Available online on 15.11.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                    Research Article

Potential Inhibitor of DENV-2 Virus Protease (NS2B-NS3): An In-Silico Studies of Anti-Viral Plants

Mhashevolu Rhakho *

Department of Botany, St. Joseph’s College (A) Jakhama, Nagaland, India.

 

 

Medicinal plants have been used in healthcare in traditional medicine since time immemorial. It has been a cornerstone of traditional medicine for centuries, providing natural remedies for various ailments. These plants are a rich source of bioactive compounds that have evolved to protect them from environmental stresses, pathogens, and insects. Humans have tapped into this chemical arsenal to develop medicines that prevent and treat diseases. Studies have been carried out globally to focus on plant-based chemicals for drug discovery which have led to the production of plant-based medicines ¹. Medicinal plants have also played a crucial role in the development of modern medicine. Many conventional drugs are derived from plants, such as aspirin from willow bark, quinine from cinchona trees, and morphine from opium poppies. Additionally, plants are a rich source of leads for drug discovery, with many pharmaceutical companies exploring plant-based compounds for new medicines ². Herbal medicines have gained its popularity in the last few decades for its therapeutic treatments like Ayurveda, Unani, Homeopathy, Sidha, etc, which use herbs as a major constituent. Plants consist of multiple alkaloids/compounds that occur naturally (as against single extracts) exhibit synergistic actions such as antiviral, antibacterial, anti-protozoa, and antioxidant ³. Medicinal plants used in traditional medicine and indigenous knowledge to treat a variety of ailments include bulbs, shrubs, ferns, and trees^4.Among the different plant parts, the leaves were most frequently used for the treatment of diseases followed by root, fruit, flower, tuber, rhizome, bark, stem⁵. Herbal medicines can be administered as a paste, decoction, infusion, juice or poultice, or taken orally with no preparation. Medicinal plants contain a wide variety of secondary metabolites or compounds such as tannins terpenoids, alkaloids, flavonoids and possess significant antibacterial, antifungal, anticancer, antidiuretic, anti-inflammatory and anti-diabetic. Pharmacognosy provides a vast understanding of potential medicinal plants applications for the prevention and treatment of numerous conditions and diseases including Dengue⁶. There is an immense ongoing interest among the researchers in developing new antiviral therapeutic agents from natural products or the extract of plants. The numbers of studies that have been undertaken to document and preserve medicinal plant knowledge in Nagaland are few. The numbers of scientific research into the efficacy and safety of phytomedicines and scientific investigations are carried out to confirm the anti- viral properties which can be used to combat various viral diseases. The following antiviral plants are considered for the study, viz., Azadirachta indica, Brassica campestris, Carica papaya, Catharanthus roseus, Euphorbia hirta, Ocimum sanctum, Psidium guajava, Allium sativum, Houttuynia cordata, and Mimosa catechu⁷.

Dengue is a virus-based infection caused by the dengue virus (DENV), a member of the genus Flavivirus in the family Flaviviridae which is transmitted to humans through the bites of infected female mosquitoes, primarily the Aedes aegypti mosquito. The virus in other species within the Aedes genus such as Aedes albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes (Stegomyia) aegypti, Aedes albopictus, etc can also act as vectors, but their contribution is secondary to Aedes aegypti^8-9. Along with the dengue virus, Flavivirus includes Japanese Encephalitis Virus (JEV), Yellow fever virus (YFV), West Nile virus (WNV) and tick-borne Encephalitis virus (TBEV). Zika virus outbreak has been reported in various countries which is a prominent human-pathogenic flaviviruses ^10-11. Dengue is endemic in more than 125 countries, a significant cause of mortality in tropical and subtropical regions. Infections range from asymptomatic self-limited, acute, febrile disease called dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). There has been a drastic increase case of Dengue and over 2.5 billion people, in more than 100 countries, are at risk of dengue infection, with millions of cases occurring around the world every year. ^12-13. According to WHO, an estimation of 390 million infections is reported annually worldwide, of which 96 million are clinically apparent. The largest number of dengue cases reported was in 2023. The WHO Region of the Americas reported 4.5 million cases, with 2300 deaths. A high number of cases were reported in Asia: Bangladesh (321 000), Malaysia (111 400), Thailand (150 000), and Vietnam (369 000). Dengue is considered to be ubiquitous throughout the tropics, with local spatial variations in risk influenced strongly by rainfall, temperature and the degree of urbanization. Dengue fever can be better framed for health interventions in terms of specific environmental features and assemblages of high-risk spaces. ^14-15. Half of the world’s population is at risk of dengue infection as there is no specific antiviral treatment that act against DENV is currently available despite considerable efforts to find potent inhibitors for the Dengue protease. The only dengue vaccine available on the market is CYD-TDV, which was developed by Sanofi Pasteur (marketed as Dengvaxia and is currently approved in 20 countries in Latin America, Asia, and Australia. While CYD-TDV is effective for the prevention of severe infection in previously infected people, it also increases the risk of severe dengue in individuals who have not been previously infected by dengue. The results highlighted were important in directing future vaccine development to identify immune correlates of protection ^16-17. 

The causative agent for dengue fever (DF) is dengue virus (DENV), an RNA virus from the Flavivirus genus belonging to the Flaviviridae family. It is roughly spherical in shape, an enveloped single-stranded positive-sense RNA virus with an icosahedral nucleocapsid covered by the lipid bilayer. It has a positive-strand RNA genome inside a protein capsid also known as nucleocapsid and 11 kb long DENV genome can function as mRNA, and, similar to that in eukaryotes, there are untranslated regions (UTRs) at both the 5′ and 3′ end flanking the open reading frame (ORF). DENV has the ability to infect a wide range of cell types including cells of the human immune system ranging from dendritic cells, monocytes, B- and T-cells, hepatocytes, endothelial cells. Dengue infections are caused by 4 different serotypes, which are: DENV 1, DENV2, DENV3, and DENV4. These serotypes share approximately 65% of their genome similarity, but there is some genetic variation in each serotype. Despite these variations, infection with each of the dengue serotypes results in the same disease and range of clinical symptoms^{13, 18}.

The pathogenic female Aedes aegypti mosquito releases the Dengue virus via saliva into the skin of the mammalian host. Salivary components of Aedes aegypti mosquitoes have been identified as significant contributors to increased viral replication. A salivary protein named “34 kDa protein” increases DENV viral titer in human keratinocytes, reducing the expression of the antimicrobial peptides LL-37 and S100A7 and type I interferons. Viral replication starts in the salivary glands of the vector which leads to virion release in the saliva that initiates binding of the virus to host cell receptors for entry via receptor-mediated endocytosis ^8,19,20. DENV after receptor-mediated endocytosis, the positive-strand viral RNAis released into the cytoplasm and translated into a polyprotein. The translated polyprotein are further cleaved by viral and host proteases into three structural (capsid: C, membrane: M, and envelope: E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins which helps in replication. The RNA genome and the capsid protein interact to form a complex, while other structural proteins form part of the virion envelope. Although the NS proteins are absent within the virion, they assist in virus replication and evasion of the immune system within an infected cell. ^21,22,23,26. C protein localized in the cytoplasm and nuclei is a foremost structural component of DENV which is thought to be crucial for its well-organized replication. The lipid bilayer of virions is formed by lipid between the nucleocapsid core and E/M outer shell. 

During the replication of DENV, a membrane-bound replication complex formation helps to incorporate host factors, viral proteins, and genomic RNA. The positive-strand (+) DENV genomic RNA acts as a template to synthesize complementary negative-strand (-) RNA, which acts as a template for the synthesis of new positive-strand viral RNAs^21,24,25. Viral replication occurs on the endoplasmic reticulum by NS proteins followed by viral assembly and trafficking of immature viral particles to the trans-Golgi network (TGN) where it undergoes pH-dependent maturation. The acidic environment of the TGN facilitates viral maturation where the pr domain of precursor membrane protein (prM) is cleaved by the host protease furin. prM shields the envelope proteins from premature fusion and pH-induced reorganization during viral secretion. Subsequently, the fully matured infectious virus particle are then released from the hostcell.^8,25,26.

Among the four serotypes of Dengue -DENV 1, DENV2, DENV3, and DENV4, DENV2 is most prevalent globally which contains a single-stranded RNA of positive polarity. The Dengue virus (DENV) is roughly spherical in shape with a diameter of approximately 50 nm, which enveloped single-stranded positive-sense RNA virus of 11 kb long which is translated into a large polyprotein during the infectious life cycle. This polyprotein is processed by cellular and viral proteases into three mature structural proteins: the capsid (C), envelope (E), and membrane (M) proteins along with other seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). These nonstructural proteins play roles in viral replication, virion assembly and attenuation of the host antiviral response ^8,27,28.

 

 

Figure 1: Dengue protease (NS2B-NS3) with catalytic site

 

NS3, a large multifunctional protein of 618 amino acids belongs to the superfamily 2 of RNA helicases/NTPases having Walker A, GK(S/T) and Walker B, DEx(D/H) motifs, along with other superfamily-characteristic conserved sequence motifs present in subdomains I and II (which possess the fold of the RecA protein) endowed with protease, helicase, nucleoside 5′-triphosphatase (NTPase), as well as 5′-terminal RNA triphosphatase activities, plays an important role in viral polyprotein processing and genome replication. The N-terminal of NS3 comprises a serine protease domain, with the protein NS2B acting as a membrane-anchoring cofactor, necessary for proteolytic activity (figure 1). Helicase activity of NS3 catalyzes the unwinding of the duplex RNA in the 3′–5′ direction releasing the single stranded RNA available to NS5 as a template for replication in the presence of a divalent cation and the energy derived from the hydrolysis of ATP ^{29,30, 31}. NS2B is a viral serine protease and, along with other NS proteins NS1, NS2A, NS4, and NS4B, plays diverse roles in viral replication, assembly, and release. The C-terminal end of the NS3 protein has three enzymatic properties: a 5′ RNA-triphosphatase (RTP), a nucleoside triphosphatase (NTPase), and a helicase. NS3 forms a complex with NS5 and assists in replication through the unwinding of viral RNA and dephosphorylation prior to 5′-end capping ^{8, 29, 32}. The dengue virus NS3 protease, a member of the flavivirin enzyme family (EC 3.4.21.91), is located in the N-terminal 184 residues of the multifunctional 69 kDa NS3 protein and contains a functional catalytic triad consisting of His51, Asp75 and Ser135 (figure 1). In addition to the serine protease, the NS3 protein contains enzymatic activities of a nucleoside triphosphatase, a 5' - RNA triphosphatase (RTPase) and a RNA - stimulated RNA helicase. The NS3 protease catalyses the post-translational cleavage of the viral polyprotein precursor in the non-structural region at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites and at additional sites within the viral capsid protein, NS2A, NS4A and within a C-terminal region of NS3 itself. The overall conformation of the dengue virus NS3 protease displays the β-barrel conformation typical for serine proteases, although the viral enzyme appears to possess higher compactness with short or absent loop structures and a relatively shallow substrate binding site ^33,34. Based on a number of studies, the methyltransferase (MTase) domain of the DV non-structural protein NS5 (NS5 MTase) is thought to be a promising antiviral target^{35, 36]} However, the closed conformation is the major form even in the unbound state, which thus represents the best model for structure-guided drug designs^{27, 37, 38}.

2.1  Selection of Phytocompounds

After comprehensive search on databases, 10 plants having antiviral properties were selected to confirm their efficiency against DENV-2. A total of 133 phytoconstituents from the selected plants were identified.

 

Table 1: List of selected phytocompounds.

Sl. No	Phytocompounds	Name of Plants
1	Nimbin	Azadirachta indica39
2	desacetylnimbin
3	desacetylsalannin
4	kaempferol 3-O-rutinoside
5	Epicatechin
6	nimbinene,
7	6-deacetylnimbinene
8	Nimbandiol
9	Nimbolide
10	ascorbic acid
11	n-hexacosanol
12	7-desacetyl-7-benzoylazadiradione
13	7-desacetyl-7-benzoylgedunin
14	7-hydroxyazadiradione
15	Nimbiol
16	Quercetin
17	ß-sitosterol
18	Rutin
19	Limbonin
20	Azadirolic acid
21	Nimbochalcin
22	Azadirachtin
23	Limbocidin
24	3-p-coumaroylquinic	Brassica campestris40
25	Caffeic
26	Ferulic
27	sinapic acids
28	kaempferol 3-sophoroside-7-glucosides
29	Citric
30	Aconitic
31	2-ketoglutaric
32	Maleic
33	Shikimic
34	fumaric acids
35	Zeaxanthin
36	Lutein
37	p-coumaric (4-hydroxycinnamic
38	Chymopapain,	Carica papaya41.42
39	Carpaine
40	Dehydrocarpaine
41	Oleic acid
42	Tocopherol
43	Squalene
44	Neophytadiene
45	butyl 9,12,15-octadecatrienoate
46	n-hexadecanoic acid
47	phytol ( tetramethyl-2-hexadecen)
48	dasycarpidan-1-methanol acetate (ester)
49	octadecenoic acid
50	D-limonene
51	bis (2-(2-chloroethoxy)ethyl) ether
52	Dimethoxydimethylsilane
53	dibenzyl ether
54	benzhydrazide,
55	o-butylisourea
56	2-chloro-5,5-dimethyl-1-phenyl-3-hexen-1-ol
57	2-methoxybenzeneacetaldehyde
58	Myricetin
59	1-methyl-2-pyrrolidinone
60	Benzonitrile
61	Nonanal
62	octanoic acid
63	1-decene
64	nonanoic acid
65	Benzene
66	Benzene, 1,3-bis(1,1-dimethylethyl)-5-methoxy
67	1-iodooctadecane
68	2-methylnaphthalene
69	2-tetradecene
70	10-undecenoic acid
71	Dodecanal
72	1,4-dimethylnaphthalene
73	9-oxononanoic acid
74	1-hentriacontane
75	2,4-di-tert-butylphenol
76	nonanedioic acid
77	dimethyl ester
78	1-octadecene
79	Ajmalicine	Catharanthus roseus43,44
80	Catharanthine
81	Tabersonine
82	Serpentine
83	Vindoline
84	Quercitol	Euphorbia hirta45
85	Alpha (α)-amyrin
86	beta (β)-amyrin
87	Friedelin
88	Taraxerol
89	Euphorbins B
90	Euphorbin E
91	Gallic
92	Geraniin
93	ellagic acid
94	Terchebin
95	Tannic
96	tartaric acids
97	lupeol fatty acid ester
98	fatty acid  phytyl     esters
99	linoleic acid
100	Triterpenoid
101	Eugenol	Ocimum sanctum46,47
102	β-sitosterol
103	Stigmasterol
104	Campesterol
105	Reynoutrin	Psidium guajava48,49
106	Guajaverin
107	Avicularin
108	Isoquercitrin
109	Hyperoside
110	2,6-dihydroxy-3,5-dimethyl- 4-O- (6 ″ - O-galloyl-β-D-glucopyranosyl) –benzophenone
111	flavonoids (naringin)
112	flavonoids ( hesperetin)
113	flavonoids (daidzein)
114	γ-glutamyl-l-cysteine peptides	Allium sativum50,51
115	Alliin (S-allyl-l-cysteine sulfoxide)
116	diallyl thiosulfinate
117	diallyl disulfide
118	Garlicin
119	diallyl trisulfide (allitridin or DATS),
120	Ajoene
121	vinyl-dithiins (3-Vinyl-4H-1,2-dithiin)
122	Chlorogenic acid,	Houttuynia cordata52,53
123	Glabranin
124	7-O-methyl-glabranine
125	4-methyl-heptane	Mimosa catechu54
126	carboxylic acid
127	methyl laurate
128	2-ethyl-3-methylbut-1-ene
129	tetra decanoic
130	4-hydroxybenzoic acid
131	Afzelechin
132	Aromadendrin
133	Baicalein

 

 

The three-dimensional structure of the phytoconstituents identified from the selected plants was obtained from the PUBCHEM⁵⁵ and the structures that are not available were drawn using ChemDRAW. The Mol2 structure of the ligands were used as the input file for screening of the drug-like properties was performed using SWISS ADME and the parameters of Lipinski Rule of 5 i.e, Molecular Weight (MW), Partition Coefficient (LogP), Hydrogen bond donors, Hydrogen Bond acceptors, Molar Refractivity (MR) were recorded.

2.3 Molecular Docking 

The structures of the compounds were further evaluated via Molecular docking using Pyrx^56,57. Structures of Ligands were minimized using Pyrx and the parameters for Energy minimization are uff for force-field and Conjugate Gradient for Optimization Algorithm. The file is then converted to pdbqt file. The structure of protease, 2FOM⁵⁸ was obtained from the Protein Data Bank. The structure of Protein was prepared using Discovery Studio where the water molecules and hetero atoms were removed. Pyrx software was employed for energy minimization and conversion to pdbqt file. Molecular Docking Analysis was performed using Autodock Vina^59,60 which is incorporated in Pyrx Graphical User Interface.  The Active site selected on the protease for docking are HIS51, VAL75, LYS73, LYS74, ASP75, THR120, LEU128, PRO132, SER135, GLY151, ASN152, GLY153, VAL154, and ALA164. The active sites are centered inside the grid box where the docking is performed.

A total of 133 phytoconstituents from 10 different plants and three reference drugs were screened using SWISS ADME. The software used for ADME Screening is SWISSADME⁶¹ where a total number of 133 ligands from the selected plants in mol2 files were fed into SWISSADME. Based on Lipinski rule of five, all the ligands were selected for docking as there were no serious violation to the rule. 

3.1 Protein Preparation

The viral Protease used for this study is Dengue Virus NS2B/NS3 Protease 2FOM. Viral Protein 2FOM structure was downloaded from PUBCHEM ⁶² in PDB format and prepared using MOE.  The catalytic triad His51, Asp75 and Ser 135 are present

3.2 Molecular Docking

Among the selected phytoconstituents, the top 5 phytoconstituents docking scores of the interactions with 2FOM and interacting active site amino acids are ranked based on their docking scores and shown in table 2, where Ligand91 (Terchebin) being the highest with -8.1 kcal/mol and Ligand12 (7-desacetyl-7-benzoylazadiradione) and Ligand20 (Azadirolic acid) being the lowest with a score of -7.7 kcal/mol each. Docking results shows that all the selected Phytoconstituents have higher docking scores as compared to the reference drugs used in this study.

 

 

 

 

 

Table 2: Top docking scores with important interactions

Sl. No	Ligand no	Ligands	Dock Score (kcal/mol)	Important interactions
1	91	Terchebin	-8.1	Trp50, Arg54, Asp75, Gly153, Tyr161, Leu128, Tyr150, Phe130, Pro132, Ile36, Val52, His51
2	13	7-desacetyl-7-benzoylgedunin	-7.8	Leu128, Phe130, Gly151, Gly153, Asp75, His51
3	100	Triterpenoid	- 7.8	Tyr150, Gly151
4	12	7-desacetyl-7-benzoylazadiradione	-7.7	Pro132, His51, Tyr161, Tyr150, Leu128
5	20	Azadirolic acid	-7.7	Gly151, His51, Lys73, Val72, Lys74
6	Ref.1	Doxycycline	-6.6	Val72, Trp50, Asp75, Gly151, Gly153, Val154
7	Ref.2	Monosdenvir	-7.5	Leu128, Pro132, Ser131, His51, Asp75, Asn152, Val154
8	Ref.3	Zanamivir	-5.6	Gly151, Pro132, Ser135, His51, Try150, Phe130, Leu128, Gly153

 

(A)	(B)
(C)	(D)
(E)	(F)
(G)	(H)
(I)	(J)
(K)	(L)
(M)	(N)
(O)	(P)
Figure 2: 2D and 3D images of Protein-Ligand interactions: (A) 2D image of protein-ligand91 interactions (B) 3D image of protein-ligand91 interactions (C) 2D image of protein-ligand13 interactions (D) 3D image of protein-ligand13 interactions (E) 2D image of protein-ligand100 interactions (F) 3D image of protein-ligand100 interactions (G) 2D image of protein-ligand12 interactions (H) 3D image of protein-ligand12 interactions (I) 2D image of protein-ligand20 interactions (J) 3D image of protein-ligand20 interactions (K) 2D image of protein-ligandRef.1 interactions (L) 3D image of protein-ligandRef.1 interactions (M) 2D image of protein-ligandRef.2 interactions (N) 3D image of protein-ligandRef.2 interactions (O) 2D image of protein-ligandRef.3 interactions (N) 3D image of protein-ligandRef.3 interactions.

 

The results of molecular docking analysis of the top binding ligands with the reference are shown in figure2. Result of the docking analysis of Protein-ligand91 Interactions shows 7 Conventional Hydrogen Bond with Amino acids – HIS 51, ILE36, PRO 132, PHE 130, TYR 150, TYR 161, GLY 15, Hydrogen Bond interaction with catalytic Traid –HIS 51 and One unfavourable donor interaction with - TRP 50 is observed. In Protein-ligand13 interaction, docking analysis shows 3 Cabon Hydrogen bond with PHE 130, GLY 151, GLY 153, Pi- Cation bond with the catalytic triad – HIS 51 and Pi-Anion bond with the catalytic triad –ASP 75. Additionally, one unfavourable interaction is observed. Docking analysis of Protein-ligand100 interactions shows 1 Conventional Hydrogen Bond with TYR 50, 1 Cabon Hydrogen bond with GLY 151. There is no interaction with the catalytic triad and no unfavourable interaction is observed. In case of Protein-ligand12 interactions, it shows 1Conventional Hydrogen Bond with TYR 150, 1 Cabon Hydrogen bond with LEU 128, Pi-Sigma interaction with catalytic triad- HIS 51 and No unfavourable interaction observed. A protein-ligand20 interaction shows 2 Conventional Hydrogen Bond with GLY 151 and VAL 72, 1 Cabon Hydrogen bond with LYS 74. There is no interaction with the catalytic triad, however, one unfavourable Acceptor-Acceptor interaction with LYS 73 is observed. Docking analysis of Protein-ligand Ref.1 interactions shows 5 Conventional Hydrogen Bond with VAL 72, TRP 50, ASP 75, GLY 151, GLY 153, Interaction with catalytic triad – ASP 75 and one unfavourable interaction is observed. In Protein-ligandRef.2 interactions, there is1 Cabon Hydrogen bond interaction with ASN 152, Pi- Cation bond with the catalytic triad – HIS 51 and Pi-Anion bond with the catalytic triad –ASP 75. The result shows no unfavourable interaction between the ligands and the drugs. In Protein-ligandRef.3 interactions, 8 Cabon Hydrogen bond interactions with GLY 151, PRO 132, SER 135, HIS 51, TYR 150, PHE 130, LEU 128, GLY 153, Interaction with Catalytic traid – HIS 51and SER 135 is seen. Also, one unfavourable interaction is observed.

 The incidence of Dengue- mosquito-borne viral fever is increasing dramatically in recent years with   repeated outbreaks from many States and newer areas around the world in recent decades. While many dengue infections are asymptomatic or produce only mild illness, the virus can occasionally cause more severe cases, and even death. To date, there is no universal vaccine available to treat or prevent dengue. However, current research efforts in the development of effective vaccines and therapeutics against dengue are emerging to develop antivirals against dengue.

The present work is based on the use of the phytocompounds extracted from 10 medicinal plants found in Nagaland to inhibit Dengue virus. A total of 133 Phytocompounds were extracted from the selected plants as an anti-dengue agent targeting 2FOM (NS2B/NS3 Protease) of DENV. Virtual screening of the phytocompound was carried out using SWISS ADME to estimate the drug likeness and medicinal potential to inhibit the target protein of DENV. Reference drugs (Doxycycline, Monosdenvir, Zanamivir) which were previously studied were considered to compare its binding affinity to a target. Molecular docking of the selected phytocompounds and reference drugs was performed using Pyrx. Based on scores, 5 top ligands were selected to investigate the most effective phytocompounds owing to their binding affinity and analyze their efficacy to inhibit the target. Compounds with stronger binding affinities and their mode of interactions between the compounds with highest affinities and their binding site indicate the potential inhibitors. 

The study reveals that Ligand 12 (7-desacetyl-7-benzoylazadiradione) and Ligand 13 (7-desacetyl-7-benzoylgedunin) can be potential inhibitors for DENV (NS2B/NS3 Protease). These phytocompounds are found in Azadirachta indica^63,64,65 commonly known as Neem which can be considered as the most potential antiviral plant selected for these studies based on its physiochemical properties. 

The future of in silico studies of phytoconstituents holds great potential for advancing our understanding of these complex molecules, bioactivity, toxicity, prediction of phytoconstituent interactions and mechanisms to increase efficiency in research and development thus, enhancing drug discovery. More anti-viral plants can be considered for comprehensive study of phytocompunds applying increased computational power to simulate complex molecular interactions and analyze large datasets, expansion of databases containing phytoconstituent information to facilitate data sharing and research and visualization of molecular interactions and simulations to enhance understanding. Combination of in silico studies with experimental techniques can also help to validate results and improve accuracy to design new drugs using phytoconstituents with optimized bioactivity and reduced toxicity.

The combined methods of In-Silico studies with other computational methods such as Molecular Dynamic (MD) simulation, can be considered for further research to optimized and give extensive and profound results to provide deeper insights of the anti-viral activity of these natural molecules which can serves as a lead to aid in designing the therapeutic drugs against Virus infections. Besides Molecular Dynamic (MD) Simulation, other computational methods such as Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics General Born Surface Area (MM/GBSA) can be employed to understand the thermodynamics of protein-ligand interactions to predict the binding affinity of small molecules, estimate the binding free energy and calculate the free energy of binding between ligands and proteins which are essential in drug design and discovery. 

Conflict of Interest

The author declares no conflict of interest.

Acknowledgements

I would like to extend my sincere gratitude to St Joseph’s College (Autonomous), Jakhama for providing the facilities to carry out this study. Heartfelt appreciation and gratitude to Dr. Peter Solo (Dean of Science), St Joseph’s College (Autonomous), Jakhama for his constant assistance and valuable insights during the whole process.

Author Contributions: All authors have equal contribution in the preparation of manuscript and compilation.

Source of Support: Nil

Funding: The authors declared that this study has received no financial support.

Data Availability Statement: The data presented in this study are available on request from the corresponding author.  

Ethics approval and consent to participate: Not applicable

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