O-β-GlcNAcylation, Chloroquine and 2-Hydroxybenzohydrazine may hamper SARS-CoV-2 entry to human via inhibition of ACE2 phosphorylation at Ser787 but also induce disruption of virus-ACE2 binding

The novel coronavirus COVID19 disease is extremely contagious and has been spread worldwide. First COVID-19 case was identified in December, 2019 and within three months, more than one million affected cases and over 65,000 deaths have been reported. SARS-coronavirus 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 April 2020 doi:10.20944/preprints202004.0390.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. (SARS-CoV-2) also known as 2019-nCoV is a causative agent of COVID-19 disease and belongs to the SARS CoV (Severe Acute Respiratory Syndrome corona virus) family. The SARS-CoV-2 enters the human body by binding its viral surface spike protein with the host angiotensinconverting enzyme 2 (ACE2) receptors and cause infection. To prevent the virus entry and its transmission in the human body, we focused on the two domains of ACE2: i) the N-terminal extracellular binding domain (18-740 residues) reported for coronavirus spike interaction, and ii) the C-terminal cytoplasmic region (762-805 residues) to prevent the virus transmission. Therefore, we proposed: i) inhibition of receptor binding domain (RBD) of SARS-CoV-2 and human ACE2 protein may prevent the virus entry to the host and ii) inhibition of phosphorylation at Ser-787 of ACE2 protein may prevent the transmission of the virus in the COVID-19 patients. In the past, the critical role of Ser 787 in human ACE2 protein has been experimentally verified in SARS-CoV transmission, that upon binding to the receptor, SARSCoV induces CKIImediated phosphorylation of ACE2 at Ser-787 that in-turn facilitate virus entry to host cells, followed by replication and activation of ACE2, initiates downstream signaling leading to lung fibrosis. Therefore, in this study, we have suggested post-translational modification (PTM) O-βGlcNAcylation, and two compounds Chloroquine and 2-hydroxybenzohydrazine might share the common pathways to prevent the COVID-19 infection in human. The addition of O-βGlcNAcylation at same or neighboring Ser/ Thr residues results in phosphorylation inhibition and a change in protein structural and functional confirmations. Thereby, using neural networking methods, we have identified Ser/ Thr residues in ACE2 that are potential sites for phosphorylation and / or O-β-GlcNAcylation. Molecular docking showed that UDP-GlcNAc has more binding affinity with Ser-787 than the phosphoryl group. Moreover, chloroquine and 2hydroxybenzohydrazine also showed great potential to bind at Ser-787 that may result in inhibition of Ser-787 phosphorylation and downstream signaling. Furthermore, O-β-GlcNAcylation, chloroquine and 2-hydroxybenzohydrazine showed their high affinity at ACE2-SARS-CoV2receptor binding domain that may prevent the entry of SARS-CoV-2 into human body. In conclusion, inhibition of human ACE2 phosphorylation at Ser-787 and ACE2-SARS-CoV-2 binding domain could be promising targets against SARS-CoV-2 infection.

This situation could be worse in near future if there is no treatment available for this infection.
SARS-CoV-2 is an enveloped virus structurally related to other severe acute respiratory syndromes-related coronaviruses. It has 96% genomic identity with chine's horseshoe bat coronavirus. It belongs to realm "Riboviria" and family coronaviridee (Li, 2016;Lu et al., 2020;Peiris et al., 2004). Coronaviruses contains four important structural proteins, including spike, envelope, membrane and nucleocapsid. Spike protein of coronavirus is responsible for the viral attachment and entry to the human cells via human ACE2 receptors (Li, 2016;Shang et al., 2020).
Chen et al showed that interaction of spike protein induced human ACE2 phosphorylation by casein kinase II followed by coronavirus infusion and activation of ERK1/2 that in turn activated fibrosis-associated chemokine ligand 2 (CCL2) regulation (Chen et al., 2010). CCL2 regulation has been found to induce severe lung inflammatory disorders followed by progressive respiratory failure and patient's death (Chen et al., 2010;Song et al., 2018). The inhibition of spike protein interaction with human ACE2 could be considered as an important target to develop a vaccine against SARS infections.
Despite phosphorylation, ACE2 may also experience modification by O-β-GlcNAc. It has been reported previously that O-GlcNAcylation could interfere with viral pathogenesis in host cells (Ahmad et al., 2011a;Ahmad et al., 2011b). During O-β-GlcNAcylation, one molecule of Nacetylglucosamine (UDP-GlcNAc) is attached to a Ser or Thr residue by the enzyme O-GlcNAc transferases (OGT) (Kamemura et al., 2002). All O-β-GlcNAc modified proteins are also phosphoproteins and exhibits both global and site-specific reciprocal relationship (Kamemura and Hart, 2003). O-β-GlcNAcylation of proteins results in temporary conformational changes that modulate many of their functions. Residues where O-β-GlcNAc and phosphorylation compete for each other are known as Yin Yang sites (Zachara and Hart, 2002). These Yin-Yang sites can be predicted using computer-assisted neural network-based programs and analyzed to predict the modification potential and functional importance of each site.
Recent reports from various hospitals around the globe highlighted the importance of malaria-drug chloroquine (CQ) for the treatment of coronavirus infection in human. Chloroquine has been proposed as a possible treatment for the coronavirus infection (Alam and Lee, 2016;Avital et al., 1994;Keyaerts et al., 2009;Kundin et al., 1964) . In the past, Kundin WD et al (1964) found Benzol hydazides and hydrazines derivatives effective against the influenza virus in the chick embryo lung tissue cultures (Kundin et al., 1964), and these compounds also have been suggested previously as anticancer agents (Alam and Lee, 2016). Therefore, in this study, we have used Chloroquine, 2-hydroxybenzohydrazide and O-GlcNAcylation as docking ligands, and we found that these compounds might inhibit the ACE2 phosphorylation and suppressing the interaction of coronavirus spike glycoprotein with human ACE2.

Materials and methods
In the present study, the proteins of interest were: i) Human Angiotensin-converting Enzyme-

Sequence Alignment of human ACE2 protein
The FASTA sequence of human ACE2 was retrieved from the Uniprot sequence database with the entry name ACE2_human and accession number Q9BYF1 (Wu et al., 2006). A homology search of the ACE2 protein sequences of all organisms in the NCBI database was done with the BLASTP algorithm using default parameters (Altschul et al., 1997). A total of 100 hits were obtained for human ACE2 with an E-value of zero. Sequences based on isoforms, predicted, hypothetical, synthetic, and unnamed proteins were neglected to ensure reduced redundancy in selection. Out of 100 retrieved sequences, fourteen were selected representing major mammalian groups. The Uniprot accession numbers for fourteen selected sequences ( Fig. 1) were Human (Q9BYF1), R pearsonii (Pearson horseshoe bat, B0LW5_9CHIR), R macrotis (Big eared bat, E2DHI9_9CHIR), R. pusillus (least horseshoe bat, E2DHI9_9CHIR), R sinicus (Chines rufos bat, E2DHI7_9CHIR), Gorgo (G3QWX4), Pantr (A0AA2J8KU96), Panpa (A0A2R9BkD8), Nomle (GIRE79), Macne (AOA2K6D1N8), Macmu (B6DUE2), Rat (Q5EGZ1) and Mouse (Q8R010). ClustalW2 (Thompson et al., 1994) (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used for multiple alignments of all the sequences of ACE2 to get the conservation status of Ser and Thr residues.

Post-translational modifications prediction
In this study, well documented prediction programs (Qian and Sejnowski, 1988;Saraswathi et al., 2012;Thompson et al., 1994) have been used to determine the post-translational modifications (phosphorylation and O-β-GlcNAcylation) on human ACE2 protein.

Prediction of Yin-Yang sites
The potential phosphorylation and O-β-GlcNAcylation sites on ACE2 protein were predicted by YinOYang

Human ACE2 protein 3D structure prediction
The experimentally determined crystalline protein structure of native human Angiotensinconverting Enzyme-related Carboxypeptidase (ACE2) is available in the protein data bank (PDB ID: IR42) with 1-615 residues. To fully understand the post-translational modifications on Ser and Thr residues and their surface accessibility, a complete 3D structure of ACE2-Human with 805 residues was required. Therefore, the complete 3D structure of ACE2-Human was predicted using I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Zhang, 2008). The best predicted ab-initio models of ACE2-Human were selected based on their 1) similarity with the experimentally verified human ACE2 protein structure, and 2) chemical correctness of protein's backbone angles using Ramachandran plots. Molprobity (http://molprobity.biochem.duke.edu/) and RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) (Davis et al., 2004) servers were used to perform geometrical evaluations and Ramachandran plot's formation.

3D structure prediction of SARS-CoV-2 (2019-ncoV) receptor-binding domain (RBD)
The SARS-CoV-2 (2019-nCoV) spike (PDB ID: 6VSB) is a trimer protein. Therefore, FASTA sequence of its chain A was only selected for sequence alignment with the FASTA sequence of Chain E of dimer SARS-CoV spike protein (PDB ID: 2AJF). The FASTA sequences downloaded from RCSB Protein Data Bank to do a comparison between the residues of the two proteins in the binding domain regions. EMBOSS Water (https://www.ebi.ac.uk/Tools/psa/emboss_water/) was used for the sequence alignment of SARS-CoV-2 and SARS CoV spike proteins.
The experimentally determined cryo-electron microscopy structure of the SARS-CoV-2spike in the perfusion conformation (PDB ID: 6VSB) lacks the residues from TYR453-LEU492 in its structural conformation (Wrapp et al., 2020). Therefore, SwissProt was used to predict the

Protein structure preparation for docking analysis
To prepare the predicted 3D structures of ACE2 or SARS-CoV-2 spike proteins individually, the highly scored I-Taseer predicted 3D protein structure of human ACE2, and the highly scored Swiss Model predicted structure of SARS-CoV-2 spike protein respectively was selected.

Ligands preparation for Docking Analysis
The ligands UDP-GlcNAc (PubChem CID: 445675), Chloroquine (PubChem CID: 2719) were fetched by their PubChem CIDs into the UCSF Chimera and saved in pdb format. On the other side, the structure of phosphate was created in the Open Bable converted into Mol format, hydrogens were added and then converted into MOL2 format. The individual pdb files of UDP-GlcNAc and Chloroquine were also opened in Open Bable and converted into Mol format, hydrogens were added and then converted into MOL2 formats. All MOL2 files were then subjected to DockPrep in UCSF Chimera at default parameters for preparing the ligands. The energy minimization and dock preparation of 2 -hydroxybenzohydrazide (PubChem CID: 13637) ligand was done in UCSF Chimera and saved as Mol2 file.

Docking Analysis:
In SwissDock program, MOL2 files of the ligands were submitted along with the prepared proteins (pdb files) for flexible docking at default parameters. The predicted docking files generated from SwissDock were visualized and analyzed in UCSF Chimera 10.1. While for docking ligand in the binding pocket of the protein, Auto Grid program of Chimera with a grid marker having dimensions of 30x30x30 was used in AutoDock Vina. To determine the surface accessibility of the residues in the binding pockets, UCFS Chimera X version 0.93 was used to generate the hydrophobicity surfaces. Our approach to select a docking poses in each analysis comprised of: binding interaction of ligand with Ser/Thr residues, the number of hydrogen bonds, high binding affinity energy (-∆G) and the experimentally reported binding pockets.

Human ACE2 has conserved Ser/Thr residues
ACE2 is highly conserved protein comprises of 805 amino acids (AA). The N-terminal of ACE2 consisted of extracellular domain (18-740 AA) and has been reported to act as a binding site for the coronavirus spike proteins whereas most of the C-terminal region of ACE2 protein is cytoplasmic (762-805 AA). ACE2 has fifty-four Ser and thirty-nine Thr residues in its whole sequence. Human ACE2 was aligned with the orthologous sequences of other mammalian species, including bats to determine the evolutionary conserved Ser/Thr residues ( Fig. 1). Absolutely conserved, conserved substitutions and semi-conserved Ser and Thr residues within each subtype were determined among the fourteen mammalian species. Fig. 1 showed 4,10,19,43,44,47,70,77,105,109,124,128,155,257,317,331,409,411,502,511,545,547,563,602,611,617,623,626,645,646,680,692,707,721,783,and 804 (37/54);27,52,129,276,282,294,324,334,347,362,365,371,414,434,449,453,496,571,519,548,567,608,686,730,763,and 803 (27/39) are highly conserved in mammals. While 420,507,118,122, and 125 were found to be conserved substitutes. On the other side, 113,167,218,280,425,607,709,776 and 787;78,92,229,445,593,698, and 798 showed semi-conserved substitutions and mainly substituted by Ala (alanine) and Asn (asparagine) residues.

3D Modelling of ACE2 protein
In this study, we predicted the complete 3D structure of ACE2 protein comprises of 805 amino acids using I-Taseer server to examine the post-translational modifications, surface accessibility (buried and exposed) of the residues for compounds to be interacted to inhibit the binding of corona virus spike protein.
The server used the available experimentally determined ACE2 3D template (PDB ID: 1R42) (1-615AA) and predicted five possible models ( Fig S1). All the predicted models were aligned with currently available partial 3D structure, and model with 100% identity and low geometrical errors was chosen for further study (named as M1-ACE2, Fig 2). We constructed Ramachandran plots against each predicted model ( Fig S1). The Ramachandran plot analysis estimates phi and psi torsion angles based on experimentally known data and standard geometrical properties of protein structure (Hollingsworth and Karplus, 2010).

High potential of phosphorylation and O-β-GlcNAcylation modifications on ACE2 protein
ACE2 has been found to be phosphorylated and being a main target in coronavirus infection. ACE2 phosphorylation could mediate host-pathogen responses (Chen et al., 2010). The phosphorylation potentials of the Ser and Thr residues and kinases involved for phosphorylation were shown in Table 1, whereas graphical representation of potential phosphorylation residues was shown in Fig 3A. Forty-seven (47) Ser/ Thr residues were predicted potential phosphorylation sites. However, among these 23 were buried or less accessible residues for modifications. The accessible potential phosphorylated sites were comprised of 24 Ser (2,3,19,47,70,105,106,109,113,124,128,218,254,317,420,425,602,617,626,646,709,721,783,787) and 20 Thr (15,20,27,55,92,118,122,125,276,324,365,434,445,496,548,593,686,698,763,803) residues. A number of kinases may be implicated in phosphorylation of Ser and Thr residues. Almost each kinase predicted was involved in phosphorylation of two or more residues (Table 1).
Both phosphorylation and O-β-GlcNAcylation could compete for Ser and Thr linkage on the same or neighboring residues. These residues are known as Yin-Yang sites. Yin-Yang sites determined by the neural networking software could be false-positive or false-negative due to the residues which are completely buried or not accessible for phosphorylation and O-β-GlcNAcylation modifications. To minimize the false predictions, UDP-GlcNAc sugar was docked with human ACE2' 3D structure, and we found a total of thirteen Ser (47, (Fig 3. E-F, Table 2). Fig 3D showed presence of a phosphoryl group in the vicinity of Ser-787 with maximum energy released (-2.1 kcal/mol). These results showed that ACE2 has high potential for O-β-GlcNAcylation binding upon competition with phosphorylation modification at the same or neighboring residues.

Chloroquine and 2-hydroxybezohydrazide may alter downstream signaling of human ACE2 protein by inhibiting phosphorylation
Recent reports from clinics caring coronavirus-infected patients showed a positive effect of malaria drug chloroquine (CQ) on eliminating/ reducing the SARS-CoV-2 symptoms. Therefore, in this study, we tested CQ along with another compound known as 2-hydroxybezohydrazide (HBH) to observe the inhibitory effect on human receptor protein ACE2. The conformation of CQ in Fig 4 (A, B, C) shows that it could be able to form two hydrogen bonds with the residues of Ser 787 (2.702 Å) and Hsd 788 (2.831 Å) of ACE2 receptor with high binding affinity (∆G= -7.111 kcal/mol).
In the past (1964), 2-hydroxybenzohydrazide (HBH) was effectively used to inhibit the growth of influenza virus in chick embryo lung tissue culture (Kundin et al., 1964). The derivates of HBH were found to have anti-microbial activities and anti-influenza activity (Alam and Lee, 2016; Kundin et al., 1964). In our study, HBH was found to bind with the ACE2 receptor via two hydrogen bonds with Ser-787 (2.385Å) and the neighboring residue Lys-788 (2.108Å) as shown in Fig 4 (D, E, F). These findings showed that both CQ and HBH might compete with the phosphorylation on Ser 787 and results in alteration in the structural and functional conformation of ACE2 receptor protein, thus inhibits the SARS-CoV-2 spike interaction and signaling to proceed with the cascade of the infection.

O-β-GlcNAcylation modification in the ACE2 and SARS-CoV-2 RBD complex
SARS-CoV-2 RBD with ACE2 receptor complex was prepared in Chimera by replacing the SARS-CoV spike with SARS-CoV-2 spike in SARS-CoV RBD-ACE2 receptor complex (PDB ID: 2AJF). We found four hydrogen bonds between SARS-CoV-2 and ACE2 residues in RBD   (Li et al., 2005). Moreover, in this study, these residues have been found in the interactions between SARS-CoV-2 spike RBD and ACE2 complex in various conformations, and binding of UDP-GlcNAc may disrupt these interactions to inhibit the binding of SARS-CoV-2 spike binding with the human ACE2 receptor.

SARS-CoV-2 -human ACE2 receptor complex
In this study, CQ and HBH have also been found to interact with the RBD of SARS-CoV-2 spike and ACE2 receptor complex. One conformation showed CQ binding via one hydrogen bond with the Glu35 residue (1.869Å) of ACE2 with high binding affinity (ΔG= -8.581 kcal/mol) in a binding pocket interacting with the RBD of SARS-CoV-2 spike (Fig 7A). The other conformation of CQ showed its binding via one hydrogen bond with the Glu471 residue (1.809 Å) of RBD of SARS-CoV-2 spike in a binding pocket interacting with the ACE2 with high binding affinity (ΔG= -8.441 kcal/mol) ( Table 2). Both CQ conformations showed that these interactions in either way may disrupt the binding of SARS-CoV-2 spike with the human ACE2 receptor to inhibit the virus entry into the host cell.
Similar to CQ, HBH also showed high binding affinity (ΔG= -6.301kcal/mol)with the Glu471 residue (1.997Å) of SARS-CoV-2 spike via one hydrogen bond, and with the Glu35 residue (2.318Å) of ACE2 via second hydrogen bond in the RBD cavity of SARS-CoV-2 spike and ACE2 receptor complex (Fig 7B, Table 2). Various conformations of HBH in the RBD cavity showed that either HBH binds with the SARS-CoV-2 spike RBD or the ACE2 receptor or both may disrupt the structural and functional conformation of the SARS-CoV-2 spike and ACE2 complex, thus inhibit the entry of virus into the host cell.

Discussion
The current global outbreak of coronavirus SARS-CoV-2 demands efficient therapeutics against this infection. Due to limited clinical and experimental data, researchers are focusing on computational analysis to examine the SARS-CoV-2 structure and function, and its interaction with the host protein to find the possible cure. In this study using computational techniques, we proposed a mechanism and compounds to inhibit the SARS-CoV-2 spike interaction with human ACE2 receptor, entry into host cells, and downstream signaling. Chen et al (2010) showed that upon binding with ACE2 receptor, SARS-CoV induces ACE2 phosphorylation at Ser-787 via activation of CKII (Chen et al., 2010). This process does not only facilitate SARS entry to host cells for reproduction but also induces CCL2 signaling to cause lung fibrosis and severe infection (Chen et al., 2010). Our results proposed that inhibition of phosphorylation of human ACE2 receptor at Ser-787 might block downstream signaling of spike protein of SARS-CoV-2. This will in turn reduce virus replication and ACE2 downstream signaling to control the COVID-19 disease spread.
To inhibit the phosphorylation of ACE2 at Ser-787, we have proposed two inhibition mechanisms i) inhibition through PTMs and (ii) inhibition through drug binding. Post translational modifications (PTMs) at specific residues cause geometrical and conformational changes in proteins to alter their functions (Duan and Walther, 2015;van der Laarse et al., 2018;Xin and Radivojac, 2012). Interestingly, both phosphorylation and O-β-GlcNAc compete for the same or neighboring Ser/ Thr residues and change protein conformation and its functional process. These Ser/ Thr residues are known as Yin-Yang sites (Leney et al., 2017). ACE2 has been found to be phosphorylated at two residues Ser-680 and Ser-787. The phosphorylation at Ser-787, present at C-terminal of ACE2 is responsible for its downstream signaling upon SARS interaction (Chen et al., 2010). Ser-787 is a conserved residue except in few mammals, including mouse and rat as shown by multiple alignments.
The first mechanism is to inhibit the ACE2 phosphorylation with the O-β-GlcNAc at Ser-787 by O-GlcNAc transferase (OGT). . Unlike phosphorylation, it is usually difficult to detect O-β-GlcNAc on proteins due to several technical and experimental limitations (Li et al., 2019;Zachara et al., 2015). To overcome this difficulty, several neural network methods have been used to predict O-β-GlcNAc on Ser/ Thr residues based on available experimental data and consensus sequences . Our results showed that ACE2 has high potential for O-β-GlcNAcylation, which can inhibit the downstream signaling of SARS-COV-2 by reducing the ACE2 phosphorylation at Ser-787. The neural network methods usually predict PTMs based on the secondary structure and homology of proteins of interest (Audagnotto and Dal Peraro, 2017;Blom et al., 2004). To add further confidence in our results, we predicted O-β-GlcNAcylation sites on 3D structure of proteins as well. We identified six potential accessible yin yang Ser/ Thr residues that could induce ACE2 conformational and functional changes upon phosphorylation and O-β-GlcNAc interplay. Interestingly, in our study, UDP-GlcNAc is found to be three times stronger than the phosphoryl group in binding with Ser/ Thr residues of ACE2. These results strengthened our hypothesis that UDP-GlcNAc might be able to inhibit Ser-787 phosphorylation of ACE2 to stop the viral infection spread in the body. The previous reports also found that SARS CoV virus was less efficient to infect mouse and rat species despite maximum homology in ACE2 structures (Li et al., 2004;Sheahan et al., 2020;Shereen et al., 2020;Wang et al., 2020). Based upon our results, we suggest this could be due to the mutation of Ser-787 in the mouse and rat with glycine as it is difficult for the glycine to be phosphorylated.
In our study, the sequence alignment of SARS-CoV-2 spike with SARS-CoV spike protein showed mutation at the residues Y442L, R426N and T487N may change SARS-CoV-2 binding with the ACE2 receptor. The comparative analysis also revealed that the other mutated residues in the SARS-Cov-2 spike have same propensity of the side chain as found in the SARS-CoV (Fig 4).
The binding position of UDP-GlcNAc in the RBD in the vicinity of Thr 487 may alter the conformation and thus inhibit the binding of the SARS-CoV-2 spike with the human ACE2 receptor to prevent the entry into the host cells (Fig5, table 2). Our results have also been supported by the previous studies that Ser481 and Thr487 of SARS-CoV were responsible to induce SARS-CoV binding with human ACE2 receptor (Li et al., 2005). Therefore, we can hypothesize based upon our results that O-β-GlcNAcylation in the RBD zone of SARS-CoV-2 spike complex with human ACE2 may disrupt the interaction of the two proteins and ultimately inhibit the viral entry into the host cells.
The other method to inhibit the interaction of SARS-CoV-2 with the ACE2 at the receptor binding domain and /or to inhibit the ACE2 phosphorylation at Ser-787 are by the compound's binding to the receptor ACE2. Therefore, in this study, we examine the role of chloroquine (CQ) and 2-hydroxybezohydrazide (HBH) as probable inhibitors against SARS infection. CQ is a wellknown anti-malarial drug and has been proposed recently as being efficient against SARS-CoV-2 infection too . Meanwhile, HBH has been found to reduce influenza infection (Alam and Lee, 2016; Kundin et al., 1964). Interestingly, we found these inhibitors showed their strong binding with Ser-787 of the ACE2 protein, thus blocking the access of the phosphoryl group at the sites or inactivate phosphatases involved to block the viral infection. Furthermore, our results also showed that both CQ and HBH have strong binding with Glu35 of ACE2, a critical residue to bind with the SARS-CoV-2 RBD. Moreover, both CQ and HBH were also able to bind at the Glu471 residue of SARS-CoV-2 spike RBD, important for binding with ACE2. The interaction of CQ and HBH either with the ACE2 or the SARS-CoV2 may inhibit the viral entry into the host cells.
In conclusion, our results showed that: i) inhibition of the receptor binding domain (RBD) of SARS-CoV-2 and interacting binding pocket of human ACE2 receptor may prevent the virus entry to the host, and ii) inhibition of phosphorylation at Ser-787 of ACE2 protein may prevent the transmission of the virus in the COVID-19 patients. We highlighted the significant importance of the inhibition ofACE2 phosphorylation at Ser-787, and the inhibition of SARS-CoV-2 RBD and interacting binding pocket of human ACE2 receptor either by molecular level via O-β-GlcNAcylation or by the use of CQ and HBH inhibitors (Fig 8) to block the entry or downstream signaling of SARS-CoV-2 infection. These findings suggest the potential targets to develop therapeutics to control the SAR-CoV-2 (2019-nCoV) infection.

Author Contributions
W. A was responsible for the study design. W.A, K. S and N. I collected and analyzed the data.

Declaration of interest
We author have no interests to declare.

Supplementary data
The supplementary data has been provided with the submitted manuscript. Zhang, Y., 2008. I-TASSER server for protein 3D structure prediction. BMC bioinformatics 9, 40.