Interactions mechanism of commonly used drugs for the treatment of COVID-19

In this study conformation analysis of seven drugs commonly used in the treatment of COVID-19 was performed The most stable conformers of the drug molecules were used as initial data for docking analysis Using the Cavityplus program, the probable most active binding sites of both apo and holo forms of COVID-19 main protease enzyme (Mpro) and spike glycoprotein of SARSCoV-2 receptors were determined The interaction mechanisms of the 7 FDA approved drugs (arbidol, colchicine, dexamethasone, favipiravir, galidesivir, hydroxychloroquine, remdesivir) were examined using the AutoDock Vina program The six of the seven drugs were found to be more stable in binding to apo form of COVID-19 Mpro and spike glycoprotein Moreover, a set of molecular mechanics (MM) Poisson-Boltzmann (PB) surface area (SA) calculations on the investigated drugs-protein systems were performed and the estimated binding free energy of remdesivir and the apo form of Mpro system was found to be the best The interaction results of FDA drugs with the apo form of COVID-19 Mpro and spike glycoprotein can play an important role for the treatment of COVID-19


INTRODUCTION
Corona viruses belong to the family of Coronaviridae, a family of enveloped-single strand positive RNA viruses. The Coronaviridae family is divided into four types (α, β, γ and δ) [1]. The corona viruses of the alpha and β strains usually infect mammals and humans, while the species γ and δ infect birds. This specification was made according to the phylogenetic analysis of corona viruses and genome structure [2]. Corona virus SARS-CoV-2 (virus that causes COVID-19) is a new corona virus of the genus β [2,3]. The novel corona virus is highly homologous to the known SARS-CoV corona virus that caused an outbreak of severe acute respiratory syndrome (SARS) in 2000-2004, however it is not the same virus. There are currently no vaccines and/or specific therapeutic drugs that target SARS-CoV-2. Until we had specific vaccines or therapeutic drugs targeting SARS-CoV-2, FDA-approved "restructured" drugs would be used to treat patients with COVID-19. [4][5][6]. The protein called angiotensin converting enzyme 2 or ACE2 "receptor" is thought to provide the entry point for SARS-CoV-2 to bind and infect a wide variety of human cells, as like as SARS CoV.
FDA approved drugs, chloroquine (CQ), hydroxychloroquine (HCQ), remdesivir (RDV) and arbidol (USA) appear as promising antivirals to fight COVID-19. In this study molecular docking of the FDA approved drugs with COVID-19 M pro were investigated. The hypothesis behind molecular docking studies is to identify the binding affinities of these drugs and identify key amino acid residues that play an important role in their mechanism of action.
In this study the interaction mechanisms of the 7 FDA approved drugs were investigated by docking of drugs with COVID-19 M pro and spike glycoprotein. The docked drugs were hydroxychloroquine, remdesivir, arbidol, favipiravir, colchicine, galidesivir and dexamethasone.
The results of trials and clinical studies on these drugs were summarized by Mohapatra et al. in 2020 [7].
The spike protein (S protein) of SARS-CoV has very important roles in viral infection and pathogenesis [1]. The main protease (M Pro ) of SARS-CoV-2 is an enzyme essential for virus replication through viral proteolytic activity and subsequent generation of infectious virus particles. [8]. The SARS-CoV-2 M Pro active site has two conformational stages: Apo (closed conformation) and holo (open conformation) stages [8].
In a study performed by Amin and Abbas [9], the in-silico interactions of chloroquine and hydroxychloroquine with the N terminal domain (NTD) of nucleocapsid protein (N protein) (NTD-N-protein) of SARS-CoV-2 were investigated and it was reported that these drugs bind efficiently to the active sites of the NTD-N-protein and reduce the efficacy.
In a study conducted by Rachakulla and Rachalulla [10], the in silico docking study was performed to reveal the potential bond affinity and binding interactions of dexamethasone remdesivir, hydroxyquinoline, favipiravir against COVID-19 M pro . It was found that dexamethasone, approved by the FDA for other medical purposes, has a high potential bond affinity and binding interactions against the SARS-CoV-2 protease compared to drugs currently used for COVID-19 treatment. However, it was noted that further clinical trials were needed to recommend as alternative medical treatments for COVID-19 [10].
Khan and Htar compared the binding affinities of the remdesivirin and dexamethasone for the SARS-COV-2 M pro to find out the most preventive potential against COVID-19 [11]. Dexamethasone was found to bind with a high affinity to the same sites of the SAR-COV-2 M pro better than remdesivir, due to having more hydrogen bonds than remdesivir antiviral drug.
In the study of Blaising et al., the physicochemical properties, pharmacokinetics, toxicity and molecular mechanisms of the arbidol drug, were investigated and predicted its possibility and suitability of a broad spectrum antiviral. It was thought that the interactions of arbidol with membranes and aromatic amino acids in proteins could be at the center of broad spectrum antiviral activity [12].
In this study, conformational analyzes of hydroxychloroquine, dexamethasone, favipiravir, arbidol, galidesivir, colchicine, remdesivir molecules were carried out to examine the energically possible conformers and to reveal their stability. In order to understand the biological activity and mechanism of these molecules against COVID-19 M pro , molecular docking study was carried out with apo and holo forms of COVID-19 M pro and spike glycoprotein. The drugs were sorted on the basis of binding strength.
The potential binding sites on the surface of the receptors were determined using the Cavityplus program [15]. Molecular docking studies were performed on the identified active sites [16], using AutoDock-Vina software. The binding free energies of the most stable ligandprotein systems that determined by molecular docking analysis, were calculated by the programs developed by Wang [17] and the ACFIS 2.0 web server [18][19][20][21].

RESULTS AND DISCUSSION
The spike protein, the main antigenic component responsible for inducing host immune responses, neutralizing antibodies, and protective immunity against virus infection, is a structural protein of SARS-CoV. Therefore, spike protein is chosen as the target for the presence of corona virus vaccine and development of anti-viral drugs [22]. The M pro is a vital role in polyprotein processing and virus maturation, for this reason COVID-19 M pro is an attractive drug target for antiviral drug design toward COVID-19 treatment. Cavities of apo and holo forms of COVID-19 M pro and spike glycoprotein of SARSCoV-2 receptors were determined using the Cavityplus program [15]. Among these cavities, the cavities with the highest probability of binding molecules were determined according to drug score and Pred Max pK d value. Respectively three and one druggable cavities were determined according to drug score value in apo form of COVID-19 M pro and spike glycoprotein, and also one cavity was determined according to Pred Max pK d value in holo form of COVID-19 M pro . Using the AutoDock Vina program [16], the molecules were docked to the active sites which are druggable cavities. The crystal structures of spike glycoprotein (PDB ID: 6VXX), apo form of COVID-19 M pro (PDB ID: 6M03) and holo form of COVID-19 M pro (PDB ID: 6LU7) were obtained from the protein database [23][24][25]. The docking for molecules was adapted by removing the water molecule from the receptors and adding polar hydrogens. The active sites of the apo and holo forms of the COVID-19 M pro and the spike glycoprotein of SARSCoV-2 were defined in the grid size of 40 Å × 40 Å × 40 Å.
In Table 1, the binding sites, binding energies of apo and holo forms COVID-19 M pro and spike glycoprotein protein with drugs are given. Figures 1-7 presents the interactions between target proteins and drugs.
The interactions between the arbidol molecule and the apo form of COVID-19 M pro are as follows (see Figure 1): 3.98 Å long alkyl interaction with the Val104 amino acid; 2.59 Å long hydrogen bond interaction between arbidol and the Gln110 amino acid; 3.33 Å long hydrogen bond interaction with the Asn151 amino acid; 3.94 Å long pi-sigma interaction with the Phe294 amino acid; 3.99 Å long pi-sigma interaction with the Val303 amino acid; 5.03 Å long pi-sulfur interaction with the Phe305 amino acid. Our results are in accord with previous findings. Recently, Sing and Flores [26] investigated the interactions between apo form of M pro (PDB ID: 6M03) and some molecules that could be used as therapeutics against COVID-19 disease including ramipril benzyl ester, propafenone dimer, lariciresinol, citrusinol, segatalin, cinobufagin and vitisnol C. Their results were in accord with our findings [26]. The molecular docking analysis performed by Sing and Flores [26] revealed that ramipril benzyl ester interacts with Asn151, Phe294 and Val 303 amino acid residues of the target protein 6M03. Moreover the results showed that, propafenone dimer interacts with Gln110, Phe294, and Phe305, citrusinol molecule interacts with Asn151 and Gln110 residues. These results are in accord with our findings. Our docking simulations showed that arbitol molecule is involved in interaction with Gln110, Asn151, Phe294, Val303 and Phe305 amino acid residues of M pro target protein. The docking outcomes suggest that arbitol binds to the same amino acid residues as the other molecules [26], which are known to be effective against COVID-19. Arbitol showed better binding affinity for the apo form of M pro (-6.5 kcal/mol) then the holo form (-6.0 kcal/mol).
Colchicine showed better binding affinity for the SARS-COV2-spike glycoprotein (-6.9 kcal/mol) then the apo form of M pro (-6.8 kcal/mol) (see Figure 2). Colchicine interacted with Arg466, Asp467 and Ile468 residues of the spike glycoprotein. In the study on the molecular interaction docking between synthetic peptides and SARS-CoV-2 spike glycoprotein [27] the presence of an interaction between Mo-CBP3-PepII peptide and through Arg466 residue of spike glycoprotein was shown. TheArg466 is the same amino acid residue of spike glycoprotein that the colchicine molecule interacts.
As a result of molecular docking analysis of dexamethasone with the apo form of COVID-19 M pro , the following interactions are yielded (see Figure 3): 5.01 Å long pi-alkyl interaction with Phe8 residue; 3.83 Å long Alkyl interaction with Arg298 residue; 4.71 Å long alkyl interaction with Val303 residue and 4.57 and 5.47 Å long pi-alkyl interactions with Phe305 residue. Comparison of our results with those of ramipril benzyl ester coupled with 6M03 [26] showed that the amino acids with which the dexamethasone molecule interacted were the same as the ramipril benzyl ester molecule, indicating that the binding active site in the apo form was the same. The dexamethasone showed a better binding affinity for the apo form of M pro (-8.6 kcal/mol) than the holo form (-7.8 kcal/mol) and the spike glycoprotein (-7.4 kcal/mol).        The favipiravir showed a better binding affinity for the holo form of COVID-19 M pro (-5.7 kcal/mol) than the apo form (-5.3 kcal/mol) and the spike glycoprotein(-5.1 kcal/mol). The favipiravir interacts with the holo form of COVID-19 M pro through H-bonds (see Figure 4). Å long hydrogen bond interaction between the molecule and the Asn151 amino acid; 2.08 Å long hydrogen bond interaction with Thr292 amino acid; 2.26 Å long hydrogen bond interaction with Asp295 amino acid are revealed. In the molecular docking analysis of some antiviral compounds against holo form of COVID-19 M pro (6LU7), it has been revealed that the antiviral compounds {niclosamide (C 13 H 8 Cl 2 N 2 O 4 ), +(-)epicatechin (C 15 H 14 O 6 ), diterpene (C 21 H 28 O 7 ), niclosamide (C 13 H 8 Cl 2 N 2 O 4 )}interact with the Asn151, Thr292 and Asp295 amino acid residues of the holo form of M pro (6LU7), which are the same amino acid residues that favipiravir interacts [28].
The binding affinity of the galidesivir to the spike glycoprotein (-7.1 kcal/mol) is better than the binding affinity to the apo (-6.4 kcal/mol) and holo (-6.2 kcal/mol) forms of COVID-19 M pro . The molecular docking study of galidesivir with spike resulted in H-bonding interactions between the drug and spike glycoprotein residues as follows (see Figure 5): 3.09 Å long hydrogen bond interaction with the Asp88 amino acid; 2.49 Å long hydrogen bond and 3.59 Å long carbon hydrogen bond interactions with the Asn196 amino acid; 2.65 Å long hydrogen bond interaction with the Asn234 amino acid; 2.54 and 2.21 Å long hydrogen bond interaction with the Ile235 amino acid and4.94Å long pi-cation interaction with the Lys462 amino acid. In a recent study molecular simulation analyses of SARS-CoV-2 spike protein with phytoconstituents of Withania somnifera Dunal Habit (including Viscosalactone B) which are known to have various bioactivities, were performed [29] and is shown that viscosalactone B (C 28 H 40 O 7 ) molecule interacts with Asp88, Asn196, Asn234 and Ile235 residues of the spike glycoprotein. These are the same amino acid residues of the spike glycoprotein that galidesivir interacts with. The results indicate that the binding active site within spike glycoprotein for viscosalactone B molecule is the same as the galidesivir molecule we studied.
The interactions of the hydroxychloroquine molecule with spike glycoprotein are as follows (see Figure 6): 1.45 Å long unfavorable donor-donor interaction between the Thr108 amino acid of the target protein and hydroxychloroquine; 5.33 Å long and 5.09 Å long pi-alkyl interactions between the Lys462 residue and the drug. The hydroxychloroquine shows better binding affinity for the spike glycoprotein (-6.4 kcal/mol) than those for the apo (-5.9 kcal/mol) and holo (-5.2 kcal/mol) forms of COVID-19 M pro .
The molecular docking studies of the remdesivir molecule with Apo form of COVID-19 M pro yielded the following interactions between remdesivir molecule and the aminoacid residues of target protein (see Figure 7): 2.92 Å long hydrogen bond interaction with the Gln110 residue; 2.83 and 3.07 Å long hydrogen bond interactions with the Thr111 amino acid residue; 2.90 Å long unfavorable acceptor-acceptor interaction between the Asn151 amino acid residue; 4.85 Å long alkyl interaction between the Pro252 residue; 4.70 Å long Pi-Alkyl interaction and 2.52 Å long hydrogen bond interaction with the Phe294 residue; 2.20 Å long hydrogen bond interaction with the Asp295 residue; 4.19 Å long Alkyl interaction with the Val297 residue; 4.86 Å long unfavorable positive-positive interaction and 2.77 Å long; hydrogen bond interaction with the Arg298 residue; 4.47 Å long pi-alkyl interaction with the Val303 amino acid residue. The remdesivir shows better binding affinity for the apo form of M pro , (-7.7 kcal/mol) than for the holo (-6.6 kcal/mol) form and spike glycoprotein (-5.7 kcal/mol).
Molecular mechanical energies combined with Poisson-Boltzmann or generalized Born and surface area continuous solving (MM/PBSA and MM/GBSA) methods are approaches used to estimate the free energy of binding small ligands to biological macromolecules. Typically they are based on molecular dynamics simulations of the receptor-ligand complex. These approaches are used to estimate the free energy of binding small ligands to biological macromolecules [17][18][19][20][21]30]. Typically they are based on molecular dynamics simulations of the receptor-ligand complex.
The predicted binding free energy of the arbidol-Apo form, dexamethasone-Apo form and remdesivir-Apo form were -17.71, -31.89 and -3.87 kcal/mol, respectively by using the MM/PB(GB)SA approaches with the GAFF2 and ff14SB force field combination and the GB6 procedure [17]. We could not perform the calculations for all studied molecules due to the insufficiency of the program, which was designed for limited size receptors.

CONCLUSION
We performed an in silico molecular docking simulations on the interaction of 7 FDA approved drugs with both apo and holo forms of the M pro enzyme of SARS-CoV-2. Moreover, the interactions of these 7 drug molecules with Spike glycoprotein protein were also investigated. The cavityplus program were used to determine the druggable cavities of the both apo and holo forms of the COVID-19 M pro s and the spike glycoprotein of the SARSCoV-2 receptors. By comparing all the compounds that interacted with COVID-19 M pro based on the binding affinity, dexamethasone (-8.6 kcal/mol) followed by remdesivir (-7.7 kcal/mol) and galidesivir (-7.1 kcal/mol) have a good binding affinity. Moreover, galidesivir has been shown to have the best binding affinity for the Spike glycoprotein (-7.1 kcal/mol) among the studied compounds. In addition a set of molecular mechanics (MM) Poisson-Boltzmann (PB) surface area (SA) calculations on the investigated drugs and apo form of COVID-19 M pro were also performed. By comparing all the compounds that interacted with COVID-19 M pro based on the binding free energy, remdesivir showed the best interaction with the apo form of the M pro {ΔG(PB): -20.61 kcal/mol; ΔG(GB): -30.09 kcal/mol}. All interactions between receptors and molecules and their binding affinities are explained in detail, these results are thought to be important for the treatment of COVID-19.