Investigation of interaction between close and open types of SARS-Cov-2 spike glycoprotein with synthesized and natural Compounds as inhibitor; Molecular Docking Study

Purpose Viral diseases are increasingly endangering universal public health because of a shortage of successful antiviral therapies. The novel pandemic 2019 n-Cov2 disease (COVID-19) is recently identi�ed as viral disorder triggered by a new type of coronavirus. This type of coronavirus binds to the host human receptors through the Spike glycoprotein(S) Receptor Binding Domain (RBD). Two types of spike protein have been identi�ed in open and closed states in which the open type causes severe infection. Thus, this receptor is a signi�cant target for antiviral drug design. Methods Totally 111*2 natural and synthetic compounds were chosen from the PubChem database as ligands. To recognize the ability of direct contact between ligands and the binding site of 2019 n-Cov 2 - ACE2 protein, we have docked all compounds to the protein using AutoDock Vina. The FaF3-Drugs, Pan Assay Intrusion Compounds (PAINS), absorption, distribution, metabolism, excretion (ADME) and Lipinski's rules were used to evaluate the drug-like properties of the identi�ed ligands. Antiviral compound prediction (AVC pred) also was used to assess antivirus properties. Results The results showed that seven ligands out of all had interactions with spike protein-angiotensin converting enzyme 2 binding site. We have found that six out of seven ligands show drug-like characteristics. We also found that the �uorophenyl and propane groups of ligands had the best interaction with the binding site of the protein. Conclusion Further, our results showed the ability of these ligands to prevent receptor binding of the spike protein SARS-CoV-2, so they would be considered as novel compounds of COVID-19 therapy drugs.


Introduction
Coronaviruses are the large family of viruses that belong to the family of coronaviridae.Based on genomic structures and phylogenetic relationships, the subfamily coronavirinae includes four sorts Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Woo et al. 2012).
Recently named extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and categorized into the genus Betacoronavirus (Hui et al. 2020), that induce animal and human respiratory and intestinal infections (Vijay, Perlman 2016).Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus(MERS-CoV), and Severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) strains share the identity of the genome sequence nearly 79% and 50%, respectively (Lu et al. 2020).However, signi cant contrasts as far as the study of disease transmission and physiopathology between these three infections have additionally been watched (Cruz et al. 2020;Huang et al. 2020;Wang et al. 2020).2019-nCoV, classi ed as SARS-CoV-2, has a higher transmission risk than other in its family.SARS-CoV-2 is quite close to the other two widely studied coronaviruses; MERS-CoV and SARS-CoV (Organization 2020;Tai et al. 2020).However, there are still no antiviral medication and vaccine approved for the treatment and prevention of SARS-CoV-2 infections.Four important structure of coronaviruses are spikes (S), envelopes (E), membranes (M), and nucleocapsid (N) (Zhou et al. 2018;Cui et al. 2019).Angiotensin-converting enzyme 2(ACE2) is a key enzyme that SARS-CoV and several SARS-related coronaviruses connect to it, in order to enter to target cells (Kirchdoerfer et al. 2018;Song et al. 2018).The most current ndings suggest that the SARS-CoV-2 attaches to the ACE2 receptor on the host cell surface utilizing the spike protein receptor-binding domain (Goswami, Bagchi 2020;Walls et al. 2020;Li et al. 2005).The glycoprotein transmembrane spike which forms homo-trimer protruding from on the viral surface, helps coronaviruses to entrance into host cells (Walls et al. 2016).
The spike glycoprotein on the coronavirus envelope synthesized nearly 1300 amino acids as a single precursor to the polypeptide chain and cleaved by host furin-like proteases into the amino (N)-terminal S1 subunit and the carboxyl (C)-terminal S2 subunit, the host cell connection, receptor binding and the stabilization of host cell membrane and viral membrane fusion during infection are four duties for which glycoprotein spikes are responsible (Du et al. 2009;Millet, Whittaker 2015).In Figures 1A, B, and 1C the homotrimer of SARS-CoV-2 S glycoproteins and a monomer protein respectively are represented.Two types of spike protein have been identi ed in open and closed states, that the closed SARS-CoV-2 S ectodomain trimer applying 3-fold symmetry with three ACE2-recognition motifs, whose location was meddled focuses on the crystal structure of SARS-CoV S in complex with ACE2(Figure1A) [16].Opened SARS-CoV-2 S is an asymmetric reconstruction of the trimmer with a single subunit B domain (Figure1B) [15].On the whole, this information indicates that S glycoprotein trimers present in profoundly pathogenic human coronaviruses seem to be in moderately opened states, although they remain mostly closed in common cold-related human coronaviruses (Guan et al. 2003;Li et al. 2004;Wan et al. 2020).Based on recent evidence the binding a nity of SARS-CoV for hACE2 with the rate of transmissibility, viral replication in distinct organisms, and seriousness of the disease is correlating [29,30], it is believed that the most pathogenic coronaviruses will indicate S glycoprotein trimers spontaneously make closed and open conformations, as is the case with SARS-CoV-2, SARS-CoV and MERS-CoV, respectively (Walls et al. 2020).S1 and S2 subunits are Two functional subunits responsible for the host cell receptor, viral, and cell membrane combination that forms the spike glycoprotein (Belouzard et al. 2009;Bosch et al. 2003;Walls et al. 2016;Kirchdoerfer et al. 2016).The S1 subunits have two duties: this subunit comprises hl;ost cell attachment domains by identifying molecules of cell surface sugar and attaching to speci c cellular receptors (Li 2015(Li , 2016)),Therefore, its receptor-binding domain (RBD) is critical in determining cell tropism, host range and zoonotic transmission of coronaviruses (Lu et al. 2015;Graham, Baric 2010).A hydrophobic fusion peptide and two heptad repeat regions compose the S2 subunit (Song et al. 2018).spike receptor-binding domain bound with the cell receptor ACE2 causes the triggers conformational changes in the S1 and S2 subunits, leading to the exposure of the fusion loop and its insertion into target cell membrane (Hofmann, Pöhlmann 2004;Lan et al. 2020).In this study, we have considered 6 different groups of ligands (Table 1): Anti-virus, Flavonoids, Fluorophenyls, Phenylpropanoids, some drugs that have been suggested for SARS-CoV-2, and compounds similar to uorophenyl groups were virtually screened by Using the PubChem database that 3 compounds nally chosen which were Propane groups.Antiviruses have been used because of their antiviral properties and checking their effectiveness against SARS-CoV-2.Flavonoids are present in nearly all fruits and vegetables, as a category of natural substances with variable phenolic structures (Panche et al. 2016).These natural products are well known for their useful health effects such as antimicrobial activity, antioxidant, anticancer, and antivirus (Cushnie, Lamb 2005;Pietta 2000;Ren et al. 2003;Zhou, Li 2007).The compounds of uorophenyl are composed of uoro + phenyl, which are different compounds.As it is shown in the previous study, 2fluorophenyl, 3-fluorophenyl, and 4-fluorophenyl have antibiotic and antifungal activity so this compounds are used to docking in our study (Saleh et al. 2010).Phenylpropanoids are an enormous class of plant secondary metabolites got from aromatic amino acids phenylalanine in many plants or tyrosine in partial monocots.(Deng, Lu 2017).By having various biological functions, phenylpropanoids are useful for human health, so the systematic research has centered on natural and biotechnologically induced phenylpropanoids for medical usage as antioxidants, anticancer, antiviral, anti-in ammatory, wound healing, and antibacterial substances during the last few decades (Korkina et al. 2011).In this study, set of all six ligand groups will be investigated for interaction with the S1 subunit of SARS-CoV-2 spike glycoprotein by molecular docking.AutoDock vina (http://autodock.scripps.edu) is a popular opensource application for performing molecular docking, the prediction of ligand-receptor interactions.In the drug discovery process, molecular docking is a computationally intensive and prominent method.In this paper, we remember the criticality of research and the development of a COVID-19 drug by attaching ligands to the S glycoprotein RDB, which could assume a compelling role in interfering with the spike refolding procedure and thus repressing viral entry into host cells.

Protein preparation:
As mentioned, the S1 subunit in the B domain is the main cause of the pathogenic difference of the SARS-CoV-2, so throughout this study, only the B chain was examined in both opened and closed types.The structure of the SARS-CoV-2 spike with closed and opened state (respectively with PDB ID: 6vxx and 6vyb) was downloaded from Protein Data Bank (Berman et al. 2000).First, we used modeller 9.2 software for modeling the missing residue that located in subunit S1 for both B chains selected (after modeling the chains to obtain the amino acid number in the 6vxx(closed type) -87 and in the 6vyb (open type) -102 positional difference is the compared to downloaded structures from PDB) (Webb, Sali 2016;Fiser, Do 2000).The B chains and ligands were then translated to pdbqt format to dock in autodock vina software (Trott, Olson 2010).Before docking, polar hydrogens, and gasteiger charges were applied to the con guration of the B chains and ligands.The autodock Vina docking tool was used to test ligand binding on the SARS-CoV-2 B chain.Additionally, blind docking of ligands was done to recognize the possible binding sites in the S1 subunit.For this, the entire protein was covered with the grid box of dimension 36.70 50 70.01Åfor opened type protein and 63.29 52.10 50.14 for closed type with grid spacing 1 Å.Finally the conformations with high negative binding energy in binding site that mentioned in the recent study are chosen (Walls et al. 2020;Lan et al. 2020;Yan et al. 2020).
Ligands preparation ligands' 3-dimensional structure was extracted from the database of ChemSpider and PubChem, and these structures were then translated to PDB format by using the molecular visualization package of Chimera (Meng et al. 2006;Pettersen et al. 2004).

Ligand receptor interaction analysis
The docking results were analyzed using Ligplot+ and Discovery Studio Visualizer tool for a clear view of the best-docked complexes' receptor-ligand interaction (Laskowski, Swindells 2011;BIOVIA 2017;Studio 2008).

Drug-like characteristic
It is important to examine the main parameters associated with absorption, distribution, metabolism, excretion(ADME) properties such as the ve rule of Lipinski, drug solubility, pharmacokinetic characteristic, molar refractivity, and drug likeliness in order to produce medicines with a good therapeutic index (Bueno 2020;Lipinski 2004).Development of drugs requires testing ADME progressively earlier in the discovery process, at a period when multiple compounds are regarded but access to physical samples is restricted, so computational prediction of the ADME identi ed ligands were predicted virtually (Daina et al. 2017).ADME pro ling of all the ligands were determined using online software instrument (http://www.swissadme.ch).The original Lipinski RO5 deals with orally active compounds and de nes four simple ranges of physicochemical parameters ranging molecular weight (MWT) ≤500, log P ≤5, Hbond donors≤10, H-bond acceptors ≤10) (Lipinski et al. 1997).Moreover, Pan Assay Intrusion Compounds(PAINS) identi es a variety of substructural features that may help recognize compounds that appear as frequent ligands (promiscuous compounds) in several high-throughput biochemical screens (Baell, Holloway 2010), FAF-Drugs3 ltering is used (Lagorce et al. 2015).Infections of SARS-CoV-2 are actively threatening worldwide general health owing to a shortage of successful antiviral therapies, so in our work antiviral compound prediction (AVC pred) for chosen ligands was used (http://crdd.osdd.net/servers/avcpred/batch.php).In the AVCpred method, Experimental percentage inhibitory from ChEMBL, as a large-scale bioactivity database for drug discovery are used to foresees antiviral compounds against HIV, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Human herpesvirus (HHV), and 26 other viruses (Qureshi et al. 2017).

Molecular docking
Identify the ligands that bind to the S B domain-ACE2 binding site were conducted by molecular docking studies.111 compounds which downloaded from the ChemSpider and PubChem databases, were subjected to molecular docking approaches.All ligands with their chemical formula, binding a nity in opened type, and S B domain residues interaction through hydrogen and hydrophobic bonds are shown in Table 1, that the amino acids in the2019 n-Cov 2 -ACE2 binding site are bolded in this table.(all this information for closed type is available as supplementary data le S1).According to molecular docking results, we selected seven identi ed ligands for drug-like ltering that have hydrogen and hydrophobic interacting in Spike 2019 n-cov2-ACE2 binding site in both closed and opened type that the amino acids in the2019 n-Cov 2 -ACE2 binding site are shown bolede in table 2. Rossicaside A has a hydrophobic binding with Tyr347 in the open state that is part of the binding area with -7.4 Kcal/mol binding energy.1,2-Ethanediol, 1,2-bis(4-uorophenyl) with a binding energy of -6.6Kcal/mol in open type forming hydrogen bonds with Gly394 and three hydrophobic bindings that Tyr393 and Tyr403 are in S protein-ACE2 binding site (Figure 2).1,2-Propanediol, 3,3,3-tri uoro-2-phenyl-, (2R) with binding energy -6.7 Kcal/mol forming a hydrogen bond with Gly394 and its hydrophobic bond were with Tyr393, Asn399, and Tyr403 residues (Figure 3).1,1-bis(3-uorophenyl)-2-methoxyethanol with a binding energy of -6.6Kcal/mol in open type forming hydrogen bonds with Gly394, Gln396, Asn399, Gly400 while other hydrophobic interacting residues were Tyr393 Tyr403(Figure 4).1,1-diphenyl propane-1,2-diol also forming two hydrogen bonds with Gly394 and Asn399 and is also two hydrophobic bonds with Tyr393 and Tyr403(Figure 5).The seventh chosen ligand is (S)-1,1-Diphenylpropane-1,2-diol with -6.2 Kcal/mol binding a nity that form hydrogen bonds with Gly394, Gln396 and Asn399 and hydrophobic bond with Tyr393 and Tyr403(Figure 6).In closed state hydrogen bonds and hydrophobic bond are mentioned in Table1(All hydrogen bonds in closed state showed as supplementary data le S2).
Drug-like characteristic of the chosen ligands ADME is the guideline that essentially speci es the different molecular characteristic of a compound and is the prime prerequisite to be a possible drug-like (Matter et al. 2001).To assess the pharmacokinetic characteristic of the chosen ligands, we evaluated the Drug Likeliness for 7 chosen ligands based on Lipinski's rule of ve (Lipinski et al. 1997).Lipinski 's rule of 5 suggests that weak absorption or permeation is more probable if more than 5 H-bond donors are involved, 10 H-bond acceptors, the molecular weight exceeds 500 Dalton, the calculated high lipophilicity (Log P ) exceeds 5 (Lipinski et al. 1997).The qualifying range for molar refractivity was between 40 and 130, with a mean value of 97 (Matter et al. 2001).As in Table 3 is shown, just Rossicaside A cannot pass the Lipinski's rule of ve, molar refractivity that is more than 130, and it is not a Drug Likeliness with 3 Violations.The remaining 6 chosen ligands passed all the parameters of the MADE (Table3).PAINS ltering to identify the existence of chemical groups belonging to the PAINS category.Six out of seven ligands were accepted as drug-like, and the physicochemical lter passed without any structural caution.The Rossicaside A is discarded which had the catechol group in the PAINS sub-structural moieties.Also, FAF-Drugs3 ltering is rejected for Rossicaside A and other ligands are Accepted by this ltering.In continue we investigated antiviral properties of them.The result of prediction antiviral for chosen compounds are show in table 5 that show respectively Etrinavir, Rossicaside A, 1,1-bis with general rate 74.78, 47.52, 41.68, 29.87, 28.41, 28.41, 25.53.A bar plot of the percentage of inhibition e cacy in each ligand for HBV, HCV, HHV, HIV, and General (26 viruses) is shown in Figure 7(A, B, C, D, and E).As is clear in gure 7D, nal ligands have the most percentage inhibition e cacy on the HIV virus with average e cacy %65/55.The minimum percentage of inhibition e cacy is shown in gure 7C which shows the HHV with average %16/81.

Discussion
In the specialized eld of computer-aided drug designing to discover a new compound, molecular docking is widely used to explore the different forms of the binding interaction between the prospective drug and the various domains or active sites, and binding sites on the target molecules (Raj et al. 2019;Hughes et al. 2011).It's been years that molecular docking is a great tool for the exploration of medicines.This is used to model atomic level binding between proteins and small molecules, which helps us to characterize the actions of small molecules at the target protein binding site (Meng et al. 2011).In viral infections due to the lack of successful antiviral therapies, there is an urgency to speed up the process of drug development to nd new and effective drug candidates.Attachment, fusion, and entry into the host cells are 3 duties that glycoprotein of the 2019 novel coronavirus plays important roles in these works that it is a SARS-CoV-2S surface protein (Yan et al. 2020).B chain in this protein is a factor that causes the formation of two open and closed forms of coronavirus that in terms of structural biology is in a heterotrimeric form with three different polypeptide chains: chain A, chain B, and chain C making each monomer (Walls et al. 2020).In this work, the B chain of spike glycoprotein CoV2 in 2 forms closed and opened (PDB ID: 6vyb and 6vxx) were modeled for modeling the missing residue and molecular docking done with about 111 combinations were screened from the ChemSpider and PubChem databases (Table 1) to nd the best ligand to block the B-chain binding site connection with ACE2.The compound ID(CID) of selected ligands from PubChem are: CID 13916145, CID 193962, CID 2755890, CID 11095754, CID 53722331, CID 555451, CID 736300, which showed have interaction with Spike 2019 n-cov2-ACE2 binding site residues with the energy of binding a nity, respectively: -7.5 Kcal/mol, -7.4Kcal/mol, -6.7Kcal/mol, -6.7 Kcal/mol, -6.6 Kcal/mol, -6.4 Kcal/mol, -6.2 Kcal/mol.Among all different types of interactions such as H-bond, π-π, amide-π interactions, etc that always analyzed, ligand e cacy refers to the ability to bind the target's binding sites has commonly set out analyzing its hydrogen-bonding pattern and the properties of residues engaged at the binding site (Raj et al. 2019;Hughes et al. 2011).7 nal ligands are: Rossicaside A is a phenylpropanoid, which phenylpropanoids and their derivatives are commonly found in fruits, vegetables, grains of cereals, beverages, spices, and herbs.It is considered that antimicrobial, antioxidant, anti-in ammatory, antidiabetic, anticancer action, and show renoprotective, neuroprotective, cardioprotective, and hepatoprotective effects are multifaceted effects of phenylpropanoids (Jia et al. 2018;Shyr et al. 2006).Rossicaside A is an antioxidant that is used in this sudy (Gálvez et al. 2006).Etravirine is a reverse transcriptase inhibitor of the next-generation, nonnucleoside, that administered orally and it is endorsed for the treatment of Infection of HIV-1 in experienced grown-up patients who have proof viral replication and are harboring HIV-1 strains resistant to other antiretrovirals (ARV) agents (Croxtall 2012).There are different combinations of this structure, 1,2-Ethanediol, 1,2-bis(4-uorophenyl) and 1,1-bis(3-uorophenyl)-2-methoxyethanol are 2 uorophenyl compounds that in our study formed hydrogen and hydrophobic interactions in S protein-ACE2 binding area so we also use three ligands (1,2-Propanediol, 3,3,3-tri uoro-2-phenyl-, (2R), 1,1-diphenyl propane-1,2diol and (S)-1,1-Diphenylpropane-1,2-diol) that had closed structure to uorophenyl compounds.As in drug-like characteristics of the chosen ligands analysis shown, we conclude that most of the important pharmacophoric characteristics required for an adequate restraint of the S B protein are accepted by six ligands recognized from the PubChem database.Moreover, their binding with chain B in both type forms a stable complex with a sturdy network of hydrogen, hydrophobic bonds and important residues, namely: Tyr347,as Phe377, Tyr393,Gly394, Gln396, Asn399, Gly400, Tyr403, Tyr408, Gly409, Gln411, Asn414 that were recently predicted as close contact residues with the human cell host receptor (Shang et al. 2020;Walls et al. 2020).Using the ADMEtox ltering, all ligands identi ed were assessed for pharmacokinetic properties.The rule of ve Lipinski theory is commonly used to determine possible reactions between medication and other non-drug target molecules.In Lipinski's rule, it is mentioned that for any compound to be chosen as a potential drug it ought to have (a) Molecular mass < 500 Dalton (b) high lipophilicity (expressed as LogP<5) (c) less than 5 hydrogen bond donors (d) Less than 10 hydrogen bond acceptors (e), also molar refractivity between 40-130 and drug Likeliness are 2 another factor that investigated in ADMEtox ltering.In the event that a compound has more than two of the previously mentioned measures, then the compound is probably going to be a potential drug candidate (Table 3).Also PAINS and FAF-Drugs 3 ltering are 2 other drugs-ltering that FAF-Drugs 3 is a program for ltering large compound libraries in silico screening experiments or related modeling studies and PAINS ltering can process hundreds and thousands of compounds in seconds and are in widespread current use to identify PAINS in order to exclude them from further analysis.In table 4 it is shown that in 7 nal ligands just Rossicaside A is rejected also by these ltering and others are accepted.Antiviral compound prediction is the nal analysis that in this study investigated for chosen ligands, which AVCpred is an instrument to accelerate the drug discovery procedure to recognize novel and e cacious antivirus drug candidates (Table 5, Figure 7).The ligands chosen have the most percent inhibition of HIV e cacy as shown in Figure 7D, and in the other groups of viruses, they have the antiviral properties that show that nal ligands can be used as antivirus drugs.Thus we suggest that all of these ligands except Rossicaside A have the potential ability to be an effective drug.In this work we used molecular docking to reveal whether there was any close interaction with 2019 n-cov2 ligands and S B proteins, the result showed acceptable binding of Etravirine, 1,2-Ethanediol, 1,2-bis(4-uorophenyl), 1,2-Propanediol, 3,3,3-tri uoro-2phenyl-(2R), 1,1-bis(3-uorophenyl)-2-methoxy ethanol, 1,1-diphenyl propane-1,2-diol and (S)-1,1-Diphenylpropane-1,2-diol of calcitriol with some amino acids of the binding site of B chain of spike protein through hydrogen and hydrophobic interactions bonds.It is noteworthy that in this study we decided to share our discoveries with all the researchers who work for experimental drug veri cation in the area of anti-SARS-CoV-2 research over the world.

Conclusion
The main objective of the present study was to nd the best ligand by molecular docking and evaluation of binding interaction against the B chain SARS-CoV-2 with ACE2.Seven compounds from the PubChem database were chosen based on interaction against the binding site of the B chain SARS-CoV-2-ACE2.Six nal ligands were selected according to drug-like characteristic (CID 193962, CID 2755890, CID 11095754, CID 53722331, CID 555451, CID 736300).These results show that these compounds could potentially be used as a drug against SARS-CoV-2.

Declarations
Acknowledgments The authors are thankful for helpful feedback and observations to the reviewers and editors, which helped enhance the paper quality.The authors do not have any relevant a liations or nancial involvement with any organization or entity having a nancial interest.
Compliance with ethical standards Tyr393, Tyr403 Figures

Figure 2 A
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Figure 7 A
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Table 1
Con ict of interest The authors declare that there are no con icts of interest.Ethical approval This article does not contain any studies with human participants and animals performed by any of the authors.Result of 6vyb molecular docking with all ligands, which are under study in this work.5 ligand groups are ranked by binding affinity.

Table 2
Summary of top seven ranked ligands screened against RBD of Spike 2019 n-cov2, with their respective classification, Chemical formula, binding affinity, hydrogen, and hydrophobic interacting residues.

Table ADME
Properties of selected ligands against S B domain.