A new proposed mechanism of some known drugs targeting the SARS-CoV-2 spike glycoprotein using molecular docking

COVID-19 is caused by the novel enveloped beta-coronavirus with a genomic RNA closely related to severe acute respiratory syndrome-corona virus (SARS-CoV) and is named coronavirus 2 (SARS-CoV-2). The receptor binding domain (RBD) of the S-protein interacts with the human ACE-2 receptor that enables the initiation of viral entry. Hence, blocking the S-protein interactions by means of synthetic compounds mark the pivotal step for targeting SARS-CoV-2. Most of the six compounds were observed to t nicely with specic noncovalent interactions, including H bonds, electrostatic, Van der Waals and hydrophobic bonds (pi and sigma bonds). Oseltamivir was found to be the most strongly interacting with the RBD, exhibiting high values of full tness and low free energy of binding. it formed multiple noncovalent bonds in the region of the active site. Hydroxychloroquine also demonstrated high binding anity in the solvent accessbility state and t nicely into the active pocket of the S-protein. The results revealed that these compounds could be potent inhibitors of S-protein that could, to some extent, block its interaction with ACE-2. It is obvious from the 3D structure of SARS-CoV-2 spike protein was changed with the interaction of different drugs, which led to the unsuitability to bind ACE2 receptor. Hence, laboratory studies elucidating the action of these compounds on SARS-CoV-2 are warranted for clinical assessments. Chloroquine, hydroxychloroquine and oseltamivir interacted well with the receptor binding domain of S-protein via noncovalent interactions and recommended as excellent candidates for COVID-19.


Introduction
Coronavirus disease 2019 (COVID-19) occurred sporadically in Wuhan City, China in December 2019, then quickly spread throughout China and the entire world. Severe acute respiratory syndrome-corona virus 2 (SARS-CoV-2) is one the most infectious evolving current pathogen, causing severe respiratory illness and morbidity [1][2][3]. In the absence of drugs or vaccines, the cornerstone for preventing the spread of infection lies in adopting good personal hygiene, maintaining social distancing, restricting travel and the use of potentially effective natural products and therapeutics [4,5]. Many arti cial and natural compounds have been researched to combat SARS-CoV-2. Most target its spike (S) glycoprotein, which facilitates host cell entry, primarily by binding to the host angiotensin-converting enzyme 2 (ACE 2) receptor, present on the surfaces of macrophages, lymphocytes and other immune cells [6][7][8]. This phenomenon is speci c to the receptor binding domain (RBD) of the S-protein that spans from 326-580 amino acids and has unique sites for interaction with ACE-2 [9][10][11].
Chloroquine and hydroxychloroquine, which have been used for malaria prevention and treatment for decades, as well as for the treatment and management of chronic in ammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis, have gained signi cant focus as possible therapies [12][13][14]. Lopinavir is an antiretroviral protease inhibitor used in the treatment of HIV-1 infection combined with other antiretrovirals [15]. Oseltamivir is used in the treatment of the u virus (in uenza), ameliorating the symptoms (such as cough, stuffy nose, fever/chills, sore throat, tiredness, aches) and shortens the recovery time within 1-2 days. Darunavir is an antiretroviral medication that is used in treating and preventing HIV/AIDS. It is generally recommended to be used with other antiretrovirals [16,17].
Ibuprofen is a nonsteroidal anti-in ammatory drug (NSAID) class medication that is used to treat pain, fever, and in ammation, which includes painful periods of menstruation, migraines, and rheumatoid arthritis [18].
In this research, we assessed the interaction of Chloroquine, hydroxychloroquine, lopinavir, oseltamivir, darunavir and ibuprofen with the active site of the RBD of the S protein, using Autodock, Swiss dock and Discovery Studio tools.

Preparation of receptor protein and active site prediction
Based on the literature, the cryo-electron microscopy structure of SARS-CoV-2 S protein (PDB ID: 6VSB) was rst analyzed for ligand interacting sites [19,20]. Spike protein is a homotrimer, so we chose chain A for the analysis containing all the respective domains. The active site of the target protein was predicted by using binding site module using Discovery studio [21,22]. The functional active site was present in the receptor binding domain (Leu 335 to Gly 526), which also comprised the speci c active site region or loop region (NAG of RCSB: 6VSB, chain A) ( Figure 1). The active site residue ASN343 was chosen as the centre of the grid with the following coordinates: spacing: 0.753 Å, XYZ values: 76 126 76, center coordinates: -36.336 23.128 21.195. For site-speci c docking, the X-ray diffraction 3D structure of the speci c RBD of S protein (PDBID: 6M0J) with resolution 2.32.Å was chosen for interaction with the ligands. The above coordinates were assigned to the 3D structure followed by docking analysis. The 3D structure was energy minimized in solvent medium in Swiss PDB viewer to a free energy level of E= -10990.533 KJ/mol using inbuilt Gromos96 algorithm to obtain the most stable structure of the protein for docking [23,24].

Ligand preparation
The PDB format of the 3D structure of all the ligands were obtained from the drug bank https://www.drugbank.ca/drugs and PubChem https://pubchem.ncbi.nlm.nih.gov/. Chloroquine, hydroxychloroquine, darunavir, lopinavir, oseltamivir, remdesivir and ibuprofen were used as ligands ( Figure 2). The ligands were prepared for docking in the Ligand Preparation tool of the Discovery studio. The root was detected for each ligand, which was eventually saved in PDBQT les similar to that of the receptor protein.

Molecular docking and post scoring analysis
The X-ray diffraction structure of the S protein of SARS-CoV-2 (PDBID: 6M0J, 30 Å) was used for the docking analysis ( Figure 1). Site speci c docking was performed at the active site of the protein prepared using DS studio client 3.5 as described earlier. Then, all the water molecules and heteroatoms were removed in the Autodock tool. Hydrogen atoms were added to the model based on an explicit all atom model. Kollmann charges were added for ensuing interaction with the ligands, and the model was energy minimized. Both the receptor spike protein and the ligands were processed in Autodock tools as described above and converted to PDBQT format. Eight sets of docking poses were exhaustively performed; each set of Autodock Vina produced 9 conformations; among them, the best pose with RMSD = 0 and lowest free energy of binding was chosen for further analysis. The interacting sites were further visualized in Discovery Studio Visualizer and the 2D interaction and nature and types of bonds were determined.
Finally, rescoring of the docked conformations was performed in Swiss dock program with the same attributes for the receptor protein and ligands for better clarity and re nement of ligand protein interactions. The full tness value was obtained for the best docked site and analyzed with that of Autodock results.

Results
Receptor protein structure and analysis The 3D conformer of the RBD of S-protein was exposed to force eld for energy minimization in the solvent state. The energy was found to be -10990.63 Kcal/mol. Secondary structure analysis using PROMOTIF revealed that the RBD mainly comprised beta strands (25.3%) and few alpha helices (11%), in addition to beta turns, helix-turn helix, beta sheets, and gamma strands (63.7%). The active site center represented residue ASN 343 and formed a loop-like structure. Hydrophobicity-hydrophilicity analysis of the RBD residues using Peptide 2.0 software revealed that neutral and hydrophobic amino acids comprised 44.85% an 38.14%, respectively of the total peptides (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php), which indicated lower water solubility and more non polar interactions with different compounds. Further analysis using the complete homotrimeric S-protein of SARS-CoV-2 also revealed that the total protein comprised of 42.14% and 41.1% of hydrophobic and neutral amino acids, suggesting that S-protein is mainly hydrophobic and can interact with non-polar compounds (Figure 3).

Docking analysis
The interaction of chloroquine with the RBD was moderately strong (ΔG = -5.5 Kcal/mol) with the speci c interacting residues positioned near the active sites (TRP 436, LEU 441). Noncovalent interactions comprised 2 hydrogen bonds, and 5 hydrophobic bonds between the ligand and the receptor residues. The ligand t into the binding pocket of the receptor with a full tness of -770.49 (Table 1, Figure 4).
The interaction of hydroxychloroquine was strong (ΔG = -5.8 Kcal/mol) relative to other compounds, with several interacting residues near the active site region, forming 4 hydrogen bonds and 5 hydrophobic interactions. This ligand t into the central binding pocket of the RBD with a full tness of -797.05 and conferred maximum stability (Table 2, Figure 5).
Oseltamivir ligand interacted well with the RBD with a binding energy of ΔG = -5.7 Kcal/mol and full tness of -794.93. Several interacting residues were in the active site (LEU 517, PHE 464 and THR 430) and formed two hydrogen bonds and four hydrophobic bonds (Table 3, Figure 6).
The ligand lopinavir did not interact with the RBD residues using either docking approach; only 1 residue (SER 469) formed hydrogen bond near the active site (Table 5, Figure 8) with low tness of -677.139 and ΔG = -3.6 Kcal/mol.
Ibuprofen also interacted less with the receptor protein. Although it had low binding energy compared to other ligands (ΔG = -6.1 Kcal/mol), the interacting residues were less, and all formed weaker hydrophobic bonds that signi ed low interaction with the receptor (Table 6, Figure 9).

Discussion
Several medical and molecular trials involving many natural compounds and synthetic compounds used for medication of other diseases and molecular trials are being pursued [25]. Repurposing previously used drugs with health-promoting effects confer several advantages, including reduced costs, faster regulatory approvals and immediate eld trials. Until a vaccine is developed, some treatment is imperative to reduce COVID19 mortality and morbidity.
Multiple studies indicate that repurposing chloroquine and hydroxychloroquine can effectively inhibit Coronaviridae infection (including SARS and SARS-CoV-2) in vitro [26][27][28][29]. Preliminary con icting clinical evidence from studies in China and France have put the brakes on their ongoing clinical trials [30,31]. Our ndings suggested that the interaction of chloroquine and hydroxychloroquine was moderately strong with residues of the RBD of S-protein and tting well into the binding pocket of the receptor. Therefore, these compounds could also be potent inhibitors of the S-protein function and should be further evaluated in clinical trials. Our study indicates that oseltamivir could be useful for combating COVID-19, in contrast to the study from Wuhan, which observed no positive results [32]. Several clinical trials are testing the e cacy or oseltamivir in treating COVID-19. Oseltamivir is also used in many combinations in clinical trials, such as with chloroquine and avipiravir [33]. Our ndings were agreement with darunavir's antiviral activity in vitro against a clinical isolate from a SARS-CoV-2 patient, where darunavir showed no activity against SARS-CoV-2 and the data did not support the darunavir use for COVID-19 treatment [34]. Our nding regarding Lopinavir was in agreement with the study on hospitalized adult patients with severe SARS-CoV-2, where no bene t was observed beyond standard treatment with lopinavirritonavir [35]. Similarly, our results for ibuprofen indicated that it may make symptoms worse in COVID-19 patients 38 . They con rmed that the binding of coronaviruses with angiotensin-converting enzyme-2, and the bioavailability of angiotensin-converting enzyme-2 will be increased by the administration of ibuprofen, thus enhancing and potentiating the process of coronavirus infection [36].
Overall, the drugs used in this study such as hydroxychloroquine, chloroquine and oseltamivir conferred highest interactions with the RBD of S-protein of SARS-CoV-2and they are promising as anti-SARS-CoV-2 compounds. Further laboratory studies and eld-based clinical trials are warranted to further extrapolate the antiviral outcomes of these compounds.

Conclusions
Hydroxychloroquine, chloroquine and oseltamivir interacted well with the receptor binding domain of Sprotein via noncovalent interactions. These compounds are promising candidates for repurposing for the treatment of ongoing coronavirus pandemic. On the other hand, darunavir, lopinavir and ibuprofen were bad candidate for either the prevention or the treatment of COVID-19.