15 3.05 HO-H--O15 Thr25, Leu27, His41, Met49, Gly143, His164,
T173 24572-24646 Sentence denotes THR 26.A O–Rutin.het O16 3.02 HO-H--O16 Met165, Leu167, Pro168, Arg188,
T174 24647-24698 Sentence denotes TYR 54.A OH–Rutin.het O14 2.91 HOH-H--O14 Gln189
T175 24699-24746 Sentence denotes ASN 142.A OD1–Rutin.het O5 3.01 HOD1-H--O5  
T176 24747-24794 Sentence denotes ASN 142.A OD1–Rutin.het O7 2.91 HOD1-H--O7  
T177 24795-24838 Sentence denotes GLU 166.A O–Rutin.het O8 3.02 HO-H--O8  
T178 24839-24884 Sentence denotes ASP 187.A O–Rutin.het O14 3.31 HO-H--O14  
T179 24885-24930 Sentence denotes THR 190.A O–Rutin.het O10 2.87 HO-H--O10  
T180 24932-24936 Sentence denotes 3.3.
T181 24938-25035 Sentence denotes Molecular dynamics simulation of spike protein and main protease in absence and presence of rutin
T182 25036-25158 Sentence denotes The binding affinity of rutin against both spike and main protease were investigated through MD simulations using GROMACS.
T183 25159-25295 Sentence denotes Separate MD simulations for spike protein and main protease with and without rutin for 100 ns were assessed through trajectory analysis.
T184 25296-25419 Sentence denotes For the course of 100 ns MD simulation, the stable trajectory was observed and the representative structures were obtained.
T185 25420-25655 Sentence denotes The deviation of the backbone atoms for simulated structures relative to the starting structures used as a reference was evaluated through RMSD for the backbone atoms (Figures 2 and 3) and C-alpha atoms (supporting material Figure S1).
T186 25656-25821 Sentence denotes A steep magnitude RMSD variation during the entire simulation can be an implication of a malleable and free instinctive protein or the alteration of the force field.
T187 25822-26065 Sentence denotes Depending upon the outcomes of the RMSD evaluation, Figure 2(A) represents that the RMSD fluctuation stabilize at about 50 ns MD simulations for both spike protein and spike protein in presence of rutin, and the simulation time was acceptable.
T188 26066-26219 Sentence denotes In the time from 50–100 ns, the RMSD for spike protein and spike-rutin complex have approximate values about 1.50–1.65 nm and 1.20–1.26 nm, respectively.
T189 26220-26391 Sentence denotes Similarly, the main protease in absence and presence of rutin for time from 20–70 ns and 20–100 ns showed RMSD values of about 0.25–0.30 nm and 0.20–0.25 nm (Figure 3(A)).
T190 26392-26555 Sentence denotes Interestingly, RMSD for both the complex systems were more stable than the native state of target receptor spike protein and main protease (Figures 2(A) and 3(A)).
T191 26556-26691 Sentence denotes The RMSD of C-alpha atoms were also attained and was found to be similar to the RMSD of backbone atoms (supporting material Figure S1).
T192 26692-26831 Sentence denotes The average RMSD for backbone and C-alpha atoms of both the protein and their complex with rutin are shown in supporting material Table S1.
T193 26832-26841 Sentence denotes Figure 2.
T194 26843-27136 Sentence denotes Representation of the MD analysis plots for spike protein (purple color) and spike-rutin complex (red color) (A) root mean square deviation (RMSD) for backbone atoms, (B) root mean square fluctuation (RMSF), (C) radius of gyration (Rg), and (D) solvent accessible surface area (SASA) analysis.
T195 27137-27146 Sentence denotes Figure 3.
T196 27148-27448 Sentence denotes Representation of the MD analysis plots for main protease (blue color) and main protease-rutin complex (green color) (A) root mean square deviation (RMSD) for backbone atoms, (B) root mean square fluctuation (RMSF), (C) radius of gyration (Rg) and (D) solvent accessible surface area (SASA) analysis.
T197 27449-27647 Sentence denotes The integral extent of the residual dynamic criterion is achieved by assessing the variations arising from shifts of each of the protein residues that majorly feature the most flexible chain frames.
T198 27648-27773 Sentence denotes Hence, we validated the residual fluctuations by calculating the mean fluctuation for stable trajectories of each simulation.
T199 27774-27929 Sentence denotes The RMSF evaluation of all the protein residues were achieved in order to check the residues that may have tend towards an enhancement in the RMSD results.
T200 27930-28172 Sentence denotes Compelling fluctuations existed in terminal residues and in various loops along β sheets and coils of the spike protein (residues 852-1000) to about 0.47 nm and spike-rutin (residues 62–90, 400–520 and 675–730) to about 0.65 nm (Figure 2(B)).
T201 28173-28492 Sentence denotes Similarly, in case of main protease and itself in complex with rutin showed fluctuations in the terminal residues and in some regions of β sheet with some coils and loops in main protease (residues 136-148) to about 0.37 nm and main protease-rutin (residues 39-52, 150-156) to about 0.37 nm, respectively (Figure 3(B)).
T202 28493-28728 Sentence denotes Also, we noticed that residues 270 to 350 in spike protein and 160 to 190 in main protease showed comparatively reduced discrepancy in RMSF values, which were the corresponding binding segment of both the complexes after MD simulation.
T203 28729-29011 Sentence denotes Interestingly, the binding region for spike-rutin and main protease-rutin before MD simulation were found to be the same after simulation, suggesting as a strong binding interaction of the rutin towards spike protein and main protease and indicating stability of both the complexes.
T204 29012-29151 Sentence denotes The radius of gyration analysis was evaluated to determine the change in compactness of protein systems used throughout the MD simulations.
T205 29152-29487 Sentence denotes The Rg plots for spike, main protease and its complex with rutin show slight fluctuations at the initial frame and attain compactness after 30 ns with Rg score of 3.85 nm and 4 nm (spike and spike-rutin) and after 20 ns with Rg score of 2.27 nm and 2.23 nm (main protease and main protease-rutin), respectively (Figures 2(C) and 3(C)).
T206 29488-29699 Sentence denotes When compared with spike protein, Rg value for spike-rutin is stabilized and remains constant, suggesting strong binding interaction of the inhibitor, and the same is observed in case of main protease and rutin.
T207 29700-29934 Sentence denotes Similar observations were determined through SASA analysis representing the solvent defined protein surface and its orientation through folding, making the alterations in the exposed and buried regions of the surface area of proteins.
T208 29935-30132 Sentence denotes The SASA values for all the simulation systems were about 460 nm/S2/N (spike), 485 nm/S2/N (spike-rutin), 154 nm/S2/N (main-protease), and 152 nm/S2/N (main protease-rutin) (Figures 2(D) and 3(D)).
T209 30133-30299 Sentence denotes Here, spike-rutin and main protease-rutin solvation profile shows a convincing SASA value suggesting a stable structure and strong binding interaction with the rutin.
T210 30300-30551 Sentence denotes Further, the cluster analysis with a RMSD based cut-off value of 0.25 nm demonstrated the development of 13,28,3 and 2 distinctive clusters for spike, spike-rutin, main protease, and main protease-rutin simulation systems respectively (Figure 4(A–D)).
T211 30552-30665 Sentence denotes The most dominant cluster attained after 100 ns of MD simulation for all four systems are shown in Figure 4(E–H).
T212 30666-30808 Sentence denotes Also, the secondary structure analyses of the stable trajectory for both the simulation systems were performed using the DSSP tool of GROMACS.
T213 30809-31055 Sentence denotes Both, spike and main protease were formed mainly of conserved β-sheet along with some connecting loops and α-helix with small coil regions as secondary structure elements infused with various small segments of bend, turn, and β-bridge (Figure 5).
T214 31056-31237 Sentence denotes Both cluster analysis and secondary structure analysis reveals the conformational changes before and after simulations for spike and main protease with and without rutin structures.
T215 31238-31428 Sentence denotes A noticeable observation through these analyses supports the rationale that spike protein and main protease binds with rutin firmly and attain similar interactions after MD simulations also.
T216 31429-31438 Sentence denotes Figure 4.
T217 31440-31647 Sentence denotes Representation of the MD analysis plots for the RMSD matrix cluster formation showing number of clusters in (A) spike protein, (B) spike-rutin complex, (C) main protease, and (D) main protease-rutin complex.
T218 31648-31814 Sentence denotes The most dominant cluster conformation after MD cluster analysis of (E) spike protein, (F) spike-rutin complex, (G) main protease and (H) main protease-rutin complex.
T219 31815-31824 Sentence denotes Figure 5.
T220 31826-31960 Sentence denotes The secondary structure analysis of (A) spike protein, (B) spike-rutin complex, (C) main protease and (D) main protease-rutin complex.
T221 31961-32184 Sentence denotes Further, hydrogen bond analysis revealed a higher number of hydrogen bonds with the surrounding water molecules in case of spike protein (2300 hydrogen bonds) than in complex with rutin (approximately, 2200 hydrogen bonds).
T222 32185-32338 Sentence denotes Similarly, in case of main protease, 700 hydrogen bonds were formed while 560 hydrogen bonds in case of main protease complex with rutin (Figure 6(A,B)).
T223 32339-32538 Sentence denotes In addition, the hydrogen bond landscape assessed against the inhibitor revealed the dynamic equilibration of the complex trajectories with a high number of hydrogen bonds, as shown in Figure 6(C,D).
T224 32539-32651 Sentence denotes The average number of hydrogen bonds within spike-rutin and main protease-rutin are 3.72 and 2.29, respectively.
T225 32652-32830 Sentence denotes The consistent numbers of hydrogen bonds were observed which contributed significantly to the proximal binding of the potent drug rutin with the spike and main protease receptor.
T226 32831-32976 Sentence denotes The superimposed structures of spike protein and main protease both in complex with rutin before and after simulations is shown in Figure 7(A,B).
T227 32977-33106 Sentence denotes Further, these results were strengthened by the vital contribution of the complex binding energies throughout the simulation run.
T228 33107-33285 Sentence denotes These calculations with consistent high binding energies and large hydrogen bonds involvement demonstrated the stable binding of rutin with both spike and main protease proteins.
T229 33286-33295 Sentence denotes Figure 6.
T230 33297-33535 Sentence denotes Representation of the MD analysis plots for the hydrogen bond with the surrounding solvent against (A) spike protein (purple color) and spike-rutin complex (red color), and (B) main protease (blue) and main protease-rutin complex (green).
T231 33536-33685 Sentence denotes The MD analysis plots representing hydrogen bonds with the potent inhibitor i.e. rutin against (C) main protease and (D) main protease-rutin complex.
T232 33686-33695 Sentence denotes Figure 7.
T233 33697-33874 Sentence denotes Superimposed structures of (A) spike protein before simulation (green) and after simulation (blue), and (B) main protease before simulation (yellow) and after simulation (pink).
T234 33876-33878 Sentence denotes 4.
T235 33880-33890 Sentence denotes Conclusion
T236 33891-34073 Sentence denotes SARS-CoV-2 which causes the deadly and highly infectious COVID-19 remains an unmet medical issue that requires urgent attention, owing to the number of deaths it is causing globally.
T237 34074-34239 Sentence denotes We report here a computational approach for a repurposing screening program against two crucial SARS-CoV-2 drug targets viz. the spike protein and the main protease.
T238 34240-34401 Sentence denotes We discovered that the drug rutin, showed a high binding potency against both the drug targets and may therefore be capable of eliciting reduction in viral load.
T239 34402-34576 Sentence denotes The current study delineates that the highly effective repurposed drug rutin has the potential to obtain strong inhibition against SARS-CoV-2 spike protein and main protease.
T240 34577-34719 Sentence denotes Rutin showed high binding efficiency against spike protein and main protease having an XP Glide score of –8.367 kcal/mol and –11.553 kcal/mol.
T241 34720-34872 Sentence denotes Further, the molecular interactions of rutin with both the drug targets were analysed thoroughly with the help of molecular dynamics simulation studies.
T242 34873-34994 Sentence denotes The simulations for spike protein and main protease provided us with the stable trajectory analysis for both the targets.
T243 34995-35334 Sentence denotes We analyzed various parameters which include RMSD, RMSF, radius of gyration, SASA, binding energies, hydrogen bond interactions, secondary structure contents, and cluster formation throughout the simulation, which proposed insights about the stable binding and compactness due to strong interaction of the rutin with both the drug targets.
T244 35335-35512 Sentence denotes Through this in silico approach, the repurposed drug, rutin depicted reliable inhibition of two vital proteins and therefore may lead to elicitation of anti-SARS-CoV-2 activity.
T245 35514-35536 Sentence denotes Supplementary Material
T246 35537-35571 Sentence denotes Supporting_Information_Revised.pdf
T247 35572-35608 Sentence denotes Click here for additional data file.
T248 35610-35626 Sentence denotes Acknowledgements
T249 35627-35749 Sentence denotes AK is grateful to Jawaharlal Nehru University and Teri School of Advanced Studies for the management and other facilities.
T250 35750-35839 Sentence denotes AK is contended to Indian Council of Medical Research (ICMR), India for the SRF position.
T251 35841-35861 Sentence denotes Disclosure statement
T252 35862-35907 Sentence denotes The authors declare no conflicting interests.