In silico Investigation of Natural compounds identified from Ocimum species as Dengue NS3 and NS5 Protein Inhibitors

Jeyabaskar Suganya¹, G. Rajesh Kumar², Mahendran Radha³*, Dhananya S⁴

¹Assistant Professor, Department of Bioinformatics, School of Life Sciences,

VISTAS, Chennai - 600117, Tamil Nadu, India.

²Assistant Professor, Department of Pharmacology, Govt. Kilpauk Medical College,

Chennai, Tamil  Nadu, India.

³Professor, Department of Bioinformatics, School of Life Sciences,

VISTAS, Chennai - 600117, Tamil Nadu, India.

⁴Student, Department of Bioinformatics, School of Life Sciences,

VISTAS, Chennai - 600117, Tamil Nadu, India.

*Corresponding Author E-mail: mahenradha@gmail.com, hodbioinfo@velsuniv.ac.in

ABSTRACT:

Nature has bestowed on us a very rich botanical wealth. Plants are the richest resource of drugs molecules for modern medicines, nutraceuticals, traditional medicine and even chemical entities for synthetic drugs. Ocimum species (Tulasi) is a well-known medicinal plant which has used in the six Indian systems of medicine from ancient times. The latest review on the Ocimum species revealed that the species holds a very good antiviral activity. For last few years the prevalence of dengue has increased dramatically in both urban and rural area of India. The current management of Dengue is mainly symptomatic and supportive treatment with fluid management. The main purpose of this study was to analyze the inhibitory action of phytochemical compounds were identified from the plant species Ocimum through literature survey by computational docking studies. Thus docking result revealed that only one compound Bornyl acetate exhibited the best binding interaction with in the active site of the dengue viral protein through hydrogen bonding interaction. Further in vitro studies on Bornyl acetate compounds from the Ocimum species can lead to the discovery of novel viral inhibitor.

KEYWORDS: Ocimum species, Phytochemical Compounds, Virtual Screening, Molecular Docking.

1. INTRODUCTION:

Dengue fever is one of the mosquito-borne infections caused by the dengue virus, which is most common in both tropical and subtropical countries¹.  In the past few decades, the rate of dengue cases was increasing drastically². In India, more than 67 thousand dengue cases were reported last year, out of which 40% of the cases were reported to death³.

Dengue virus is the member of Flaviviridae family, having four sero types of viruses (DENV1, DENV2, DENV3, and DENV4)⁴. Their viral genome is transformed into three structural proteins (Capsid, Pre-Membrane and Envelope) and seven non-structural (NS) proteins (NS1, NS2, NS3, NS4 and NS5)^5,6. The latest report on Dengue stated that the most of the cases were caused by NS3 and NS5 proteins, which have now become as the major targets for designing of antiviral drug molecules⁷. The report revealed that the NS3 helicase, NS3 proteases and RNA polymerase of NS5 are the vital proteins in dengue virus for causing dengue in humans, for past 4 years various researchs were carried out to discover the new antiviral inhibitors for the above viral proteins.NS3 directly interacts with human (GAPDH) GlycerAldehyde-3-Phosphate Dehydrogenase which reduces the glycolytic activity and also initiate the viral replication process⁸. NS5 replicate the viral genome which in turn down-regulate the human immune interferon response through signal transducer and transcription process⁹.

Despite the numerous clinical researches was carried out for identification of novel anti-dengue drug, till now no clinically approved significant dengue drugs were available for the treatment of the Dengue^10-12. Therefore, there was an urgent need in discovery of new antiviral drug molecules^13,14. Many researches on medicinal plant, reported that the plant might possess a new antiviral phytochemical which can be present in the various parts of the plant^15-19. These phytochemicals bear very high pharmacological properties and when they induced in human also, they cause less adverse effects^20-23.

From the Literature Survey, it was revealed that the Ocimum species belonging family Labiatae possesses various pharmacological properties like antiviral activity, analgesic activity, anti-ulcer activity, antiarthritic activity, immunomodulatory activity, antiasthmatic activity, antifertility activity, anticancer activity, anticonvulsant activity, antidiabetic activity, antihyperlipidemic activity, anti-inflammatory activity, antioxidant activity, antistress activity^24-30.

In review on Ocimum species, it was reported that there was 6 different species of Ocimum endures high viral properties and these are the 6 species with its Tamil name (Ocimum sanctum or Ocimum tenuiflorum- Thulasi, Ocimum basilicum –Thiruneetru Pachai, Ocimum canum - Kukka-tulasi, Ocimum gratissimum - Teruntulasi, Ocimum killimandscharium, Ocimum lamiifolium)^31-33.

The aim of the current study is to identify the phytochemicals from 6 different species of Ocimum, which might act as dengue inhibitors for the NS3 and NS5 proteins through Computer-aided drug design^34-37.

MATERIALS AND METHODS:

Small molecules retrieval:

Through literature survey, 111 phytochemical compounds³ were identified from the plant species Ocimum (Tulasi) were shown in the Table 1. Using PubChem database (https://pubchem.ncbi.nlm.nih.gov/), the 2 dimensional structures was retrieved for 111 compounds and converted to .mol file format using Pymol software for the screening process.

Table 1: The Phyotochemical compounds of Ocimum species

SPECIES: Ocimumbasilicum			SPECIES: Ocimum killimandscharium		SPECIES: Ocimum sanctum(or) OOcimumtenuiflorum
Linoleic acid Isoquercitrin Linolenic acid Oleic acid Rutin Ursolic acid Linalool Geraniol Methyl cinnamate Methyl eugenol Neral Beta-sitosterol Caffeic acid Salvigenin	Apigenin Acacetin Genkwanin Ladanein Quercetin Palmitic acid P-coumaric acid Rosmarinic acid Stearic acid Nevadensin Vicenin-2 Eriodictyol Xanthomicrol Cirsiliol		Bicyclogermacrene Delta cadinene CubenolCubebol Beta-bourbonene Globulol Beta-elemene Alpha-gurjunene Spathulenol Caryophyllene oxide Beta-cubebene Delta-cadinene Beta-copaene (E)-beta-farnesene Alpha-Capaene Alpha Cadinol P-Cymen-8-ol 3-Octanol 1-Octen-3-ol Germacrene B Linolenic acid	Myrcene Limonene Terpinen-4-ol Alpha-terpineol Alpha-mumulene Borneol Isoborneol Alpha-campholenal Myrtenol Carvestrene Alpha-thujene Tricyclene Beta-terpineol Terpinolene Trans-sabinene hydrate (Z)Beta-ocimene Alpha-terpinene	Eugenol Luteolin-7-O-glucoside Carvacrol Cirismaritin Luteolin Isothymusin Apigenin-7-O-glucoronide Orientin Vicenin Molludistin Bornyl acetate Camphene	Camphesterol Cholesterol Stigmasterol Estragole Camphor Tannins Triterpene Oleanolic acid Gallic acid Protocatechuis acid Vanillic acid Vanillin 4-Hydroxybenzaldehyde Chlorogenic acid
SPECIES: Ocimum gratissimum
Arachidonic acid Thymol P-cymene Gamma-phellandrene Beta-phellandrene Dipentene Cis-beta-ocimene		Beta-caryophyllene Gamma-mucerolene Alpha-farnesene (E)-beta-ocimene Beta-pinene Germacrene D			SPECIES : Ocimum canum	SPECIES : Ocimum lamiifolium
					Geranial Alpha-pinene Alpha-phellandrene	Sabinene Cirsilineol Eupatorin

Screening for Drug Likeness of Small molecules:

Further 111phytochemical compounds were screened for its pharmacological properties using QED³⁸, VEGA^39,40 and Molinsipration database^41,42. The Quantitative Estimate of Drug-Likeness (QED) tool was performed for predicting the compound can be used for oral drug. VEGA tool predicts the carcinogenicity, mutagenicity, toxicity of the compounds which satisfies the oral drug properties of the drug for the compounds which pass the VEGA tool.

Retrieval of protein structures and its active Sites:

The protein NS3 protease-helicase and NS5 RNA dependent RNA polymerase were found to be most important viral protein in causing dengue fever. The three dimensional structure of theNS3 protein and NS5 protein was retrieved using Protein Data Bank (PDB) database (http://www.rcsb.org/pdb/)^43-45. The Active sites for the binding of small molecules present in the protein were identified using a MetaPocket database (projects.biotec.tu-dresden.de/metapocket/)⁴⁶. The server identifies the ligand binding sites on the surface of the protein, which is essential for NS3 and NS5 inhibitor to bind to their respective dengue targets.

In silico Molecular docking studies:

Docking  studies  were  performed  to  analyse  the  structural  relationshipbetween  NS3 (2VBC) and NS5 (2J7W) Protein and 5 compounds of Ocimum species which clears all the screening tests using Autodock 4.2.6⁴⁷. AutoDock 4.2.6 is docking software which predicts how small molecules (drug candidates) bind to a known 3D structure of the receptor (protein). The NS3 and NS5 protein were loaded and its active sites were selected for the docking process. Finally the 5 small molecules of Ocimum species were loaded in the auto dock software. Docking calculation was allowed to run using shape-based search algorithm and AScore scoring function. The scoring function is responsible for evaluating the energy between the ligand and the protein target. The best docking model was selected according to the lowest AScore calculated by Autodock4.2.6⁴⁷. The most suitable binding interaction was selected on the basis of hydrogen bond interactions between the small molecules and protein near the substrate binding site. The best docking result was analysed using PyMOL^48,49 which is an open source molecular visualization tool to view the hydrogen bond interactions between the protein and ligand.  The bonding between the ligands and the protein can be clearly viewed for predicting the distance of hydrogen formation. The predicted distance reveals that binding interaction was stable one and small molecule could inhibit the function of the protein.

RESULTS AND DISCUSSION:

Preparation of Small Molecules:

The 111 phytochemical compounds retrieved from the plant species Ocimum were screened for its pharmacological properties. Using QED (Quantitative Estimation of Drug Likeness) database predicted that out of 111 compounds only 29 compounds satisfies the drug-like properties and the value of polar surface area (PSA) for the entire compound shows positive, which implies that the predicted compounds could be easily absorbed by the targets proteins. These 29 compounds were screened for its carcinogenicity, mutagenicity, toxicity properties using VEGA tool and the tool predicted only the 6 compounds satisfies all the three properties. Finally, 6 compounds were analyzed for its Bioactive properties, the result of the database revealed that 5 compounds clears the bioactivity properties (Table 2). The 5 compounds which satisfy all the pharmacological properties were further carried out for the docking studies⁵⁰.

Table 2: Compounds satisfies the druglikness properties from various tools

Drug like compounds that predicted using QED
Compounds		Pubchem ID	LogP	Mol.Wt	PSA	HbA	HbD		Prediction
SPECIES : Ocimum basilicum
Linalool		6549	2.347	154.249	20.23	1	1		Drug like
Geraniol		637566	2.347	154.249	20.23	1	1		Drug like
Methy eugenol		7127	2.995	178.228	18.46	2	0		Drug like
p-coumaric acid		637542	1.220	164.158	57.53	3	2		Drug like
Nevadensin		160921	3.125	344.315	98.36	7	2		Drug like
Xanthomicrol		73207	3.125	344.315	98.36	7	2		Drug like
Salvigenin		161271	3.602	328.316	78.13	6	1		Drug like
Cirsiliol		160237	2.592	330.289	109.3	7	3		Drug like
Apigenin		5280443	2.518	270.237	90.90	5	3		Drug like
Acacetin		5280442	3.033	284.263	79.90	5	2		Drug like
Genkwanin		5281617	3.033	284.263	79.90	5	2		Drug like
Ladanein		3084066	3.071	314.289	89.13	6	2		Drug like
SPECIES : Ocimum sanctum (or) Ocimum tenuiflorum
Eugenol		3314	2.480	164.201	29.46	2	1		Drug like
Carvacrol		10364	2.786	150.218	20.23	1	1		Drug like
Cirsimaritin		188323	3.071	314.289	89.13	6	2		Drug like
Isothymusin		630253	2.592	330.289	109.3	7	3		Drug like
Bornyl acetate		6448	2.663	196.286	26.30	2	0		Drug like
Vanillic acid		8468	0.734	168.147	66.76	4	2		Drug like
Vanillin		1183	1.492	152.147	46.53	3	1		Drug like
SPECIES : Ocimum gratissimum
Thymol		6989	2.786	150.218	20.23	1	1		Drug like
SPECIES : Ocimum killimandscharium
Cubenol		11770062	3.222	222.366	20.23	1	1		Drug like
Cubebol		11276107	3.222	222.366	20.23	1	1		Drug like
Globulol		12304985	3.022	222.366	20.23	1	1		Drug like
Spathulenol		92231	3.274	220.350	20.23	1	1		Drug like
Alpha Cadinol		10398656	3.222	222.366	20.23	1	1		Drug like
P-Cymen-8-ol		14529	2.500	150.218	20.23	1	1		Drug like
Trans-sabinene hydrate		12315151	2.438	154.249	20.23	1	1		Drug like
SPECIES : Ocimumlamiifolium
Cirsilineol		162464	3.125	344.315	98.36	7	2		Drug like
Eupatorin		9724	3.125	344.315	98.36	7	2		Drug like
Compounds that predicted VEGA
Compounds		Mutagenicity Model		Carcinogenicity Model		Toxicity Model			Skin Sensitivity Model
SPECIES : Ocimum basilicum
Linalool		NM		NC		NT			S
Geraniol		NM		NC		NT			S
Methy eugenol		NM		NC		NT			S
SPECIES : Ocimum sanctum (or) Ocimum tenuiflorum
Eugenol		NM		NC		NT			S
Bornyl acetate		NM		NC		NT			S
Vanilliic acid		NM		NC		NT			S
Compounds that predicted Bioactivity
Compounds	GPCR		ICM	KI	NRL	PI		EI
SPECIES : Ocimum basilicum
Linalool	-0.73		0.07	-1.26	-0.06	-0.94		0.07
Methy eugenol	-0.81		-0.38	-1.06	-0.80	-1.14		-0.43
SPECIES : Ocimum sanctum(or) Ocimum tenuiflorum
Eugenol	-0.86		-0.36	-1.14	-0.78	-1.29		-0.41
Bornyl acetate	-0.32		-0.33	-1.33	-0.59	-0.44		-0.12
Vanillic acid	-0.85		-0.42	-0.99	-0.61	-1.12		-0.35
Note:Mol.Wt – Molecular weight; PSA -Polar surface area; HbA – Hydrogen bond acceptor; HbD - hydrogen bond donar; NM – Non Mutagenicity; NC –NonCarcinogenicity;  NT –NonToxicity; S - Skin Sensitivity; GPCR - G protein-coupled receptors; ICM-Ion channel modulator; KI -Kinase inhibitor; NRL-Nuclear receptorligand; PI-Protease inhibitor; EI-Enzyme inhibitor

Preparation of Protein:

The three dimensional crystal structure of the NS3 protease-helicase from Dengue virus were retrieved from the Protein Data Bank with PDB ID: 2VBC (Chain A) determined by X-Ray crystallography at a resolution of 3.15 (Å) with 618 amino acids. The three dimensional crystal structure of the NS5 RNA dependent RNA polymerase of dengue virus were retrieved from the Protein Data Bank with PDB ID: 2J7W (Chain A) determined by X-Ray crystallography at a resolution of 2.6 Å (Å) with 635 amino acids. The active sites of the NS3 and NS5 proteins for the binding of small molecules were identified using Metapocket along with its secondary structure (Table 3).

Docking Interaction:

Molecular docking^51-53 was carried out for 5 compounds (Linalool, Methyl Eugenol, Eugenol, Bornyl Acetate, Vanillic Acid) of species Ocimum and the proteins of NS3(2VBC) and NS5 ( 2J7W) using AutoDock 4.2.6  software^54-57. The predicted active residues of the NS3 and NS5 proteins were taken as the catalytic sites for above 5 compounds. All the five compounds docked with the proteins 2VBC and 2J7W exhibited the good binding interactions between them (Table 4).

Table3: Active sites for the proteins NS3 and NS5 along with their Secondary structure

Position of amino acid along with its secondary structure
NS3 (2VBC)		NS5 (2J7W)
THR450  – Alpha Helix ARG 463 – Alpha Helix LEU479  – Extended strand LYS 480  – Extended strand ASN481 – Random coil ASP482  – Random coil	CYS 428  – Random coil LYS430  – Random coil ILE447 – Random coil PRO448  – Random coil VAL449  – Extended strand	ARG737 – Random coil GLN742 – Extended strand TYR 758  – Extended strand THR793  – Extended strand THR794 – Random coil TRP795  – Random coil	SER796  – Random coil LEU511  – Random coil ASN 533 – Alpha Helix LYS 689 – Alpha Helix SER710  – Random coil ARG729  – Extended strand

Table4: Docking Interactions between the natural compounds and NS3, NS5 Proteins

Compounds	NS3(2VBC)kcal/Mol	NS5(2J7W)kcal/Mol
Linalool	-6.01	-6.12
Methyl Eugenol	-5.49	-5.32
Eugenol	-5.49	-5.35
Bornyl Acetate	-6.98	-6.66
Vanillic Acid	-4.40	-3.47

Table 5: Docking interactions between NS3  and NS5 protein with Bornyl Acetate

The docking result revealed that only one compound (Bornyl acetate) possesses the least binding interaction of -6.98 kcal/mol for NS3 protein and -6.66 kcal/mol for NS5 protein with good hydrogen bond conformation. On further analysing the bonding conformation through PyMol, it was clearly revealed that the Bornyl acetate bounded to the respective proteins by forming 2 hydrogen bonds interaction. The binding interactions are as follows: The OH atom present in the Arginine 463 formed 2 hydrogen bond linkages with bond length of 2.1 Å, 2.4 Å in NS3 protein and the OH atom present in the Asparagine 533, Lysine 689 formed 1 hydrogen bond linkage with bond length of 2.4 Å and 2.25 Å in NS 5 Protein (Table 5).

CONCLUSION:

Docking studies play a vital role in the designing and development of rational drugs. In this work, the secondary metabolites of Ocimum sanctum are the potential leads to progress as novel antiviral drugs. From the above virtual screening and docking results, it was revealed that out of 111 phytochemicals present in the plant Ocimum species, only one compounds Bornyl acetate exhibited best viral inhibitory activity. The previous report on the compound Bornyl acetate, revealed that the compound has been used for treating inflammatory, analgesic, sedative and also used as an antibiotic drug. The current work strongly recommends the compound Bornyl acetate from the Ocimum sanctum for further in vitro and in vivo studies to explore the functions and molecular mechanisms of the compound toward the dengue viral proteins, which may lead to the development of the bioactive compound as a potential antiviral drug.

CONFLICT OF INTEREST:

The authors declare they have no competing interests.

ACKNOWLEDGEMENT:

We acknowledge Vels Institute of Science, Technology and Advanced Studies (VISTAS) for providing us with required infrastructure and support system needed.

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Received on 04.09.2020            Modified on 29.12.2020

Accepted on 17.02.2021           © RJPT All Right Reserved

Research J. Pharm.and Tech 2021; 14(12):6621-6626.

DOI: 10.52711/0974-360X.2021.01144