In silico Molecular Docking Analysis of some Terpenoids against 3CLpro of SARS-CoV-2

 

Kushagra Nagori¹, Madhulika Pradhan⁶, Kartik T. Nakhate⁴, Hemant R. Badwaik³,

Reena Deshmukh¹, Ayushmaan Roy¹, Rashnita Sharma², Shobhit P. Srivastava⁷,

Sonia Chawla⁸, Vishal Jain⁵, Mukesh Sharma¹*

¹Rungta College of Pharmaceutical Sciences and Research, Bhilai, 490024, India.

²Rungta Institute of Pharmaceutical Education and Research, Bhilai, Chhattisgarh, 490024, India.

³Shri Shankaracharya Institute of Pharmaceutical Sciences and Research,

Junwani, Bhilai, Chhattisgarh, 490020 India.

⁴Department of Pharmacology, Shri Vile Parle Kelavani Mandal’s Institute of Pharmacy,

Dhule, Maharashtra 424001, India.

⁵Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, 492010, India.

⁶Gracious College of Pharmacy, Abhanpur, Chhattisgarh, 493661, India.

⁷Dr. M. C. Saxena College of Pharmacy, Lucknow, Uttar Pradesh, 226101, India.

⁸Dev Bhoomi Uttrakhand University, 248001, Uttrakhand.

 

ABSTRACT:

The recent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised global health concerns. The main viral protease called 3-chymotrypsin-like cysteine protease (3CLpro) plays an important role in viral replication by polyproteins processing that are translated from viral RNA. Therefore, the present in silico docking study aimed to assess the inhibitory actions of various terpenoids against 3CLpro of SARS-CoV-2. Molecular docking was performed using ArgusLab 4.0.1. a computational docking program and the protein-ligand interaction was visualized by using Pymol 1.7 software. The inhibitory activity of terpenoids like abietic acid, ferruginol, rosmarinic acid, zingiberine, sugiol, kaempferol and betulinic acid was tested against 3CLpro (PDB ID: 6M2N) using molecular docking paradigm while antiviral drugs- remdesivir, darunavir and hydroxychlorquine- were used as standards for comparison. All phyto-constituents showed an effective binding interaction with 6M2N, and the binding affinity was ranged from –8.854 to –13.398 as compared to remdesivir, darunavir and hyroxychlorquine. Amongst tested compounds, abietic acid, ferruginol and betulinic acid exhibited promising enzyme interaction. Results indicate that based upon the binding energy of abietic acid, ferruginol and betulinic acid could be efficient SARS-CoV-2 3CLpro inhibitors. This is supported by the fact that the effects of some terpenoidal phytochemicals especially abietic acid, ferruginol and betulinic acid showed promising enzyme interaction as compared to remdesivir and darunavir. Therefore, further studies are warranted to confirm the effectiveness of abietic acid, ferruginol and betulinic acid for the therapy of COVID-19.

 

 

 

INTRODUCTION: 

The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) triggered coronavirus disease 2019 (COVID-19) is currently the rapidly spreading disease across the globe. The virus has been mainly emerged as human-to-human contact-transmitted pathogen. Moreover, it can also be spread through the air if respiratory droplets of infected person reach the mouth, nose or eyes of persons in close contact¹. In spite of significant recovery rate amongst patients, several thousands of deaths are occurring due to COVID-19. Various antiviral drugs and other supportive medications have been used for the treatment of COVID-19; however, the results are disappointing². Therefore, the need for better therapeutic strategies to fight with COVID-19 seems necessary.Terpenoids are the main constituents of secondary plants that contain many medicinal properties including antiviral potential^3,4. Some well-established terpenoids with antiviral activity include, abietic acid⁵, limonene⁶, kaempferol⁷, zingiberine⁸, sugiol⁹, isorhamnetin¹⁰, alpha cadinol¹¹, alpha pinene¹², linalool¹³, rosmarinic acid¹⁴, ferruginol¹⁵ and betulinic acid¹⁶. Recent studies have shown that SARS-CoV-2 genome sequence is much similar to that of SARS-CoV. In SARS-CoV, the main viral protease called 3-chymotrypsin-like cysteine protease (3CL^pro) plays an important role in viral replication by polyproteins processing that are translated from viral RNA. The 3CL^pro served as a proven target for the drug discovery against SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV)^17,18. In this background, the aim of the present study was to apply insilico screening methodology for evaluating antiviral activity against SARS-CoV-2of some terpenoidal phytoconstituents (abietic acid, ferruginol, rosmarinic acid, zingiberine, sugiol, kaempferol and betulinic acid) using 3CL^pro as a target protein. Drugs like remdesivir, darunavir and hydroxychloroquine were used to compare the antiviral activity of terpenoids. These drugs have recently been shown inhibitory effect on MERS-CoV, ebola virus RNA-dependent RNA polymerase and SARS-CoV-2 3CL^pro19,20.

 

METHODS:

Preparation of ligands and receptor:

The 2D structures of terpenoidal ligands like abietic acid, ferruginol, rosmarinic acid, zingiberine, sugiol, betulinic acid and kaempferol and the drugs like remdesivir, darunavir and hydroxychlorquine was constructed using Chem Draw Ultra 12. The ligands 2D structures were then converted to 3D structures with molecular mechanic optimising by Chem3D Pro12. The structure of SARS-CoV-2 3CL protease (3CL pro) (PDB ID: 6M2N) was retrieved from the Protein Data Bank (RCSB)²¹.

 

Docking studies:

Molecular docking is performed to obtain a population of possible orientations and conformations for the ligand at the binding site. The docking was performed using ArgusLab 4.0.1. The grid center for docking was set X= 85, Y= 85 and Z= 80A^o. After validation of the docking protocol, virtual screening was conducted by flexible molecular docking into the active site of proteins. Finally, the result of binding energy was extracted from the software. The protein-ligand interaction was visualized by using Pymol 1.7 software²¹.

 

RESULTS AND DISCUSSION:

Molecular docking is widely used in order to explore the various types of binding interaction of the prospective drug with the individual areas or active sites of the target in compatible drug design, in particular to identify the lead compound²¹. The binding effectiveness of a ligand molecule and the active sites of a target were widely explained by the evaluation of their hydrogen binding pattern, and the nature of the residues that are present at the active site among all different kinds of interactions. The most affinity ligand can be selected as the possible medication for further studies. SARS-CoV-2 3CL^pro plays a key role in the replications of virus particles, and is situated at the 3"end, which is excessive in variability, unlike structural/accessory protein-encoding genes. Consequently, it is a potential target for screening anti-coronaviruses drugs.

 

In this regard, we have selected potential phytoconstituents with previously reported antiviral activity and drugs under clinical trial for carrying out the docking studies with the enzyme SARS-CoV-2 3CLpro (PDB ID: 6M2N).Among the docking studies performed on terpenoidal phytoconstituents, all the analogues had effective binding interactions with 6M2N. Binding affinity range from -8.854 to -13.398.From the results it reveals that phytoconstituents with highest docking binding affinity were betulinic acid, abietic acid, ferruginol and rosmarinic acid with binding affinity values -13.398, -11.565, -11.439 and -11.04 (kcal/mol) respectively. Whereas, zingiberine and sugiol with binding affinity values -10.7696 and-10.6559 (kcal/mol) respectively, show moderate binding affinity against the target protein. Remaining analogues showed lower binding affinity towards SARS-CoV-2 3CL^pro. The number of hydrogen bond and the number of amino acid residues of SARS-CoV-2 3CL^prointeracting with each phyto-constituents are given in Table 1.While2 D structure and 3D model of selected phyto-constituents and drugs (remdesivir, hydroxychlorquine, darunavir) bonded with 6M2N (SARS-CoV-2 3CL^pro) are tabulated in Table 2.

 

In case of standard drugs, remdesivir shown highest affinity towards the SARS-CoV-2 3CL^pro. It was found that remdesivir interact with chain A of SARS-CoV-2 3CL^pro, where 105 ARG, 151 ASN, 203 ASN, 153 ASP, 245 ASP, 295 ASP, 160 CYS, 110 GLN, 109 GLY, 246 HIS, 106 ILE, 152 ILE, 200 ILE, 249 ILE, 8 PHE, 294 PHE, 108 PRO, 293 PRO, 111 THR, 292 THR, 104 VAL, 202 VAL were the major amino acids involved  in  hydrogen  bonding,  hydrophobic  and  van  der  Waals  interaction (as shown in Fig.1). Remdesivir form six hydrogen bonds with amino acids (111 THR, 110 GLN, 110 GLN, 292 THR, 294 PHE, and 293 PRO) of receptors (Table 1).

 

While, darunavir bind with 11 amino acid residue of the chain A (151 ASN, 153 ASP, 295 ASP, 294 PHE, 293 PRO, 297 VAL, 298 ARG, 300 CYS, 302 GLY, 8 PHE, 301 SER)  and 4 amino acid residue of chain D (991 GLN, 981 HIS, 984 LEU, and 983 PHE) of receptor (Table1 and Fig.2).  It has been found that 5 amino acid binding residue is common (151 ASN, 153 ASP, 295 ASP, 294 PHE, and 293 PRO) with that of remdesivir.

 

Hydroxychlorquine bind with the SARS-CoV-2 3CLpro receptor in totally different manner to that of remdesivir and darunavir. Hydroxychlorquine shown to interact with 16 amino acids (457 ASN, 459 ASP, 601, ASP, 416 GLN, 458 ILE, 408 LYS, 314 PHE, 600 PHE, 417 THR, 411 ARG, 604 ARG, 413 GLN, 433 GLN, 412 ILE and 464 SER) of chain B (Fig.3). 

 

When we compared the docking studies of phytoconstituents with remdesivir, it was found that abietic acid and ferruginol shows highest similarities in binding pattern with the receptor. Abietic acid and ferruginol has shown 4 common amino acid binding residues (295 ASP, 249 ILE, 294 PHE, and 293 PRO) to that of remdesivir binding site (Table 1, Figs.4 and 5). Abietic acid and ferruginol also shows binding similarity with Darunavir. Where abietic acid shown 6 common binding residues with darunavir out of which 5 amino acid (298 ARG, 297 VAL, 295 ASP, 294 PHE and 293 PRO) of chain A and one amino acid (991 GLN) of chain D.

 

In case of ferruginol, 4 amino acids (298 ARG, 295 ASP, 294 PHE and 293 PRO) of chain A and one amino acid (984 LEU) is common with Darunavir binding residue. Betulinic acid shown highest binding affinity (-13.398 kcal/mol) with receptor with 7 amino acid binding residue common with darunavir [5 amino acid (298 ARG, 297 VAL, 294 PHE, 293 PRO, and 249 ILE) of chain A and 2 amino acid (991 GLN, 984 LEU) of chain D] (Fig. 6).

 

Kaempferol shown similarity in binding with receptor as that of hydroxychlorquine, where it binds to chain B of SARS-CoV-2 3CLpro.  Eight amino acid binding residue i.e. 457 ASN, 459 ASP, 601 ASP, 416 GLN, 458 ILE, 314 PHE, 600 PHE, and 417 THR are found similar (Fig.7) to hydroxychlorquine.

 

 

Fig. 1. Docking of remdesivir in to SARS-CoV-2 3CLpro. 

 

 

Fig. 2. Docking of darunavir into SARS-CoV-2 3CLpro.

 

 

Fig. 3. Docking of hydroxychlorquine in to SARS-CoV-2 3CLpro

 

 

Fig. 4. Docking of abetic acid in to SARS-CoV-2 3CLpro.

 

 

Fig. 5. Docking of ferruginol in to. SARS-CoV-2 3CLpro

 

Fig. 6. Docking of betulinic acid in to SARS-CoV-2 3CLpro

 

Table 1: Docking results of ligands against SARS-CoV-2 3CL^pro (6M2N).

Compound Name	Binding Energy (kcal/mol)	No. of Hydrogen Bonds	Bond Length of H-Bonds in Å	H-Bond with Receptor residue	Enzyme's Binding Site residue
					Chain A	Chain B	Chain C	Chain D
Abietic acid	-11.565	4	2.569, 2.505, 2.684, 2.903	991 GLN, 343 TYR, 394 LYS, 409 PHE	298 ARG (Alpha Helix), 295 ASP (Alpha helix), 295 ASP (Alpha helix), 249 ILE (Coil) 253 LEU (Alpha helix), 294 PHE (Alpha helix), 252 PRO (Alpha helix) 293 PRO (Alpha helix), 297 VAL (Alpha helix)	340 ASP (coil), 389 GLN (Beta strand), 484 GLU (Coil), 338 LEU (Coil), 394 LYS (Beta strand), 396 LYS (Beta strand),  408 LYS (Beta strand), 408 PHE (Beta strand), 343 TYR (Beta strand), 407 TYR (Beta strand), 341 VAL (Beta strand)	Nil	986 GLN (Beta strand), 991 GLN (Beta strand), 984 LEU (Beta strand)
Kaempferol	-9.39	4	2.42, 2.902, 2.347, 2.999	458 ILE, 601 ASP, 598 THR, 417 THR	Nil	457 ASN (Beta strand), 459 ASP (Beta strand), 601 ASP (Alpha helix), 416 GLN (Beta strand),  415 GLY (Coil), 458 ILE (beta strand),  314 PHE (Beta strand), 600 PHE (Alpha helix), 417 THR (Bata Strand), 598 THR (Coil)	Nil	Nil
Rosmarinic acid	-11.04	8	2.463,2.521,2.865, 2.805,2.847,2.901,2.899,2.489	3PHP, 282 LEU, 207 TRP, 4 ARG, 4 ARG, 617 LYS, 739 GLN, 616 ARG	4 ARG (Beta strand), 288 GLU (Coil), 282 LEU (Coil), 5 LYS (Beta strand), 3 PHE (Beta strand), 291 PHE (Coil), 207 TRP (Alpha helix)	Nil	616 ARG (Beta strand), 739 GLN (Beta strand), 617 LYS (Beta strand), 738 TYR (Beta strand)	Nil
Betulinic acid	-13.398	1	2.537	991GLN	298 ARG (Alpha Helix), 249 ILE (Coil), 250 LEU (Coil), 294 PHE (Alpha helix), 250 PRO (Alpha helix), 293 PRO (Alpha helix), 297 VAL (Alpha helix)	Nil	Nil	986 GLN (Beta strand), 991 GLN (Beta strand), 984 LEU (Beta strand)
Ferruginol	-11.439	0			298 ARG (Alpha Helix), 295 ASP (Alpha helix), 249 ILE (Coil), 294 PHE (Alpha helix), 252 PRO (Alpha helix), 293 PRO (Alpha helix), 297 VAL (Alpha helix)	Nil	Nil	984 LEU (Beta strand)
Zingiberine	-10.7696	0			NIL	NIL	NIL	1215 ARG (alpha helix), 1070 ASP (beta strand), 1212 ASP (alpha helix),925 PHE (beta strand), 1068 ASN (beta strand), 1069 ILE beta strand), 1211 PHE (alpha helix), 1222 PHE (coil), 1221 THR (coil), 1071 TYR (coil), 1214 VAL (alpha helix), 1220 VAL (coil)
Sugiol	-10.6559	2	2.899915, 2.679172	407 TYR, 343 TYR	NIL	389 GLN(beta strand), 484 GLU (coil), 483 LEU (coil), 394 LYS (beta strand), 408 LYS (beta strand), 409 PHE (beta strand), 343 TYR (beta strand), 407 TYR (Beta strand), 392 VAL (beta strand)	NIL	NIL
Remdesivir	-10.3449	6	2.073, 2.637, 1.829, 2.153, 2.03, 2.996	111 THR, 110 GLN, 110 GLN, 292 THR, 294 PHE, 293 PRO	105 ARG (Beta Strand), 151 ASN (Beta Strand), 203 ASN (Alpha helix), 153 ASP (Beta Strand), 245 ASP (Alpha helix), 295 ASP (Alpha helix), 160 CYS (Coil), 110 GLN (Beta Strand), 109 GLY (Coil), 246 HIS (Alpha helix), 106 ILE (Beta strand), 152 ILE (Beta strand), 200 ILE (Beta strand), 249 ILE (Coil), 8 PHE (Beta strand), 294 PHE (Alpha helix), 108 PRO (Coil), 293 PRO (Alpha helix), 111 THR (Beta strand), 292 THR (Coil), 104 VAL (Beta strand), 202 VAL(Coil)	Nil	Nil	Nil
Darunavir	-10.621	1	2.632     2.747, 2.887	302 GLY	298 ARG (Alpha Helix),  151 ASN (Beta Strand), 153 ASP (Beta Strand), 295 ASP (Alpha helix), 300 CYS (Coil), 302 GLY (Coil), 8 PHE (Beta strand), 294 PHE (Alpha helix), 293 PRO (Alpha helix), 301 SER (Coil), 297 VAL (Alpha helix)	Nil	Nil	991 GLN (Beta strand), 981 HIS (Coil), 984 LEU (Beta strand), 983 PHE(Beta strand)
Hydroxychlorquine	-8.688 2			604 ARG, 411 ARG	NIL	411 ARG (beta strand), 604 ARG (alpha helix),457 ASN (Beta strand),459 ASP (Beta strand),601 ASP (Alpha helix), 413 GLN (beta strand),416 GLN (beta strand),433 GLN (beta strand),412 ILE (beta strand),458 ILE (beta strand), 408 LYS (Beta strand),314 PHE (Beta strand),600 PHE (Alpha helix),464 SER (beta strand),417 THR (Bata Strand), 410 VAL (beta strand)	NIL	NIL

 

Table 2: 2D structure and 3D model of abietic acid, kaempferol, betulinic acid, ferruginol, remdesivir, hydroxychlorquine, darunavir bonded with 6M2N (SARS-CoV-2 3CL protease).

Compound name and its 2D structure	Cartoon representation of binding pocket	Orientation of molecule with solid surface binding pocket representation
Abietic acid
Betulinic acid
Ferruginol
Remdesivir
Hydroxychlorquine
Darunavir
6M2N Receptor

 

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Accepted on 01.09.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4791-4798.