In silico approach: docking study of oxindole derivatives against the main protease of COVID-19 and its comparison with existing therapeutic agents

Introduction

The outbreak of COVID-19 recognized as severe acute respiratory syndrome coronavirus 2 arises from Wuhan, Hunan seafood wholesale market, China. The genome of the SARS-CoV-2 is similar to the bat genome which is in a proportion of 96% similarity [1]. In Guangdong City of China confirmed the first case of coronavirus through the local transmission [2]. On 4th March 2020, an international health emergency declared by the WHO (World Health Organization), in this year on 11th August 2020, a total of 19,936,210 verified cases worldwide, with 732,499 deaths data received by WHO. COVID-19 cases are still surging due to their quick transmission [3]. The virus is made by two layers in which one is an outer layer known as protein crown and another one is the inner layer that is the core of the virus called genetic material [4]. The symptoms of this virus vary from the severe cold to lots greater extreme ailments like SARS and MERS [5]. Furthermore, to prevent the pandemic of coronavirus, until today there is no targeted vaccine or drug available (except Favipiravir, this is a drug used to treat mild symptoms of infected patients, which recently launched by Glenmark). Indeed, there is an urgent need to identify and develop effective medicine against COVID-19 to fight this deadly virus [6]. In silico based study is one of the most important and advanced for the invention of a new drug. To eliminate the high cost of clinical trials and heavy time consumption several bioinformatics methods are nowadays used. The Virtual screening of various compounds on the protein-related study is the most obligatory bioinformatics technique used for the implementation of new medicines design [7].

Since heterocyclic compounds are well known for its medicinal activities [8]. Among them, one of the significant choices is oxindole, in which the benzene ring is incorporated with a pyrrole ring, and on the second position carbonyl group is present, The IUPAC name of the oxindole nucleus is 1,3-dihydro-2H-indole-2-one. To add with this, oxindole derivatives represent plethora medicinal activities, various pharmacology reports show that the modified oxindole derivatives encompass different activities, for instance, antiviral, anticancer, antimicrobial, anti-inflammatory, analgesic, antioxidant, and antidiabetic activities due to these activities it attracts researchers for working on this compound [9]. Moreover, the synthesis procedure of oxindole derivatives is Eco-friendly as well as affordable, there is a surplus of green routes available to synthesis such compounds. Some merits that can be highlighted which varies from high yields to safe, cheap, and simple workup procedures [10]. Henceforth, 30 oxindole derivatives have been chosen for molecular docking through Molegro Virtual Docker version 7.0.0 Software (MVD). In this particular, these compounds run over a COVID-19 main protease (Mpro) which is a possible drug target macromolecule for the inhibition of COVID-19 replication. The protein which has been selected (PDB ID: 6LU7) is a key macromolecule for the chemical action of the drug molecule. Among all 30 oxindole derivatives, four oxindole derivatives exhibit moderate to excellent results via simulation. In addition to that comparative study with existing drugs has also been incorporated.

Materials and methods

Ligand preparation

SDF format (*sdf) had been chosen to save 3D structures of oxindole derivatives and drugs under clinical trials (obtained through pubchem), the conversion of 2D to 3D structures was done by Chembiodraw v. Ultra 12.0 software followed by energy minimization by using MM2 force field method in the same software, these procedures are essential before ligands import for docking process. 2D and 3D structures of all ligands are shown in Table 1. To get perfect results of docking study, it is significant that the imported structures must be well prepared like the atom connectivity and bond orders are right if, ligand file saved in PDB format usually have the bad or lacking undertaking of express hydrogens, and the PDB file structure would not adapt bond order information. Addition of H-atom and all essential valency checks done by the implemented algorithm in the MVD.

Table 1:

List of oxindole and its derivatives used for docking studies with COVID-19.

Ligand no.	Ligand name	2D structure	3D structure	Active against	Reference
OD-0	Oxindole			Antiviral, antifungal, antibacterial, antiproliferative, anticancer, anti-inflammatory, antihypertensive, and anticonvulsant activities.	[9b]
OD-1	Strychnofoline			inhibitory effects against melanoma and Ehrlich tumor cells	[14]
OD-2	Citrinadin A			anticancer	[15]
OD-3	4′-(4-bromophenyl)-5-methyl-3′-(3-methyl-4-nitroisoxazol-5-yl)-5′-phenylspiro[indoline-3,2′-pyrrolidin]-2-one			inhibitory activities against K562, A549, and PC-3 cancer cells	[16]
OD-4	4′-(3-bromophenyl)-5-methyl-3′-(3-methyl-4-nitroisoxazol-5-yl)-5′-phenylspiro[indoline-3,2′-pyrrolidin]-2-one			Anticancer	[16]
OD-5	5-methyl-3′-(3-methyl-4-nitroisoxazol-5-yl)-4′,5′-diphenylspiro[indoline-3,2′-pyrrolidin]-2-one			inhibitory activities against K562, A549, and PC-3 cancer cells	[16]
OD-6	1-benzyl-4′-(4,6-diethoxypyrimidin-5-yl)-5-fluoro-1′-methylspiro[indoline-3,3′-pyrrolidin]-2-one			anticancer activity	[17]
OD-7	1-tert-butyl 5′-ethyl 2′-methyl 2′-(2-methoxy-2-oxoethyl)-4′-(4-nitrophenyl)-2-oxospiro[indoline-3,3′-pyrrolidine]-1,2′,5′-tricarboxylate			cytotoxicities against the mouse breast cancer cells 4T1, colon cancer cells CT26, human liver cancer cells HepG2, and lung cancer cells A549	[18]
OD-8	2′-(1H-indole-3-carbonyl)-1′-(m-tolyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one			cytotoxic activity against HCT-116 cells	[19]
OD-9	1′-(3-bromophenyl)-2′-(1H-indole-3-carbonyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one			cytotoxic activity against HCT-116 cells	[19]
OD-10	2′-(1H-indole-3-carbonyl)-1′-(4-(trifluoromethyl)phenyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one			cytotoxic activity against HCT-116 cells	[19]
OD-11	1′-(2,4-dichlorophenyl)-2′-(1H-indole-3-carbonyl)-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one			cytotoxic activity against PC-3and HepG2	[20]
OD-12	6′-(1H-indole-3-carbonyl)-7′-(4-(trifluoromethyl)phenyl)-3′,6′,7′,7a′-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one			cytotoxic activity against liver and prostate cancer cells	[21]
OD-13	(E)-7′-(4-chlorophenyl)-6′-(3-(2,4-dichlorophenyl)acryloyl)-3′,6′,7′,7a′-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one			Anticancer HCT-116, HepG2, and PC-3 cells	[22]
OD-14	7′-(2,4-dichlorophenyl)-6′-(4-fluorobenzoyl)-3′,6′,7′,7a′-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one			Anticancer against Hela cell lines	[23]
OD-15	1′-benzyl-6-fluoro-5′-methyl-1,3,3a,3b,9a,10a-hexahydrospiro[chromeno[3′,2′:3,4]cyclopenta[1,2-b]pyrrole-10,3′-indoline]-2′,4(2H)-dione			cytotoxic effect against K562 cancer cells	[24]
OD-16	1′-(benzo[d][1,3]dioxol-5-ylmethyl)-4′-bromo-3-phenyl-7,8-dihydrospiro[furo[3,4-b]pyrazolo[4,3-e]pyridine-4,3′-indoline]-2′,5(1H)-dione			Anticancer	[25]
OD-17	1,2′-dibenzyl-5-fluoro-2′,3′,4′,9′-tetrahydrospiro[indoline-3,1′-pyrido[3,4-b]indol]-2-one			most active agent against cancer cell A549	[26]
OD-18	3′-benzoyl-5′-(furan-2-yl)-4′-phenylspiro[indoline-3,2′-pyrrolidin]-2-one			antibacterial activity	[27]
OD-19	3′-benzoyl-5′-(furan-2-yl)-4′-(p-tolyl)spiro[indoline-3,2′-pyrrolidin]-2-one			antifungal effect	[27]
OD-20	diethyl 1-benzyl-3′-ethoxy-2-oxospiro[indoline-3,4′-pyrazole]-1′,5′(5′H)-dicarboxylate			activities against Gibberella zeae and Pellicularia sasakii	[28]
OD-21	5′-benzyl-3′-(4-chlorophenyl)-4′-nitrospiro[indoline-3,2′-pyrrolidin]-2-one			the highest activity against Escherichia coli ATCC 10536	[29]
OD-22	2′-(1,3-diphenyl-1H-pyrazol-4-yl)-1′-(1H-indole-3-carbonyl)-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carbonitrile			antimicrobial activity	[30]
OD-23	Suitinib			Treatment of gastrointestinal stromal tumors, and metastatic renal cell cancer.	[31]
OD-24	arylsulfoanilide-oxindole			activity against lung cancer cells	[32]
OD-25	1′-benzyl-5′-bromo-2,3-dihydro-1H,1″H-[3,3′:3′,3″-terbenzo[b]pyrrol]-2′(1′H)-one			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]
OD-26	1′-benzyl-2,3-dihydro-1H,1″H-[3,3′:3′,3″-terbenzo[b]pyrrol]-2′(1′H)-one			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]
OD-27	3,3-Bis(2-methylindolyl)oxindole			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]
OD-28	1′-benzyl-2,2″-dimethyl-2,3-dihydro-1H,1″H-[3,3′:3′,3″-terbenzo[b]pyrrol]-2′(1′H)-one			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]
OD-29	1′-benzyl-5′-bromo-3,3″-dimethyl-[2,3′:3′,2″-terindolin]-2′-one			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]
OD-30	1′-benzyl-3,3″-dimethyl-5′-nitro-[2,3′:3′,2″-terindolin]-2′-one			Antibacterial, antiprotozoal, anti-inflammatory activities, and progesterone receptors (PR) agonists.	[10]

Cavity (or active sites) detection and selection

The Cavity detection algorithm is an inbuilt function of MVD v.7.0.0 software, which automatically detects the possible binding sites (cavities) the cavities found inside 30 × 30 × 30 Å³ cube, which uses for ligand binding. After cavities are recognized through the cavity detection algorithm, in this process the algorithm focus on pursuit the specific area or volume during the simulation process. Here, the program detected four different binding sites of COVID-19 protein structure (Figure 1). From these four anticipated cavities the one with the utmost volume (131.072 Å³), surface area (417.28 Å²) and coordinates values of highest volume (X = −10.6241, Y = 15.4211, and Z = 68.2623). Hence, this particular cavity had been considered for the ligand-binding process.

Figure 1:

MVD detects four cavities in the protein structure of COVID-19 (PDB ID: 6LU7), (cavity volume represents in Å), detected cavity representation: color with specifications, green: cavity, blue and red: cartoon model of COVID-19 Mpro.

Results

During the docking study, each ligand is selected as the best position to determine the MolDock score and Re-rank score. The MolDock score, Re-rank score, H-bonding and steric interaction between protein and ligand are represented in Table 2 (30 oxindole derivatives) [33]. Through literature it was found that compounds or ligands which exhibits lowest or minimal energy that can creates better binding with the protein [12]. Moreover, as the lowest energy needed for better binding so in this work, −150 kcal/mol energy or minimum has been set up as the lowest energy for 30 screened ligands and from those 30 compounds 4 has been explained in depth as those compounds have the very less binding energy than the set up value. Therefore, it can be assumed that Compounds having an energy score of −150 kcal/mol or minimal can be considered as a better inhibition agent in opposition to COVID-19.

All these 30 oxindole derivatives have binding energy less than −150 kcal/mol, but out of these 30 derivatives OD-22 ligand, OD-16 ligand, OD-4 ligand, and OD-9 ligand revealed the most lowest MolDock score which is −200.409 kcal/mol, −184.135 kcal/mol, −176.181 kcal/mol and −174.999 kcal/mol respectively, whereas oxindole(OD-0) revealed MolDock score −77.7729 kcal/mol. Moreover, the other 26 derivatives also exhibited better binding energy as compared to OD-0.

Interaction of core oxindole with protein has been shown in Figure 2(OD-0). While, Interactions of these four oxindole derivatives which are OD-22, OD-16, OD-4 and OD-9 shown in Figures 3–6, respectively. Each figure demonstrates the optimum score of the docking, Amino acids in the active site which are presented in wireframe with element color (where oxygen is red, carbon is gray, nitrogen is blue, hydrogen is white and sulfur is yellow) and the ligand is represented in Stick lines with fix color yellow. Hydrogen bonds illustrate between ligand and active site of Covid-19 Mpro by the blue lines.

Figure 2:

Oxindole ligand docked against the crystal protein structure of COVID-19 main protease (Mpro).

Figure 3:

OD-22 ligand docked against the crystal protein structure of COVID-19 main protease (Mpro).

Figure 4:

OD-16 ligand docked against the crystal protein structure of COVID-19 main protease (Mpro).

Figure 5:

OD-4 ligand docked against the crystal protein structure of COVID-19 main protease (Mpro).

Figure 6:

OD-9 ligand docked against the crystal protein structure of COVID-19 main protease (Mpro).

Ligand OD-22 has the lowest MolDock score, Re-rank score, and steric interaction energy in comparison with other ligands, with COVID-19 main protease. These four (OD-22, OD-16, OD-4, OD-9) ligands well fit in the active sites of the protein. Figure 7, illustrates hydrogen bonding with amino acids of coronavirus protein, Figure 8 shows, steric interaction with residues of COVID-19 protein, and Figure 9 represents the comparison of the binding of Oxindole, OD-22, OD-16, OD-4, and OD-9 ligands in the active sites of COVID-19 main protease(Mpro). It depicted clear binding of Oxindole(OD-0), OD-22, OD-16, OD-4, and OD-9 ligands. In this work, a comprehensive analysis of the ligand–protein interaction of OD-22, OD-16, OD-4, and OD-9 ligands are presented in Table 3 which includes protein–ligand interaction score, residues, and distance of interaction energy. Moreover, Table 4 elicits the steric interaction of the four oxindole derivatives with amino acids and atoms, furthermore, the interacting distance and strength also represented in the same table. Overall, these four derivatives show the highest steric interaction and H-bonding which provides better MolDock score and hence, among the 30 oxindole derivatives these four derivatives are the best. Along with this study, comparative study of oxindole derivatives against Drugs under clinical trials and some already given to the infected patients, the name of the drugs are favipiravir, hydroxychloroquine sulfate, lopinavir, and remdesivir have been done on the same protein of COVID-19 through MVD v.7.0.0 [34]. The docking results of these four drugs are illustrated in Table 5 which is a Moldock score and Tables 6 and 7 elicits hydrogen bonding and steric interactions respectively. These four drugs shows poor results in docking as compared to oxindole derivatives.

Figure 7:

(A–E): Hydrogen bond interaction with Covid-19 of oxindole (A), OD-22 ligand (B), OD-16 ligand (C), OD-4 ligand (D) and OD-9 ligand (E).

Figure 8(A–E):

Steric interaction with Covid-19 of oxindole (A), OD-22 ligand (B), OD-16 ligand (C), OD-4 ligand (D) and OD-9 ligand (E).

Figure 9:

(A–E): Binding of oxindole (A), OD-22 ligand (B), OD-16 ligand (C), OD-4 ligand (D) and OD-9 ligand (E) in the binding sites of COVID-19 main protease (Mpro) (PDB ID: 6LU7) with clear surfaces.

Table 5:

Moldock score and re-rank score (kcal/mol) for existing drugs docked against Covid-19 crystal structure.

Structure and name	Energy, kcal/mol or MolDock score	H-bond energy, kcal/mol	Re-rank score, kcal/mol	Steric interaction, kcal/mol
	−66.2854	−3.5692	−46.3477	−70.1253
	−97.2365	−1.47927	−72.0991	−115.542
	−121.56	−3.4934	−27.2975	−142.699
	−161.435	−3.08106	−93.7058	−169.918

Discussion

To begin with the depth explanation of each of these ligands with COVID-19 protein(PDB:6LU7). Amino acids Gln 189, Cys 145, and Phe 140 are strongly bound with OD-22 (Figure 3). Whereas, OD-16 tightly interacts with Ser 144, Cys 145, Gly 143, and Tyr 54 (Figure 4). Furthermore, H-bond formation has been seen with ‘–NH’ and Carbonitrile group of compound OD-22 which consists of amino acids Gln 189, Phe 140, and Cys 145 respectively. Apart from this, OD-16 produces H-bond with ‘–NH’ group and the amino acids are Ser 144, Cyr 145, and Gly 143. In the OD-16 one noticeable observation seen that is an amino acid, Tyr 54 interacts with ‘O’ atom (Figure 7B, C). Besides this, steric interactions with both of these ligands have been observed, in which interacting residues Met 49, Gln 189, Met 165, His 41, Phe 140, Asn 142, Glu 166, and Gly 143 for OD-22 ligand and Gly 143, Asn 142, Cys 145, His 163, Ser 144, His 164, Met 165, Asp 187, Tyr 54, Met 49, Gln 189 and His 41 for OD-16 ligand (Figure 8B, C). Here, OD-22 ligand has the lowest energy as compared to OD-16 due to less steric interactions and H-bonding in OD-16 ligand.

In addition to this, another two ligands OD-4 and OD-9 showcase satisfactory energies but not less than OD-22 and OD-16. Moreover, three H-bonding occurs between active residues Cys 145, Ser 144, and Glu 166 with ‘O’ atom present in OD-4 also, H-bond interaction seen with amino acids Glu 166 with ‘–NH’ group of the same ligand. While considering the H-bonding of OD-9, observed only two H-bond interaction with active residue Cyr 145 with ‘O’ atom and His 41 ‘–NH’ group of OD-9 (Figure 7D, E). To add with this, steric interactions of OD-4 with residues His 164, Gly 143, His 163, Glu 166, Asn 142, and Met 165 involved in the interaction and OD-9 exhibits steric interactions with active residues Asn 142, Glu 166, Cys 145, His 41, and Gly 143 (Figure 8D, E). The binding interactions of OD-4 and OD-9 have been illustrated in Figures 5 and 6, respectively.

However, the simulation study of these existing drugs through MVD on COVID-19 protein reveals the energy −66.2854 kcal/mol, −97.2365 kcal/mol, −121.56 kcal/mol, and −161.435 kcal/mol respectively (Table 5), these energies(Moldock scores) are very high as compared to energies received by four oxindole derivatives. Moreover, these drugs elicit very less H-bonding and steric interaction (Figure 11 and Tables 6 and 7) as compared to four oxindole derivatives this is because the interacting distance of the bond is high so, these drugs cannot bind tightly with protein as compared to four ligands of oxindole. Here, the favipiravir forms two H-bonds with residues Cys 145 and Ser 144 and only one amino acid Leu 141 shows steric interaction with protein (Figure 11A). Whereas, Hydroxychloroquine sulfate produces only one H-bond and two steric interactions with residues Arg 188 and Cys 145, Thr 26 respectively. Apart from this, Lopinavir and remdesivir both exhibit more H-bonding and steric interactions as compared to favipiravir and hydroxychloroquine sulfate but not more than OD-22, OD-16, OD-4 and, OD-9 ligands. Amino acids Glu 166, Cys 145, and Gln 189 are responsible to form H-bonding also residues Met 165, Phe 140, His 163 and, Met 49 are for steric interaction with lopinavir (Figure 11C). Furthermore, remdesivir forms three H-bond and three steric interactions with amino acids Asn 119, Cys 145, Ser 144 and, Gly 143, Thr 26, Asn 142 respectively (Figure 11D). To add with this, after analyzing the docking data of these drugs one common outcome observed that is amino acid ‘Cys 145’ of COVID-19 protein forms H-bond with ligands whether ligand is oxindole derivatives or existing drugs. Overall, the binding interaction pattern is represented in Figure 10. Indeed, as per the data received through the docking of these four drugs with COVID-19 protein, it can be claimed that oxindole derivatives would be a better option to treat COVID-19 patients. In addition to this, Table 8 illustrates the comparative data in terms of Moldock scores of recent existing drugs for coronavirus prevention and the proposed work.

Figure 10:

(A–D): Binding of Favipiravir (A), Hydroxychloroquine sulfate (B), Lopinavir (C) and Remdesivir (D) in the binding sites of COVID-19 main protease (Mpro) (PDB ID: 6LU7), in which amino acids in wireframe with element color, ligands in stick with element color, and cartoon model as backbone of protein.

Figure 11:

(A–D): Hydrogen bond interaction (blue line) and steric interaction (red line) with Covid-19 of Favipiravir (A), Hydroxychloroquine sulfate (B), Lopinavir (C) and Remdesivir (D).

Conclusions

All in all, it can be stated that the rapid spreading of coronavirus at every hook and corner of the world attracts every researcher for the invention that can use to cure this outbreak of novel coronavirus. Henceforth, this research related to molecular docking based on virtual screening was conducted to classify heterocyclic compounds (oxindole derivatives) having the potential to bind the main protease crystallized protein structure of COVID-19 (PDB ID: 6LU7). In addition to this, a comparative study has been conducted on existing drugs that are under clinical trial for the prevention of this threatening virus with oxindole derivatives. Based on the molecular docking study, results that obtained it can be highlighted that the oxindole derivatives able to interact with all the significant amino acids of COVID-19 protein structure. According to MolDock binding score oxindole derivatives bearing excellent binding potency against COVID-19 due to its pharmacology properties. In this presented work, among 30 compounds, four oxindole derivatives (OD-22, OD-16, OD-4, OD-9) exhibit the lowest binding energy (−200.409 kcal/mol, −184.135 kcal/mol, −176.181 kcal/mol, −174.999 kcal/mol), as well as compounds, fit well in the binding sites of COVID-19 Mpro crystallized protein structure and also interact with the residues in the active sites, which are essential for their biological activity. Apart from this, these derivatives are proven to be more efficient than existing drugs in terms of Molock scores and other parameters. Thus, oxindole derivatives might be an effective inhibitor of the main protease of COVID-19 also it would be used as an antiviral drug agent. Furthermore, in the upcoming years, studies should be conducted for the validation of these compounds using in vitro and in vivo models which will be useful for new drug inventory against coronavirus.