Tarlac State University

College of Science
Department of Chemistry

A MOLECULAR DOCKING SIMULATION AND DRUG

DESIGN FOR POTENTIAL INHIBITOR OF
NONSTRUCTURAL PROTEIN 1 RECEPTOR

IN FULFILLMENT
OF THE REQUIREMENTS FOR THE SUBJECT
THESIS

BY

Bagayan, Czarina Joyce C.

Diaz, Alissandro P.
Taguines, Stephanie D.
BS CHEMISTRY
Abstract

Abstract

Dengue is a viral illness and a huge problem which cause outbreaks and high fatality
and morbidity rate in the world which demands an urgent and priority development of a
new potential drug to slow it down the dengue virus (DENV). The cause of these severe
dengue cases is by the nonstructural protein (NS1) leakage. NS1 is the key viral protein
required for the pathogenesis of both DENV replication and dengue diseases. NS1 glyco-
protein of DENV is the cause of the activation of macrophages and disruption of endothe-
lial cells that cause NS1 protein leakage which is Dengue Hemorrhagic Fever (DHF) and
Dengue Shock Syndrome (DSS) and NS1 is also involve in genome replication of RNA
which is a good target for screening of potential drugs. The protein NS1 has binding site
ASN 130, and ASN 207. In this study, 884 phytochemical ligands of PubChem Database
were screened for potential drug through Molecular docking using Autodock vina. The
study focuses on finding a ligand which has a high binding affinity to glycosylate to NS1
active site receptor to block its glycosylation to other infected protein or cells. Molecular
Docking is used as an approach in this study. Molecular Dynamics is used to have dif-
ferent structures in docking of ligands which help to have more precise and much more
choices or finding higher binding affinity. 3DQSAR will also be used for this study. The
Top 10 ligands which has the highest negative binding affinity will be modified to in-
crease its binding affinity and it will be subjected to ADMET if it can further develop to
potential drug candidate.

Keywords: Molecular Docking, Dengue, NS1, Glycosylation, DHF, DSS, Phytochemicals, AD-
MET, MD, 3DQSAR
Table of Contents i

Table of Contents
1. INTRODUCTION 1
1.1 Dengue 1
1.1.1 Dengue’s Diseases 2
1.1.2 Dengue’s NS1 2
1.2 Importance of the Study 4
1.3 Objective of the Study 5
1.4 Scope and Delimitation 5
2. REVIEW OF RELATED LITERATURE 6
2.1 Non-Structural Protein 1 6
2.1.1 Binding sites of NS1 7
2.2 Protein Leakage 8
2.3 Ligands 8
2.4 Materials and Molecular Docking 9
2.4.1 Drug Design or SBDD 11
2.5 Drug Modification 12
2.6 ADMET 13
2.7 QSAR 13
3. SYSTEMS AND METHODS 15
3.1 Ligand Preparation 15
3.2 Protein Preparation 15
3.2.1 Detemining Binding Site and Grid Box 16
3.3 Molecular Docking 16
3.4 Molecular Dynamics 16
3.5 QSAR 17
3.6 Drug Modification 17
List of Figures and Tables ii

List of Figures

1 Gridbox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

List of Tables
Chapter 1 : The Problem and Its Background 1

CHAPTER 1

Introduction
1.1 Dengue

Dengue is a mosquito-borne viral illness, and it is a huge problem these days because it
affects up to one half of the world’s population, particularly in tropical countries in Asia
where outbreaks occur regularly. Around 390 million infected annually and every year
about 100 million can go on to develop breakbone fever [37]. This breakbone fever also
known as dengue fever experience severe pain and muscle and bone aches. This dengue
virus can be fatal within 24 to 48 hours.

When a mosquito infects a person, it releases saliva containing the dengue virus. It enters
the bloodstream and then affects the white blood cells, not only the WBC but also affects
the immune cells in the skin tissue and enters to the lymphatic system. The viral infection
can trigger inflammatory reactions.

There is no anti-viral drug is available for the treatment of infection with DENV. There
are also some restrictions to the use of an approved dengue vaccine in naı̈ve individu-
als not previously infected with DENV and in children younger than 9 years of age[49].
Consequently, it is still important to establish an alternative strategy to tackle DENV in-
fection and extreme dengue. DENV has 4 distinct serotypes (DENV-1, DENV-2, DENV-3
and DENV-4) and is a positive, single-stranded, enveloped RNA virus belonging to the
family Flaviviridae[15].
Chapter 1 : The Problem and Its Background 2

1.1.1 Dengue’s Diseases

Only one viral protein is release from infected cells and this is called NS1. This protein
by itself can cause vascular leakage which means fluids escaping from your bloodstream
that causes Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) that
leads to potentially death.
Dengue including Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS)
is one of the major public health concerns causes by Dengue Virus Nonstructural Protein
1 (NS1). DENV are consists of positive RNA genome : Capsid (C), membrane protein
(M) and Envelope protein (E ) and seven nonstructural protein namely NS1, NS2A, NS2B,
NS3, NS4A, NS4B and NS5 which are responsible for viral replications. Moreover, among
the members of the family Flaviviridae which includes Zika virus, Yellow fever virus,
Tick-borne encephalitis virus, JEV, and Hepatitis C virus (HVC) and DENV, DENV gives
the highest rate impact on the burden of global diseases.[27] Dengue is also one of the
leading top 10 threats in global health

1.1.2 Dengue’s NS1

NS1 is a key viral protein required for the pathogenesis of both DENV replication and
dengue disease.

The soluble form of NS1 contributes to improved development of DENV, DENV immune
evasion, proinflammatory cytokine development activation, endothelial barrier destruc-
tion, and vascular leakage. Also, NS1 is responsible for the excessive release of inflamma-
tory immune molecules. It is suspected that an excessive inflammatory response plays a
direct role in the pathogenesis of serious dengue disease. Studies have shown that serious
dengue patients exhibit unregulated activation of immune cells and increased develop-
ment of inflammation mediators. The dengue virus (DENV) non-structural protein or
NS1 contains two residues for the binding sites namely the Asn-130 and Asn-207 where
potential glycosylation occurs[31].
Chapter 1 : The Problem and Its Background 3

At each of these sites, NS1 formed in infected cells is glycosylated. More likely, NS1
protein is competitive inhibition.

NS1 protein is consider as a part of the causal factors in dengue. The NS1 glycoprotein
dengue 2 virus (DENV-2) contains two potentially N-linked glycosylation sites at Asn-130
and Asn-207[31] At each of these sites, NS1 formed in infected cells is glycosylated. More
likely, NS1 protein is competitive inhibition.[51]

This is a computational study that determines the interaction of receptor specifically the
NS1 protein against different ligands. There are several methods in determining the
molecular properties of proteins, one of those is the Molecular Docking wherein that
method is used in this study to determine and describe the binding and interaction of
protein-ligand complex[51, 4]. This study will provide possible or potential drugs that
could prevent the leakage of NS1 protein in dengue virus.
Chapter 1 : The Problem and Its Background 4

1.2 Importance of the Study

Dengue cases has been an emerging problem of public health and its morbidity rate is in-
creasing. Adults can be a risk of the disease, but mostly children. It could lead to Dengue
hemorrhagic fever and shock causing a potential death, that shock is the Dengue shock
syndrome if not treated beforehand. It is important to know the symptoms and how the
virus could be managed by means of protein susceptible in the growth of virus. This
study will work on the computational docking of the protein NS1 on possible drugs or
ligands good for binding, depending on their affinity. This study will be significant to the
sectors as follows:

Patients. This study will look for any possible drugs that can treat or prevent the leakage
of NS1 that will lead to more serious cases of Dengue fever. The researchers will examine
and compare the effectiveness of different ligands with the protein NS1.

Students. This study will give students the knowledge about the disease, its symptoms
and management. In addition, the study will provide students the knowledge about the
computational simulation through this study.

Future Researchers. This study would be helpful for other studies regarding the protein
NS1 and its role in Dengue virus. Furthermore, this study may serve as a basis or a refer-
ence on how computational simulation is done.
Chapter 1 : The Problem and Its Background 5

1.3 Objective of the Study

The main purpose of this study is to find a ligand with high binding affinity and modi-
fied it which can leads to potential drug candidate to inhibit against leakage of NS1 which
cause severe dengue cases

Specific Objectives:

• Molecular Docking of the ligands to the active site receptors to get the top 10 highest
binding affinity among the ligands.

• Determine structural relationship of top 10 ligands using QSAR.

• Modification of the top 10 ligand candidate with highest binding affinity to obtain
the most favourable characteristics or results.

• Using ADMET to get the result of the top binding affinity if its approved for poten-
tial drug candidates.

1.4 Scope and Delimitation

The main focus is to find a 10 highest binding affinity ligand to bind to the active site
receptors of NS1 that could potentially prevent the leakage of NS1. This research is lim-
ited to Molecular docking, ligand modification,3DQSAR and drug scanning. This study
is focused on the protein-ligand binding and the drug analysis and modification.
Chapter 2 : Review of Related Literature 6

CHAPTER 2

Review of Related Literatures

This chapter presents the related literature after the thorough and in-depth search done
by the researchers. This also present the previous and current studies on the molecular
docking of non-structural protein 1 or NS1, to fully understand the research to be done
and lastly to understand the ability of NS1 protein for better comprehension of the study.

2.1 Non-structural Protein 1

Dengue virus Non-structural protein 1 is a glycoprotein secreted at the surface of infected

cells, it can be in hexameric form or as a soluble molecule.[29] The NS1 in hexameric
form has been hypothesized to take a huge part in dengue physiopathology as it con-
tains triacylglycerides, cholesterol and Phospholipids which are present in High Density
Lipoprotein.[21] In a study, it is said that the present of High Level NS1 in a blood serum
of person allows the early detection of the dengue fever.[13] Moreover, recent studies have
found out that Non-structural protein 1 activates macrophages through TLR4 or Toll-Like
Receptor 4 and causes a disruption in the endothelial cell ,which is the main cause of vas-
cular leakages that attributes to Dengue Hemorrhagic Fever (DHF) and Dengue Shock
Syndrome (DSS)[18]

Dengue Hemorrhagic Fever and Dengue Shock Syndrome are severe cases of dengue
virus caused by plasma leakage that occurs to patients for 24-48 hours with several episodes
of shock. Severe cases of dengue are developed due to several triggers like activation
of T cells and apoptosis, causing activation and damage.[43] NS1 is a toll-like receptor
agonist in which it activates T cells to secrete cytokines contributing to the pathology
of dengue.[34] Toll-like receptors are a vital component of innate immunity system that
serves as a protection against antigens. It functions with NS1 to direct a T cell immune
response to pathogens.[53]
Chapter 2 : Review of Related Literature 7

Other studies shows that NS1 protein when bonded with toll-like receptor 4 (TLR4)
secretes immune cells that cause endothelial damage resulting to leakage.[8] As T cells
activate it produce a wide number of proinflammatory cytokines such as Tumor necro-
sis factor (TNF), interleukin-2 (IL-2), IL-6, interferon- Y (IFNY), and chemokine ligand 4
(CC4), IL-8 that alters vascular permeability. The number of cytokines induced differs
depending on how severe the disease gets.[29, 34]

2.1.2 Binding sites of NS1

NS1 protein has two N-linked glycans at ASN 130 and ASN 207 at each of these sites the
infected cells are glycosylated. In addition, NS1 drives the viral RNA replication within
the host cell. The N-linked glycan at ASN 130 comprised of complex oligosaccharide
while ASN 207 consist of high mannose glycan [52]. Glycosylation on NS1 plays a vital
role in secretion, binding to specific host and mutation of dengue virus, it prevents the
immune recognition by mutation on glycan in the host [32]. The glycosylation site at ASN
207 is required for the secretion of the protein and the stability of protein [45]. By blocking
the glycosylation of either N-linked glycans the inhibition of viral RNA replication would
be possible. Thus induces the effect of NS1 protein in dengue virus.

Dengue virus Non-structural protein 1 is a glycoprotein which is a conjugated carbo-

hydrate chain and protein. It goes through glycosylation that is the process of proteins or
lipids binding to oligosaccharide through the help of enzymes, it extends the complexity
and range of protein depending on the sugar’s covalent attachment to NS1.[52] N-linked
glycosylation is one of the two types of glycosylation, it is where oligosaccharides cova-
lently bind to asparagine residue of proteins, extending the viral infection [9]. N-linked
glycosylation plays an important role in the function of protein, it is responsible for the
entry, viral growth, secretion, and accumulation of dengue virus NS1. [45] First the gly-
coprotein is deglycosylated and then bind by the endoplasmic reticulum chaperone to
undergo protein folding [23].
Chapter 2 : Review of Related Literature 8

Protein folding is essential for the function of protein, maintain parameters and pro-
duction of proteins. [26]. After the deglycosylation, NS1 will be reglycosylated, that
process is done until the folding is correct, and it will be transported to Golgi. It is then
where the protein is matured, by blocking one of the N-linked glycosylated part of the
protein this maturation and further processing of NS1 protein could be reduced. Dengue
virus NS1 protein is engaged at the early stage of viral RNA replication. It has a very
important role from replication to the severity of dengue virus but a study in 2016 stated
that NS1 protein’s effect on the process of replication to virulence is indefinite.[23]

2.2 Protein Leakage

According to Byron E. et. Al., 2009, with Human liver and mouse described or explain
that there are implicated the DENV infections from the increased level of enzymes in liver
and possibilities of continuous bleeding. In this respect, it is worth noting that it has been
shown that DENV ’s main nonstructural protein 1 (NS1) binds preferentially to lung and
liver tissue EC. It has been hypothesized that anti-NS1 antibodies could then lead to se-
lective pulmonary vascular leakage by recognizing NS1[15].

2.3 Ligands

For the past decades used of active medicinal plants have been known to be a practice in
the field of medicine as it pays a very significant role in treating diseases, where in most of
all people around the world solely rely with the use of medicinal plant for their primary
Health care.
Chapter 2 : Review of Related Literature 9

As reported by World Health Organization (WHO) around 80% of the world’s popu-
lation rely on the use of traditional medicinal plant as their primary health care [20]. Phy-
tochemicals are found to have a great a potential as they contain antimicrobial and other
beneficial property against several viruses and other infectious/contagious diseases [14].
These phytochemicals are said to have a great contribution in safeguarding the human
body as it establishes eminent possibilities or potential, specifically antiviral properties,
which can be used in the treatment of several viral infections and diseases including the
dengue viruses[12]. Phytochemicals includes limonoids, alkaloids, flavonoids, polyines,
coumarins, thiophenes, peptides, flavonoids, terpenoids, polyphenolics and saponins
which can all be trace in Medicinal plants. [40] According to Senthilvel P et al. Flavonoids
plays a huge part in the cure of dengue viruses due to their anti-viral activity capability
[38].

Furthermore, Phytochemicals including flavonoids are reported to be an antiviral com-

pound against DENV viruses [33]. In a study conducted by Qamar et al. entitled “ 2, 200
flavonoids of 405 antiviral medicinal plants against Dengue virus NS1 protein were used
and subjected to molecular Docking with DENV NS1 Protein. The result of the study
showed that out of 100 selected flavonoids 6 of them binds deeply in the binding pocket
of the Protein and Flavonoids have shown to have a strong interaction in the Glycosy-
lation site (ASN-130) of the DENV NS1 Protein. In addition, the result of the study also
showed that flavonoids could really be a strong drug candidate against NS1. [40]

2.4 Materials and Molecular Docking

In a study conducted by Qamar MT, et. Al. 2014, Dengue virus NS1 glycoprotein is in-
volved in its RNA replication and can be a good target for drug screening against dengue
virus. Using NS1 as a receptor the researchers performed molecular docking of protein
NS1 to 2200 flavonoids of many antiviral medicinal plants using the software Molecular
Operating Environment (MOE).
Chapter 2 : Review of Related Literature 10

The researchers selected and isolated the Asn 130 to dock in the flavonoids. The re-
sult shows six flavonoids (Deoxycalyxin A, 3,5,7,3’,4’- pentahydroxyflavonol-3-O-beta-D-
galactopyranoside, (3R)- 3’,8-Dihydroxyvestitol, Sang-genon O, Epigallocatechin gallate
and Chamaejasmine) that have the highest interaction against the protein’s residue Asn
130. In conclusion, those six flavonoids could be a drug candidate against the dengue
virus[51].

In another study conducted by Ahmad A. et. al. 2015 molecular docking was performed
in NS1 protein, obtaining the receptor in Protein Data Bank with the PDB: ID 2GX9. The
ligand used in the conducted study was the chemical compound of the neem leaves or
the Azadirachta indica. Molecular Operating Environment was used as a software for the
docking. Docking was done and result showed that at different residues (R21, V18, W16,
C13, etc.) there are certain chemicals of neem leaf are interacting on each residue. The
interacting chemicals of neem leaves was found to be an antiviral compound[4].

There are several methods in developing drugs nowadays but the most widely used is
molecular docking. Molecular docking is used to determine structures of proteins and
its interaction to another protein or ligands. It has been an effective tool to know the
drug ability of substances and their interaction to target proteins. Molecular docking
performs search algorithm and has a scoring function to which the result will depend
on. Scoring function is the quantified expression of the protein-ligand interaction, their
binding affinity. Determination of protein-ligand binding is important for the screening
of drugs that could help for the optimization of drug-like molecules [42]. There are dif-
ferent softwares available for molecular docking, in other studies they have successfully
list potential drugs for dengue virus NS1 using Molecular Operating Environment. An-
other software is the Autodock vina, it is said that it is more efficient to use, its binding
prediction is improved for its accuracy and execution is sped up.
Chapter 2 : Review of Related Literature 11

It uses the file format PDBQT an extension of PDB and alongside Autodock vina is
the use of Autodock tools for setting the protein’s grid box and for the optimization of
proteins and ligands [50].

Molecular docking is use in predicting method of the likely rotation or orientation of

one molecule to another molecule to form a stabilize complex, more frequently a macro/sub-
molecule which like the receptors and a small molecule which are ligands. Molecular
docking is widely-used on most structure-based drug design. [30] A simple analogy of
its definition is the “puzzle with a missing piece” in which the ligand which acts as the
missing piece, structures itself into a correct rotation/orientation on which it will fit in
the “puzzle hole missing” which are the binding site to either inhibit or activate a certain
enzyme target. [48] To do molecular docking, the protein of the target would be the first
requirement then the protein structure is optimized and will be use to docking together
with the ligands.[28] The success of the molecular docking depends on the search algo-
rithm and the scoring function. [46] There are many molecular docking softwares, like
AutoDock 4.2[11] and AutoDock vina[3].

2.4.1 Drug Design or Structure-Based Drug Design

The great importance in pharmaceutical research and development (R & D) lies at the
knowing the principles by which small-molecule ligands recognize and interact with
macromolecules which are the receptors [24]. Forming of ligands with specific electro-
static and stereo chemical attributes to achieve high receptor binding affinity is the pur-
pose of Drug Design. The Drug Designing methods that allows to contain the necessary
features for modulation of the interested receptor for the design of the ligands [24, 41].
Selective modulation of a validated drug target by high affinity ligands interferes with
specific cellular processes, ultimately leading to the desired pharmacological and thera-
peutic effects [44].
Chapter 2 : Review of Related Literature 12

DD is a repeating process consisting of step-by step knowledge which starts from a

known molecule(target) structure, to know the identity of the potential ligands they are
conducted with in silico studies. These molecular modeling step is followed by the syn-
thesis of the top 10 ligand which are the most promising or highest affinity compounds
[17]. Then the next step is, evaluating of biological properties, such as affinity, efficacy,
potency, and etc. [16]. The three-dimensional structure of the ligand-receptor complex
which is a problem if given that the active compounds are known or has been identified
then it can be solved.[5] When the ligand-receptor complex has been identified and cho-
sen, the biological activity data and the structural information have mutual relationship
[6]. In this step, the DD process repeat to the start but with new steps to incorporate
molecule modifications which has a chance to increase the affinity of new ligands for
the binding site. A very essential factor that must be considered throughout the mod-
eling phase is the flexibility of the intended receptor. To address this issue of flexibility
we may use this technique which is Molecular Docking which are very useful in this
situations.[7, 25]

2.5 Drug modification

In a study conducted by Péter Csizmadia, Marvin is a package of applets used to draw

and imagine chemical structures and substructures. Anyone with a web browser can
use Marvin without the need for any special programs or plugins. In Java 1.0, Mar-
vinSketch/AWT and MarvinView/AWT are written, so they function even in older web
browsers.The reason is that Marvin is the only modularized chemistry applet kit, more
features are external modules that are only downloaded when required. An example of
modularization in Marvin is import/export into different file formats. It can handle also
molecule file in many formats such as SMILES, CML, SMARTS etc. It also supports stere-
ochemistry, valence checking, bonds etc[10]. Similarly, this study will use marvin sketch
in drug modification and save it in SMILES format.
Chapter 2 : Review of Related Literature 13

2.6 ADMET

In another study conducted Daina A. et.al. A potent molecule must meet its target in the
body in appropriate quantities to be successful as a drug, and remain there in a bioactive
form long enough for the expected biological events to occur. Drug growth requires the
measurement of absorption, delivery, metabolism and excretion (ADME). In a stage when
considered compounds are numerous, but access to the physical samples is limited, pro-
gressively earlier in the discovery process[1]. In the same way, this study will explain the
predictive models for physicochemical properties, pharmacokinetics, drug-likeness and
medicinal chemistry friendliness.

ADMET is an acronym in pharmacology and pharmacokinetics for Absorption, distribu-

tion, metabolism, excretion, and toxicity, ADMET also explains the pharmaceutical com-
pound disposition within an organism. The five criterias will determine a compound’s
solubility, liphophilicity, hydrophilicity, pharmacokinetics, druglikeness, and medicinal
chemistry friendliness. [19] Roughly 50% of potential therapeutic compounds were mark
as failures during their clinical trials and also were removed from the market due to poor
ADMET properties.[22]

2.7 QSAR

3DQ-SAR / QSAR is a computational method used for prediction and analysis of molecu-
lar interaction fields. [36] It is the analysis of relationship between the biological activity of
a set of compounds and their three-dimensional properties. 3D-QSAR is prevalent among
drug design method because it identifies the active conformation of each molecules, it can
identify the pharmacophore, can employ comparative molecular field analysis (COMFA)
and other approaches, and build molecules. 3D-QSAR includes molecular topological
differences (MTD), molecular shape analysis (MSA), comparative molecular movement
analysis (COMMA), comparative molecular field analysis (COMFA), and self organizing
molecular field analysis (SOMFA).
Chapter 2 : Review of Related Literature 14

Open3D-QSAR is one of the most convenient 3D-QSAR software, because of its high
computational performance, scriptable interface and flexibility. [39]

3D-QSAR has no restrictions to molecules of the same structural class, has a predictive
capability, doesn’t have reliance on experimental values, and unusual substituents it can
be applied to molecules. However, the problems for 3D-QSAR includes the molecules
must also be seen aligned and the flexibility of molecules[47]
Chapter 3 : Methodology 15

CHAPTER 3

Systems and Methods

This study uses the software Auto dock Vina to perform docking. Multiple docking was
done on drugs approved by different jurisdiction against the receptor Dengue virus non-
structural (NS1) protein. Several software in Windows 10 are used to perform the docking
with multiple ligands along with the Auto dock vina, QSAR and Open Babel GUI. Open
Babel GUI is a downloadable software that converts different chemical structures into de-
sired format, in this case ligands in SDF format were mass converted to PDBQT format
[35].

3.1 Ligand Preparation

The method on this study will involve the 884 ligands that will be downloaded in PUB-
CHEM in its SDF format[11] . The 884 ligands will be downloaded in SDF and then
convert to PDBQT format using the open babel GUI.

3.2 Protein Preparation

The crystal structure of Dengue virus type 2 nonstructural protein or NS1 will be ac-
quired from RCSB and will be downloaded in PDB format with the PDB ID: 4O6B [51].
The protein will be then prepared by removing the unnecessary atoms leaving only the
chain A of the molecule where the residues of the active site are located. Active site of the
protein has the residues ASN 130 and ASN 207 where the glycosylation of the receptor
happens[2].Furthermore, the receptor in Protein Data Bank (PDB) format will be modi-
fied into PDBQT format using the Autodock tools, removing the water molecules, that
may interfere with the binding of ligands, and then adding the polar hydrogens.
Chapter 3 : Methodology 16

3.2.1. Determining Binding Site and Grid Box

The researchers defined the binding site by selecting the residues Asn 130 and Asn 207
in Auto dock tools. A grid box will be place at the active site by setting its coordinates
according to which the active site is filled in the box, the researchers will set grid box each
on both active sites and done molecular docking on the said active sites where ligands
will bind. The grid box will be set to 1.000 angstrom. Also, the sizes of dimensions x, y,
and z were all set to 20 angstroms

Figure 1: Gridbox

3.3 Molecular Docking

Molecular docking will be done in all 884 ligands to the protein. The top 10 ligands that
gives the highest affinity (most negative) with the protein will be taken.

3.4 Molecular Dynamics

Molecular Dynamics will be done to further analyze the protein of interest, where in this
study the Non-structural protein 1(NS1). Each protein will be extracted on a timescale of
100 nanoseconds (ns) in production run in molecular dynamics (MD). After performing
the MD, another 11 structures will be acquired and docked to the 884 ligand. In getting
the top 10 ligands from all 11 structures, the researchers will get the mean of all the 884
ligands for each 11 docking binding affinity, after getting the mean, ligands that shows
the most negative affinity will be chosen to be further modified.
Chapter 3 : Methodology 17

Molecular Dynamics (MD) will be done to further analyze the protein of interest, where
in this study the Non-Structural Protein 1 (NS1) . Each protein will be extracted on a
timescale of 100 nanoseconds (ns) in production run in Molecular Dynamics (MD). After
performing the MD , another 11 structures will be acquired and docked to the 884 ligand.
In getting the top 10 ligands from all 11 structures, the researchers will get mean of all
the 884 ligands for each 11 docking binding affinity , after getting the mean, ligands that
shows the most negative affinity whill be chosen.

3.5 QSAR

In this study Quantitative Structure Activity Relationship (QSAR) will be use to deter-
mine the relationship between the structure, properties and biological activities of the
compounds. Moreover, this will tell what functional group must be added and other nec-
essary changes must be done in order to obtain a more negative binding affinity.

3.6 Drug Modification

The chosen top 10 most negative ligands will be modified using Marvin sketch. Sub-
stituents of the ligands will be modified by adding or replacing groups, with the structure
of flavonoids as the basis to get a more negative affinity.[20] The modified ligands will be
docked again to the protein NS1 using Autodock vina to assess their binding affinity. The
modified ligands will be subjected to ADMET to assess their toxicity, the criteria of AD-
MET that did not meet by the modified ligand will be adjusted according to what satisfied
the conditions. The modified ligand that does not contribute on the toxicity and that had
a decrease in affinity (more negative) will be chosen as a drug candidate for NS1.
Bibliorgaphy 18-23

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