Evaluation of Natural Compound as a Potential Drug Against DENV Non-structural
Proteins: In silico Study
Dengue fever is one of the most threatening epidemics of this era and there
is no targeted vaccine and therapy. The present study was designed to elucidate
the natural compounds as therapeutic targets for dengue virus. In this study,
it is proposed that target approach for dengue drug discovery based on natural
ligands obtained from plant extract of C. papaya. Nonstructural proteins
were retrieved from the protein data bank and natural compounds were drawn using
drawing tool, before docking. All of them were subjected to drug and absorption
distribution metabolism toxicity analysis which shows satisfying results which
leads to docking studies. Series of 8 compounds have been screened and docked
for binding energy prediction and on the basis of lowest binding energy, the
potential ligands like 2-Methoxy-4-vinylphenol, 9-Octadecyne and 9, 12, 15-Octadecatrienoic
acid, (Z, Z, Z) are recommended for further studies.
June 14, 2013; Accepted: March 07, 2014;
Published: May 13, 2014
Dengue shock syndrome is the alarming threat worldwide. It is caused by airborn flavivirus
transmmited through female mosquitoes. Increasing technology and urban development allows this
epidemic condition to grow and transmit. Human to human transmision dramatically increase the
risk. Very recent estimate in middle and South India approximately 500 cases met with drastic fate. Only symptomatic
treatment is available to deal with the disease. Understanding the mechanism
and mode of viral replication cycle is neccesary for target identification.
Totally four serotypes existed among these, depending on severity, they are
classified by WHO (2009) and Bhattacharjee
and Bhattacharjee (2011).
The pathogenesis of severe Dengue Hemorrhagic Fever (DHF) is still at nascent
stage, no vaccine is yet available for inhibition and the vector control measures
are not promising. Dengue virus was isolated in India in 1944 but there are
very few research centres involved in the core pathogenesis mechanism finding,
drug discovery and development is also the tedious process until the mechanism
is explained (Gupta et al., 2012). The natural
herbal therapies have been tried against such an unknown infections since ancient
times in India. Targets were selected from most prevalent strains of DENV2.
Full genome of the dengue shows that the virus has structural and non-structural
proteins; among this non-structural (NS) enzymes of the replication complex
include the NS3 protease and with its NS2B, cofactor, the NS3 helicase/nucleoside
triphosphatase (NTPase)/RNA 5 triphosphatase (RTPase) and the NS5 methyltransferase/RNA-dependent
RNA polymerase. Murthy et al. (1999), Erbel
et al. (2006), Augustine Ocloo et al.
(2012) hence these proteins were selected as target for the study.
The need of the hour is to develop natural antiviral drugs for dengue. Previously
published data and evidences say local plant extract of C. papaya having
the inhibitory effects on dengue, without scientific background. Papaya is also
called pawpaw in English an herbaceous succulent plants that posses
self supporting stems. Extensive research has been done on the medicinal properties
of the leaves of Papaya and has found to be more effective as a antiseptic,
blood purifier like biological activities (Gross, 2003;
Ayoola and Adeyeye, 2010).
On the base of literature, this pilot study was aimed to evaluate the medicinal
natural of compounds from C. Papaya and to study their in silico
antiviral activity using docking studies.
MATERIALS AND METHODS
Preparation of protein structure: Selected 3 protein targets were downloaded
from database Protein Data Bank (PDB). (PDB: http://www.rcsb.org/pdb/home/home.do).
All water molecules were removed and on final stage hydrogen atoms were added
to receptor molecule before docking.
Preparation of ligand structure: Total 8 important natural compounds
available in the plant extract of C. papaya were selected on the basis
of their biological activity reported. Structures of the phytochemicals were
drawn using Chemsketch drawing tool and saved as structure data format.
Protein ligand interaction: Target proteins were then carried out for
binding site studies for better understanding of interaction among the small
and large molecules. This interaction is based on the geometry of target and
flexibility of the ligand.
ADMET analysis: Small molecules obtained from the plant material are
always screened for the potential drug like activity predictions, in this study
all the compounds were screened for the drug likeness calculation using the
pre-ADMET software (Lipinski et al., 1997).
Absorption, Distribution, Metabolism and Excretion (ADME) were screened to
analyse the drug like properties of the phytocompounds, calculation was performed
by using the admet-SAR tool. SMILES of ligand were used as input and using the
module different parameters were calculated such as blood brain barrier, aqueous
solubility, metabolism and carcinogenicity (Cheng et
Computational docking studies: Docking calculations were carried out
using Docking Server (Bikadi and Hazai, 2009). Gasteiger
partial charges were added to the ligand atoms. Non-polar hydrogen atoms were
merged and rotatable bonds were defined. Docking calculations were carried out
on protein model. Essential hydrogen atoms, Kollman charges and salvation parameters
were added affinity (grid) maps of 23 Å grid points and 0.375 Å
spacing were generated using the auto grid program. Auto Dock parameter set-and
distance-dependent dielectric functions were used in the calculation of the
van der waals and the electrostatic terms, respectively.
Docking simulations were performed using the Lamarckian Genetic Algorithm (LGA)
and the Solis and Wets local search method (Solis and Wets,
1981). Initial position, orientation and torsions of the ligand molecules
were set randomly. All rotatable torsions were released during docking. Each
docking experiment was derived from 10 different runs that were set to terminate
after a maximum of 250,000 energy evaluations. The population size was set to
150. During the search, a translational step of 0.2 Å and quaternion and
torsion steps of 5 were applied.
Three-dimensional structures of all the three proteins of DENV2 were downloaded
from PDB shown in Table 1.
The GCMS analysis results of the plant gives some important phytochemicals
(data not shown) which were selected for the docking purpose, structure and
details given in Table 2.
ADMET analysis shows aqueous solubility of all compounds. Citronellyl butyrate
and 9 octadecyne show level of good solubility. The Blood Brain Barrier (BBB)
analysis generally used for penetration studies and in this study all the compounds
except 2-Methoxy-4-vinylphenol and L-Arabinitol shows high penetration levels
which mean they can cross the barrier and acts on Central nervous system. Cytochrome
P (CYP450) is the important for drug metabolism.
|| Protein retrieved from PDB
|| Isolated phytochemicals from C. papya used as a ligand
In this study, N-Aminomorpholine and 9-Octadecyne shows qualifies the test
for inhibition. On the otherhand Citronellyl butyrate found to be more lethal
due to its carcinogenic activity (Table 4 and 5).
In docking studies, NS3 and NS5 non-structural proteins were found to be more
targeted by ligand, shows lowest binding energies, 2-Methoxy-4-vinylphenol,
9,12,15-Octadecatrienoic acid, (Z,Z,Z), 9-Octadecyne shows less binding energy
less than -4.0 kcal mol-1 i.e., (-6.15) kcal mol-1 etc.,
scores shown in Table 3. While docking interaction of ligand
with respective proteins shown in Fig. 1-3(a-h).
|| Protein ligand interaction docking scores
NS3 protein docked with following ligand
(a) 9, 12, 15-Octadecatrienoic acid, (Z, Z, Z) (b) 9 Octa decayne, (c)
Citronyll butyrate, (d) 4-Hydroxybenzyl alcohol, bis(pentafluoropropionate),
(e) 2-Methoxy-4-vinylphenol (f) L-Arabinitol and (g) Phytol and (h) N-Aminomorpholine
NS5 protein docked with following ligand
(a) Citronyll butyrate, (b) 2-Methoxy-4- vinylphenol, (c) 9, 12, 15-Octadecatrienoic
acid, (Z, Z, Z), (d) N-Aminomorpholine, (e) 9-Octa decayne, (f) Phytol,
(g) L-Arabinitol and (h) 4-Hydroxybenzyl alcohol bis(Pentafluoropropionate)
||ADMET Properties of the compounds
NS2+NS3 complex protein docked with following
ligand (a) 4-Hydroxybenzyl alcohol, (b) 9-Octa decayne bis(Pentafluoropropionate),
(c) L-Arabinitol, (d) Citronyll butyrate, (e) 2-Methoxy-4-vinylphenol,
(f) 9, 12, 15-Octadecatrienoic acid, (Z, Z, Z), (g) N-Aminomorpholine
and (h) Phytol
|| ADME analysis values
Eight naturally available compounds were critically evaluated against target
and for drug likeliness activity using different parameters. Analysis shows
that all compounds are suitable for the drug Lipinski Rule Five, but out of
8 and 3 compound such as 2-Methoxy-4-vinylphenol, 9,12,15-Octadecatrienoic acid,
(Z,Z,Z) and 9 octadecyne were strictly qualifies the parameters which can refer
the compound as a drug molecule.
There are number of recent high quality research reporting drug designing in
dengue. Most are relevant and sharing the knowledge about the Flavivirus
and the cell infection mechanism but yet except some of the leading pharma industries
no plant based antiviral drug development is initiated. In the present day,
there are no drugs against dengue as far as stated (Halstead,
2008; Gubler et al., 2007; Noble
et al., 2010).
DENV having the Non-structural protein 3 (NS3) is responsible for proteolysis
of the dengue viral RNA polyprotein as well as carrying out various enzymatic
reactions that are mandatory for replication of the dengue virus. In addition,
NS5 can stimulate the NTPase activity of NS3 which is necessary for unwinding
of dsRNA substrates by helicase activity during viral replication. Arakaki
et al. (2002) and Salonen et al. (2005)
hence, the different non-structural dengue proteins were selected as a target
which always plays a role in viral replication. Therefore, C. papaya was
selected for evaluation of its natural antiviral active compounds. Previous
studies stated the medicinal properties of the plant against dengue at pathological
level but without scientific evidence. Since, the ancient times, C. papaya
recommended for viral infection, wound healing etc. Hence, this in silico
attempt was made to elucidate the mechanism and to develop a hypothesis explaining
inhibitory activity of compounds available in C. papaya against virus
at cellular level (Saklani and Kutty, 2008, Ayoola
and Adeyeye, 2010; Dawkins et al., 2003;
Current docking results confirms that isolated compounds having medicinal properties
and can bind more effectively with the respective targets in dengue virus type
2. Among the 8 compounds 3 show promising result against the NS3 and NS5 proteins.
2-Methoxy-4-vinylphenol, 9, 12, 15-Octadecatrienoic acid, (Z,Z,Z) interact with
pockets available in target proteins. Both the compounds interact mainly with
RNA dependent polymerase which directly inhibits the viral replication. This
would open the doors for new drug discovery and drug development in the field
of dengue. The proteomics based studies are important in targeted drug delivery
system hence this study reveals the docking part after the complete proteomics
of targeted proteins of dengue virus.
Dengue has emerged as a most important viral infection in the 21st century.
The complexity of dengue infection and the difficulties in vaccine design has
prompted us to study the role of antiviral compounds in C. papaya. The
crude extract of the plant has been used in treatment of dengue however, without
enough scientific evidence. This study will throw some light on the antiviral
role of the compounds present in the extract which will play an important role
in drug development.
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