LIGAND BASED DRUG DISCOVERY OF NOVEL DENGUE-2 NS2B …eprints.usm.my/31902/1/MOHAMED_SUFIAN_BIN_MOHD_NAWI.pdf · 2015 . LIGAND BASED DRUG DISCOVERY OF NOVEL ... 1.7.2 Determination

LIGAND BASED DRUG DISCOVERY OF

NOVEL DENGUE-2 NS2B-NS3 PROTEASE

INHIBITORS

MOHAMED SUFIAN BIN MOHD NAWI

UNIVERSITI SAINS MALAYSIA

2015

LIGAND BASED DRUG DISCOVERY OF NOVEL

DENGUE-2 NS2B-NS3 PROTEASE INHIBITORS

by

MOHAMED SUFIAN BIN MOHD NAWI

Thesis submitted in fulfilment of the requirements

for the degree of

Doctor of Philosophy

August 2015

ii

ACKNOWLEDGEMENT

All praise is due to Allah S.W.T because of His bounty I can complete this research

towards fulfilling the requirements for my degree of Doctor of Philosophy.

Foremost, I would like to express my sincere gratitude to my supervisor Professor

Habibah A. Wahab for the continuous support of my PhD study and research, for her

patience, motivation, enthusiasm and immense knowledge. Her guidance helped me

in all the time of research and writing of this thesis.

My utmost gratitude to both my field supervisors, Professor Noorsaadah Abd.

Rahman and Associate Professor Shafida Abdul Hamid. Their scientific advice,

knowledge, many insightful discussions and suggestions make my way to complete

this thesis. Special thanks to Prof Mutasem for teaching me insightful knowledge on

preparing pharmacopore model and Professor Rohana Yusof for providing space,

instrumentation and general lab equipment together with helpful suggestions for my

enzyme preparation.

I thank my fellow lab mates in PhDs lab: Choi Sy Bing, Yam Wai Kit, Maywan,

Yusuf, Razip, Belal, Muchtaridi, Rosfatihah, Saera, Lee, Lim, Ban Hong, Nadia and

Azizairul for the endless helps, stimulating discussions, encouragement, insightful

comments and for all the fun we have had in the last four and half years.

My sincere thanks also go to the lab members of Prof. Rohana Yusof Research

Laboratory, Department of Molecular Medicine, UM: Dr. Shatrah, Dr. Heh Choon

iii

Han, Tan, Christina, Hani, Mimi, Amar and Dr. Husin for offering me the

opportunities to work in their group and helping me on enzyme expression,

purification and in-vitro works. I am grateful to Dr. Narendra Babu from USM, Dr.

Anjan Patail from Kulliyyah of Science IIUM, Dr Lee Yen Kee from Chemistry

Department UM and Maywan from PhDs lab for helping me with the synthesis

works.

I also thank my friends and supporting staff at the School of Pharmaceutical Sciences

USM; Department of Molecular Medicine and Department of Chemistry UM; as well

as Kulliyyah of Pharmacy and Kulliyyah of Science of IIUM.

To IIUM scholarship and study leave committee, Ministry of Higher Education and

Public Service Department of Malaysia, thank you for the financial support that was

awarded to me throughout my PhD studies. I acknowledge Ministry of Science,

Technology and Innovations (MOSTI) for the funding of the research.

I would like to express my utmost gratitude to my wife, Siti Hadijah Shamsudin and

my children: Afifah, Amirul and Amirah for their moral support that gave me the

confidence, will and strength to endure the pressure and tension in pursuing my

ambition and to them I dedicate this thesis.

Finally but not least, thank you to my mother, my late father and mother-in-law for

their prayers, understanding and inspiration throughout my study. May Allah give

His blessing in their lives in this world and in the hereafter.

iv

TABLE OF CONTENT

ACKNOWLEDGEMENT……………………………………………………. ii

TABLE OF CONTENT………………………………………………………... iv

LIST OF TABLES……………………………………………………………... ix

LIST OF FIGURES……………………………………………………………. xii

LIST OF ABBREVIATIONS………………………………………………….. xviii

LIST OF PUBLICATIONS…………………………………………………… xxi

ABSTRAK……………………………………………………………………... xxii

ABSTRACT…………………………………………………………………… xxiv

CHAPTER 1 - INTRODUCTION AND LITERATURE REVIEW

1.1 STATEMENT OF THE PROBLEM……………………………………... 1

1.2 DENGUE…………………………………………………………………. 4

1.3 DENGUE VIRUS………………………………………………………… 7

1.3.1 The structure and genome of dengue virus………………………... 8

1.3.2 Life cycle of Flavivirus……………………………………………. 9

1.3.2.1 Entry to the cell………………………………………….. 10

1.3.2.2 Translation and polyprotein processing………………….. 11

1.3.2.3 RNA replication………………………………………….. 12

1.3.2.4 Viral assembly and release of new progeny virion………. 13

1.4 PROTEINS OF DENGUE VIRUS……………………………………… 13

1.4.1 Structural proteins and functions………………………………….. 13

1.4.1.1 Capsid protein……………………………………………. 14

1.4.1.2 E and M proteins………………………………….. 14

1.4.2 Non-structural proteins and functions…………………………….. 17

v

1.4.2.1 Dengue virus NS2B-NS3 protease………………………. 18

1.4.2.2 Dengue virus NS2B-NS3 protease as a potential drug

target……………………………………………………...

24

1.4.2.3 Expression of dengue NS2B-NS3 protease……………… 33

1.5 COMPUTER AIDED DRUG DESIGN……………………………….…. 35

1.5.1 Pharmacophore……………………………………………………. 35

1.5.1.1 Pharmacophore modelling using Discovery

Studio (DS 2.5) ……………………………………..........

37

1.5.1.1.1 Assessment of the generated hypotheses…….. 38

1.5.2 Dengue-2 Virus NS2B-NS3 protease pharmacophore……………. 41

1.6 NATURAL PRODUCT IN DRUG DISCOVERY………………….…… 41

1.7 ENZYME KINETIC AND MECHANISME……..……………………… 45

1.7.1 Steady state analysis of enzyme kinetics………………………….. 46

1.7.2 Determination of Vmax, Km and kcat………………………………… 49

1.7.3 Reversible modes of inhibitor interaction with enzymes…………. 51

1.7.3.1 Competitive inhibition…...………………………………. 53

1.7.3.2 Non-competitive inhibition……………………………… 53

1.7.3.3 Uncompetitive inhibition………………………………… 55

1.7.4 Determination of α and Ki values using nonlinear regression

mixed model inhibition equation in GraphPad Prism 6.0 software..

58

1.8 GOAL AND OBJECTIVES OF THE STUDY…………………… 60

CHAPTER 2 - MATERIALS AND METHODS

2.1 EXPRESSION AND PURIFICATION OF DENGUE-2 NS2B-NS3

PROTEASE………………………………………………………………

62

2.1.1 Materials………………………...………………………………… 62

2.1.1.1 Materials and instrument for protein overexpression and

purification……………………………………………….

62

vi

2.1.1.2 Lysogeny Broth (LB) medium (media for bacterial

cell growth)……………………………………………….

63

2.1.1.3 Stock solutions…………………………………………… 63

2.1.1.4 Buffers for protein purification……...……… 64

2.1.1.5 Solution for SDS-PAGE (sodium dodecyl

sulfate polyacrylmide gel electrophoresis)……………

65

2.1.1.6 Buffer for bioassay (200 mM Tris - HCl, pH 8.5)………. 67

2.1.2 Methods…………………………………………………………… 67

2.1.2.1 Starter scale culture and glycerol stock preparation……... 68

2.1.2.2 Large scale protein over-expression, harvesting

and extraction……………………………………………..

68

2.1.2.3 Protein purification………………………………………. 69

2.1.2.4 Verification of protease using SDS-PAGE……………… 70

2.1.2.5 Bio-Rad protein assay……………………………………. 71

2.1.2.6 Bovine serum albumin (BSA) standard curve…………… 72

2.1.2.7 7-Amino-4-methylcoumarin (AMC) standard plot

for protein assay…………………………………………..

72

2.1.2.8 Preparation of fluorogenic peptide substrate…………….. 72

2.1.2.9 Determination of protease activity……………………….. 73

2.2 EXPLORATION OF DENGUE-2 VIRUS NS2B-NS3 PROTEASE

PHARMACOPHORIC SPACE…………………………………………..

75

2.2.1 Software………………………………………................................ 75

2.2.2 Preparation of ligand set…………………………………………... 75

2.2.3 Selection of training subsets………………………………………. 80

2.2.4 Pharmacophore generation………………………………………... 81

2.2.5 Ligand pharmacophore mapping and 3D - QSAR modeling……... 82

2.2.6 NCI Database screening…………………………………………... 84

vii

2.2.7 Inhibition assay of NCI compounds……………………………… 84

2.3 DEN-2 NS2B-NS3 PROTEASE INHIBITORS DERIVED FROM THE

COMPOUNDS IN NADI DATABASE…………………………………

87

2.3.1 Screening of NADI database……………………………………… 87

2.3.2 Design of new inhibitors from the active hit compounds in

NADI…… 87

2.3.3 Synthesis of the designed compounds…………………………….. 88

2.3.3.1 Scheme 1: Synthesis of S1……………………………….. 89


2.3.3.3 Scheme 3: Synthesis of S3……………………………..… 91


2.3.4 Inhibition assay of the synthesised compounds…………………… 92

2.3.5 Structure-Activity Relationship (SAR) study……………………... 93

CHAPTER 3 - RESULTS AND DISCUSSION 3.1 EXPRESSION AND PURIFICATION OF DENGUE-2 NS2B-NS3

PROTEASE……………………………………………………………….

94

3.1.1 Expression, purification and quantification of DEN-2 NS2B-NS3

protease……………………………………………………………

94

3.1.2 Protease activity assay…………………………………………….. 98

3.2 EXPLORATION OF DENGUE-2 VIRUS NS2B-NS3 PROTEASE

PHARMACOPHORIC SPACE…………………………………………..

102

3.2.1 Pharmacophores generation……………………………………….. 102

3.2.2 Ligand pharmacophore mapping and QSAR modelling………….. 103

3.2.3 Pharmacophore screening and inhibition assay of the compounds

from NCI database…………………………………………………

107

3.3 DEN-2 NS2B-NS3 PROTEASE INHIBITORS DERIVED FROM

THE COMPOUNDS IN NADI DATABASE……………………………

127

3.3.1 Screening of NADI database……………………………………… 127

viii

3.3.2 Design of new inhibitors from active hit compounds in NADI…… 128

3.3.3 Synthesis of the designed compounds…………………………….. 138

3.3.4 Inhibition assay of the synthesised compounds…………………… 160

3.3.5 SAR study of non-competitive inhibitors…………………………. 171

3.3.6 Further discussion…………………………………………………. 173

CHAPTER 4 - CONCLUSION

4.1 ACHIEVEMENT OF THE OBJECTIVES OF THE PROJECT…………. 177

4.2 LIMITATION OF STUDY………………………………………………. 178

4.3 FUTURE STUDIES……………………………………………………… 179

REFERENCES………………………………………...……………………... 180

APPENDICES

LIST OF PUBLICATIONS

ix

LIST OF TABLES

Page

Table 1.1 WHO classification of dengue haemorrhagic fever. Source:

(Howard, 2005)

7

Table 1.2 Diagnostic signatures of reversible inhibition modalities

58

Table 2.1 Experimental bioactivities and pharmacophoric features of

the compounds in the ligand set. The molecular structures

are reported in Appendix 1

77

Table 2.2 Training subsets employed in exploring the pharmacophoric

space of DEN-2 NS2B-NS3 protease inhibitors; numbers

correspond to compound ID in Table 2.1 and Appendix 1

81

Table 3.1 Best fit values for Vmax and Km for protease activity assay

using nonlinear regression Michaelis-Menten equation in

GraphPad Prism 6.0 software

101

Table 3.2 Experimental activities of the compounds in the training

subsets, fit values and correlation statistical (F-stat) against

hypothesis S1T1H01, S1T1H2, S1T1H3 and S1T1H4. ID

numbers correspond to compounds in Table 2.1 and

Appendix 1

104

Table 3.3 Pharmacophoric features corresponding weights, tolerances

and 3D coordinates of S5T5H06 and S2T3H01

107

Table 3.4 Best-fit values of global (shared) parameters, α and Ki for

different concentrations of Panduratin A, fitted using a

nonlinear regression mixed model inhibition equation

implemented in GraphPad Prism 6.0 software

110


different concentrations of compound N1, fitted using a



112





113




114

x






115





116





117





118





119





120

Table 3.14 Fit value on pharmacophore models S5T5H06 and

S2T3H01, α value, type of inhibition and Ki value towards

DEN-2 NS2B-NS3 protease of Panduratin A and the

virtually active hit compounds from NCI database

121

Table 3.15 Active fragments extracted from of the molecular structure

of active hits from NADI Database that fit against

pharmacophore model S5T5H06. Active fragment F1 and F2

were extracted from part A and active fragment F3 and F4

were from part B

133

Table 3.16 The analogues of second phase compound containing

different R1 and R2 to fit the pharmacophoric features of

S5T5H06 model: HBD as violet sphere, HBA as light green

sphere, Hbic as blue sphere and Ring Aromatic as orange

sphere

137

xi


different concentrations of compound S1, fitted using a



162

Table 3.18 Best-fit values of global (shared) parameters; α and Ki for




163


different concentrations of compound S3. fitted using a



164





165

Table 3.21 2D molecular structure with schematic pharmacophoric

features, fit values on the model S5T5H06, α and Ki of the

synthesised compounds and Panduratin A towards DEN-2

NS2B-NS3 protease. HBD designated as violet sphere, HBA

as light green sphere, Hbic as blue sphere and Ring

Aromatic as orange sphere

166

xii

LIST OF FIGURES

Page

Figure 1.1 Several structures of potential anti-dengue compounds

reported by Kampmanna et al., 2009 ((a) and (b)), Wang et

al., 2009 (c) and Tomlinson et al., 2009 ((d) and (e))

2

Figure 1.2 WHO classification of symptomatic dengue infection

(Source: Deen et al., 2006)

6

Figure 1.3 Schematic representation of flavivirus genome organization

and polyprotein processing. Sites of polyprotein cleavage

mediated by the viral NS2B-NS3 and by host signalase are

shown. The enzymatic activities of NS3 and NS5 are also

indicated. Source: (Sampath & Padmanabhan, 2009)

8

Figure 1.4 Flavivirus life cycle. Numbers shown in boxes refer to the

pH of the respective compartments. Source: (Perera,

Khaliq, & Kuhn, 2008)

10

Figure 1.5 Structure of flaviviruses. (A) A surface shaded view of the

cryo-EM reconstruction of immature DENV-2 showing the

spiky surface features. (B) A surface shaded view of the

cryo-EM reconstruction of mature DENV-2 showing the

relatively smooth surface features (C) Fit of the atomic

coordinates of the E protein into the immature virus and (D)

Mature virus showing the arrangement of the E proteins on

the surface of the virion. Source: (Perera et al., 2008)

16

Figure 1.6 Schehter and Berger System of Nomenclature

19

Figure 1.7 Charge relay system for activation of an active site serine

residue

20

Figure 1.8 Structures of NS2B–NS3 protease (Blue, NS3pro; Green,

NS2B). Residues of the catalytic triad Histidine 51 (HIS

51), Aspartate 75 (ASP 75) and Serine 135 (SER 135) are

shown as red-colored stick models. Source: (Wichapong et

al., 2009)

21

Figure 1.9 Sequence alignment of DENV and WNV NS2B and NS3

domains. Stars indicate residues that are identical whereas

dots and colons indicate similar residues. Residues which

are drawn italic and underlined are missing residues in the

X-ray structures. Residues located in the S1, S2, S3, and S4

pockets are colored cyan, yellow, green, and magenta,

respectively, and catalytic triads are displayed in bold red.

Source: (Wichapong, 2009)

23

xiii

Figure 1.10 Structures of several peptide inhibitors against DEN-2

NS2B-NS3 protease reported by Leung et al., 2001 ((a) and

(b)), Chanprapapah et al., 2005 ((c), (d), (e) and (f)), Yin et

al., 2006a ((g), (h) and (i)) and Yin et al., 2006b ((j), (k)

and (l))

26

Figure 1.11 Structures of several small molecule (non-peptidic)

inhibitors against DEN-2 NS2B-NS3 protease reported by

Ganesh et al., 2005 ((a), (b), (c) and (d)), Kiat et al., 2006

((e), (f), (g) and (h)), Mueller et al., 2008 ((i), (j) and (k))

and Bodenreider et al., 2009 ((l) and (m))

31

Figure 1.12 The structures of the examples of the active natural

products against DENV

43

Figure 1.13 A time course for the appearance of product in the enzyme-

catalysed reaction shown on scheme 1.1

47

Figure 1.14 Plot of initial velocity as a function of substrate

concentration for a typical enzyme

48

Figure 1.15 The reciprocals of initial velocities are plotted against the

reciprocals of substrate concentration to yield a

Lineweaver-Burk plots

51

Figure 1.16 Equilibrium scheme for enzyme turnover in the presence

and absence of reversible inhibitors. Source: Copeland

(2005)

52

Figure 1.17 Cartoon representations of the three major forms of

reversible inhibitor interactions with enzymes: (A)

competitive inhibition; (B) non-competitive inhibition; (C)

uncompetitive inhibition. Source: Copeland (2005)

56

Figure 1.18 Theoretical double-reciprocal plots (1/V vs. 1/[S]) for an

enzyme in the presence of various concentrations of: (A)

competitive inhibitor, (B) non-competitive inhibitor (α = 1)

and (C) uncompetitive inhibitor

57

Figure 1.19 Workflow of the project

61

Figure 2.1 Flowchart of protocols in the production of DEN-2 NS2B-

NS3 protease

68

Figure 3.1 SDS-PAGE of fractions obtained from purification of

DEN-2 protein precursor on Ni2+

-NTA-agarose column; (a)

Fractions from column and (b) The pooled fractions. Lanes:

M, protein molecular weight marker; lane 1, 2, 3, 4, 5, 6

and 7 are the fraction 1, 2, 3, 4, 5, 6 and 7 eluted from Ni2+

-

NTA column; TS2 is pooled enzyme

95

xiv

Figure 3.2 SDS-PAGE of fractions obtained from purification of


-NTA column; Lanes: M,

protein molecular weight marker; lane 1, 2, 3, 4, 5 ,6, 7and

8 are the fraction 1, 2, 3, 4, 5, 6, 7 and 8 eluted from Ni2+

-

NTA column

95

Figure 3.3 Western blot membrane profile of Ni2+

-NTA-agarose

purified CF40.gly(T).NS3pro. Purple band indicates the

binding of His-tagged CF40.gly(T).NS3pro, and it was

observed at ~38 kDa (Source: Han, 2012).

96

Figure 3.4 Bovine serum albumin (BSA) standard curve (values are

mean ± SD, n = 3). This plot was used for to culculate the

DEN-2 NS2B-NS3 protease complex concentration

97

Figure 3.5 Elution profile of fractions obtained from purification of


-NTA-agarose affinity

column

98

Figure 3.6 The fluorescence intensity as a function of AMC

concentration (values are mean ± SD, n = 4)

99

Figure 3.7 Michaelis-Menten plot, the enzyme-catalysed reaction

velocity (V) as a function of the substrate concentration at

DEN-2 NS2B-NS3 protease enzyme concentration of

0.5µM (values are mean ± Std. Error, n = 3)

101

Figure 3.8 Pharmacophoric features of S5T5H06 and S2T3H01: HBD

shown as violet vectored spheres, HBA as light green

vectored spheres, Hbic as blue spheres, Positive ionisable

as red spheres, Ring Aromatic as orange spheres and

excluded volume as grey spheres

106

Figure 3.9 Structure of the compounds, which virtually active on the

pharmacophore models S5T5H06 and H2T3H01 that were

obtained with courtesy from National Cancer Institude

108

Figure 3.10 Lineweaver-Burk plot for Panduratin A as inhibitor. [I]

denoted the concentration of the inhibitor

111

Figure 3.11 Lineweaver-Burk plot for N1 as inhibitor. [I] denoted the

concentration of the inhibitor

112



113



114

xv



115



116



117



118



119



120

Figure 3.20 Mapping active hit N1 (NSC66742, Ki = 17 µM) against

S5T5H06 (A), S2T3H01 (B) and the chemical structure of

N1 (C). Mapping active hit N9 (NSC673801, Ki = 26 µM)

against S2T3H01 (D) and the chemical structure of N9 (E).

HBD shown as violet vectored spheres, HBA as light green

vectored spheres, Hbic as blue spheres, Positive ionisable

as red spheres, Ring Aromatic as orange spheres and

excluded volume as grey spheres

125

Figure 3.21 Most active hit compound from NADI (MSC2020:

Cinnamatannin A) on hypothesis S5T5H06 (Fit value 5.08).

HBD as violet vectored spheres, HBA as light green

vectored spheres, Hbic as blue spheres, Ring Aromatic as

orange vectored spheres and excluded volume as grey

spheres

127

Figure 3.22 Examples of the 2D molecular structures of the active fits

from NADI database that mapped with the pharmacophoric

features of the model S5T5H06. HBD as violet ring, HBA

as light green ring, Hbic as blue ring and Ring Aromatic as

orange ring

128

Figure 3.23 Two-dimentional structure of compound MSC2008 divided

into Part A and Part B. Each part consists of an active

fragment. HBD as violet ring, HBA as light green ring,

Hbic as blue ring and Ring Aromatic as orange ring

132

Figure 3.24 Two-dimentional and three-dimentional molecular

structures of the first phase design compounds map on

pharmacophore model S5T5H06. HBD as violet ring, HBA

as light green ring, Hbic as blue ring and Ring Aromatic as

orange ring

134

xvi

Figure 3.25 Molecular structure of the first phase design, G3D2

modified to second phase design. The functional groups

that map on pharmacophore model S5T5H06 are shown;

HBD as violet ring, HBA as light green ring, Hbic as blue

ring and Ring Aromatic as orange ring

135

Figure 3.26 The reaction mechanism of Schiff base formation. a)

nucleophilic attack from primary amine to the electrophilic

carbon of aldehyde, b) the first proton shift from amine to

the oxyanion, c) the second proton shift from the amine to

the hydroxyl group, d) the water elimination, e) Schiff base

product

138

Figure 3.27 The full 1H-NMR spectrum of S1

140

Figure 3.28 The expanded 1H-NMR spectrum of S1

141

Figure 3.29 The full 13C-NMR spectrum of S1

142

Figure 3.30 The FTIR spectrum of S1

143

Figure 3.31 The COSY spectrum of S1 144

Figure 3.32 The NOESY spectrum of S1

145

Figure 3.33 The HSQC spectrum of S1

146

Figure 3.34 The mass spectrum of S1 showed the ion peak of 375.2380

as M + H of corresponding compound

146


147


148

Figure 3.37 The full 13C-NMR spectrum of S2

149


149

Figure 3.39 The COSY spectrum of S2. 150

Figure 3.40

The NOESY spectrum of S2 151

Figure 3.41

The HSQC spectrum of S2.

152

Figure 4.42

The ms spectrum of S2.

152


154

Figure 3.44 The expanded 1H-NMR spectrum of S3 154

xvii


155

Figure 3.46 The mass spectrum of S3

155


156


156

Figure 3.49

The mass spectrum of S4. 157

Figure 3.50 Lineweaver-Burk plot for S1 as inhibitor. [I] denoted the


162



163



164



165

Figure 3.54 Mapping of potential inhibitors from NADI guided

compounds on pharmacophoric model S5T5H06: (A) S1,

C, Ki = 78 µM; (B) S2, NC, Ki = 232 µM; (C) S3, NC, Ki =

97 µM and (D) S4, NC, Ki = 29 µM. HBD shown as violet

sphere, HBA as light green sphere, Hbic as blue sphere,

Ring Aromatic as orange sphere and excluded volume as

grey spheres. C and NC signified competitive and non-

competitive, respectively

169

Figure 3.55 Suggested potential lead structure for the design of non-

competitive DEN-2 NS2B-NS3 protease inhibitor

173

xviii

LIST OF ABBREVIATIONS

(p-Me)Phe : Para methyl phenyl

µL : Microliter

µM : Micromolar

µm : Micrometer

2D : Two-dimensional

3D : Three-dimensional

3D-QSAR : Three dimension quantitative structure activity relationship

AMC : 7-Amino-4-methylcoumarin

APS : Ammonium persulphate

BSA : Bovine serum albumin

C : Capsid

CBB : Coomasie Brilliant blue

CBB R-250 : Coomasie Brilliant blue R-250

CFR : Case fatality rate

CHARMm : Chemistry at HARvard Macromolecular Mechanics

COSY : Correlation spectroscopy

DEN-1 : Dengue Virus Type 1




DENV : Dengue virus

DHF : Dengue haemorrhagic fever

DMSO : Dimethyl sulfoxide

DS : Discovery Studio

DSS : Dengue shock syndrome

E : Envelope

EI : Enzyme-inhibitor complex

ER : Endoplasmic reticulum

FV : Fit value

g : Gram

GFA : Genetic function algorithm

http://en.wikipedia.org/wiki/Macromolecular

xix

HBA : Hydrogen bond acceptor

HBD : Hydrogen bond donor

Hbic : Hydrophobic

HCl : Hydrochloric acid

HEPES : 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HSQC : Heteronuclear single quantum correlation

IC50 : Half maximal inhibitory concentration

IPTG : Isopropyl–β–D–thiogalactopyranose

JEV : Japanese Encephalitis Virus

kDA : Kilo Dalton

Ki : Dissociation constant for the EI complex

Km : Michaelis constant

LB : Lysogeny broth

M : Membrane

m : Meta

mM : Millimolar

mm : Millimeter

mmol : Millimole

MAct : Most Active Compound

MD : Molecular dinamics

MLR : Multiple linear regression

mRNA : Messenger ribonucleic acid

MS : Mass spectroscopy

NaCl : Sodium chloride

NADI : Natural Product Discovery System

NC : Nucleocapsid core

NCI : National Cancer Institute, USA

Ni2+

-NTA : Nickel-nitrilotriacetic acid

nm : Nanometer

NOESY : Nuclear Overhause effect spectroscopy

NS2B : Non-Structural Protein 2B

NS3 : Non-Structural Protein 3

NS5 : Non-Structural Protein 5

xx

NTPase : RNA nucleoside triphosphatases

o : Orto

OD : Optical density

ORF : Open reading frame

p : Para

PDB : Protein Data Bank

prM : Pre membrane protein

prM-E : Pre membrane envelope

QTOF : Quadrupole time-of-flight

RNA : Ribonucleic acid

rpm : Round per minute

RTPase : RNA 5'Triphosphatase

SAR : Structure activity relationship

SDS : Sodium dodecyl sulphate

SDS-PAGE : Sodium dodecyl sulfate polyacrylmide gel electrophoresis

TBEV : Tick-borne encephalitis virus

TEMED : N,N,N,M’-tetramethylethyldiamine

TLC : Thin layer chromatography

TMS : Tetramethylsilane

Tris : Tris(hydroxymethyl)aminomethane

Tris-HCl : Tris(hydroxymethyl)aminomethane – HCl buffer

Trp : Tryptophan

Tyr : Tyrosine

UTR : Untranslated region

Vmax : Maximum velocity

WHO : World Health Organisation

WNV : West Nile virus

YFV : Yellow fever virus

α : Alpha

π-π : Pi-pi

π-cation : Pi-cation

π-σ : Pi-sigma

xxi

LIST OF PUBLICATIONS

Mohamed Sufian M. Nawi, Noorsaadah Abd. Rahman, Shafida Abd. Hamid and

Habibah A. Wahab (2010). Pharmacophore Modeling and in Silico Screening

of NADI Database for the Discovery of Dengue Virus NS2B-NS3 Protease

Inhibitors. International Seminar and Expo on Jamu 2010. Universitas

Padjadjaran, Bandung, Indonesia, 5-6th November 2010.

Habibah A Wahab, Suriyati Muhamad, Mohamed Sufian Mohd Nawi, Mohd Razip

Asarudin, Maywan Hariyono and Ezatul Ezleen Kamarulzaman (2013).

Nature, the Source of Lead Compounds for Drug Discovery. International

Conference on Medicinal Chemistry and Timmerman Award 2013.

Universitas Indonesia, Jakarta, Indonesia, 28-29th

October 2013.

Habibah A Wahab, Suriyati Muhamad, Mohamed Sufian Mohd Nawi, Nur Kusaira

Ikram, Maywan Hariyono and Ezatul Ezleen Kamarulzaman (2013).

Interoging Nature for Lead Compounds for Drug Discovery Using

Computational Techniques. The 5th

Seminar and Workshop on

Computer-Aided Drug Design. Bandung, Indonesia, 9-13th

December 2013.

Muhammad Asyraf Abduraman, Mohamed Sufian Mohd Nawi, Habibah A.

Wahab, Rohana Yusof and Tan Mei Lan (2013). The Development of Dengue

Virus NS2B/NS3 Protease Inhibition Assay for Lead Compound Optimization

In Drug Discovery Process. International Conference on Medical and Health

Science. Kota Bharu, 22-24th

May 2013.

Muhammad Asyraf Abduraman, Mohamed Sufian Mohd Nawi, Habibah A.

Wahab, Rohana Yusof and Tan Mei Lan (2013). The Development of

Specific NS2B/NS3 Protease Inhibition Assay Using AlphascreenTM

Technology. National Biotechnology Seminar. Penang, Malaysia, 6-8th

June

2013.

xxii

PENEMUAN UBAT BERASASKAN LIGAN UNTUK PERENCAT

NOVEL PROTEASE NS2B-NS3 DENGGI-2

ABSTRAK

Kes demam denggi dilaporkan meningkat setiap tahun, namun sehingga kini

belum ada ubat anti-denggi boleh didapati di pasaran. Oleh itu, pencarian ejen anti-

denggi adalah kritikal. Pemotongan poliprotein pelopor oleh enzim protease NS2B-

NS3 merupakan proses yang sangat penting untuk replikasi flavivirus. Oleh itu

enzim protease NS2B-NS3 sesuai dijadikan sasaran untuk membangunkan ubat anti-

denggi. Di Malaysia, Denggi-2 adalah jenis sera yang paling lazim. Dalam kajian ini,

pendekatan berasaskan ligan telah dilaksanakan dalam mencari perencat enzim

protease NS2B-NS3 Denggi-2 baharu yang berpotensi. Model farmakofor telah

dihasilkan daripada pelbagai struktur perencat enzim protease NS2B-NS3 Denggi-2

yang pernah dilaporkan, yang terdiri daripada molekul peptida dan bukan peptida.

Model farmakofor terpilih telah digunakan untuk menyaring senarai sebatian dalam

pengkalan data National Cancer Institute, AS (NCI) untuk mencari perencat protease

NS2B-NS3 Denggi-2 yang baharu secara maya. Produk semulajadi dalam pengkalan

data Natural Product Discovery System (NADI) juga disaring dan struktur yang aktif

secara maya telah dieksploitasi untuk merekabentuk molekul perencat baru, yang

kemudiannya disintesis. Aktiviti-aktiviti perencatan sebatian yang aktif secara maya

dari pengkalan data NCI dan yang disintesis telah diuji secara biocerakinan in-vitro

terhadap enzim protease NS2B-NS3 Denggi-2 menggunakan substrat peptida Boc-

Gly-Arg-Arg-MCA. Kewajaran pemilihan model farmakofor S5T5H06 dan

S2T3H01 telah disahkan secara ujikaji dengan penemuan 3 perencat enzim protease

NS2B-NS3 Denggi-2 baharu dari pengkalan data NCI dengan nilai-nilai Ki 17 ± 6

xxiii

μM (perencat tidak kompetitif), 26 ± 4 μM (perencat kompetitif) dan 95 ± 35 μM

(perencat kompetitif). Disamping itu, 1 perencat kompetitif (Ki = 78 ± 41 μM) dan 2

perencat tidak kompetitif (Ki = 29 ± 6 μM dan Ki = 97 ± 49 μM) telah diperolehi dari

molekul yang disintesis berasaskan struktur sebatian daripada pengkalan data NADI.

Penemuan menarik ini mendedahkan bahawa produk semulajadi dari kepelbagaian-

bio Malaysia yang terdapat dalam pengkalan data NADI adalah sumber yang

berharga untuk pembangunan ejen anti-denggi. Daripada keputusan ujikaji ini juga,

satu andaian dapat dibuat iaitu ligan yang mempunyai farmakofor seperti model

S5T5H06 boleh terikat kepada tapak ikatan aktif atau tapak ikatan alosterik dengan

mod ikatan yang sama. Daripada kajian “SAR”, satu struktur pemula yang berpotensi

untuk membangunkan perencat tidak kompetitif terhadap protease NS2B-NS3

Denggi-2 telah dicadangkan. Struktur ini mempunyai nukleus pusat yang terdiri

daripada di-fenilmetana. Nukleus pusat ini dihubungkan oleh kumpulan imina di

kedudukan 1 dan 1' ke dua tangan fenil, R1 dan R2. Selain itu, struktur pemula ini

perlu mempunyai kumpulan hidrofobik dan penerima ikatan hidrogen (HBA) sebagai

rantaian sampingan kepada R1 dan penderma ikatan hidrogen (HBD) sebagai

rantaian sampingan kepada R2. Berdasarkan keputusan-keputusan yang diperolehi,

dapat disimpulkan bahawa farmakofor model S5T5H06 dan S2T3H01 boleh

digunakan untuk pencarian perencat enzim protease NS2B-NS3 Denggi-2 yang

berpotensi daripada pengkalan data kimia. Farmakofor model S5T5H06 dan

S2T3H01 juga boleh dijadikan panduan untuk merekabentuk perencat enzim

protease NS2B-NS3 Denggi-2 berpotensi yang baru berdasarkan struktur sasaran

yang aktif.

xxiv

LIGAND BASED DRUG DISCOVERY OF NOVEL DENGUE-2

NS2B-NS3 PROTEASE INHIBITORS

ABSTRACT

The reported dengue cases are increasing yearly, yet no anti-dengue agent is

available in the market. Therefore, the search for anti-dengue is critical. In Malaysia,

Dengue-2 (DEN-2) is the most prevalent serotype. NS2B-NS3 protease is the

enzyme for the cleavage of polyprotein precursor, which is crucial for the flavivirus

replications. This makes it a potential target for the development of therapeutics

against the dengue virus. In this study, ligand-based approach was implemented in

searching for the new potential DEN-2 NS2B-NS3 protease inhibitors.

Pharmacophore models were developed from diverse reported structures of DEN-2

NS2B-NS3 protease inhibitors, comprising peptide and non-peptide molecules. The

selected pharmacophore models were employed to screen the US National Cancer

Institute (NCI) list of compounds to search for new DEN-2 NS2B-NS3 protease

inhibitors. The list of natural products in Natural Product Discovery System (NADI)

database was also screened and the structures of active hits were exploited for the

design of new inhibitor molecules. The designed molecules were then synthesised.

The in-vitro assay were conducted to examine the inhibitory activities of the selected

hits from NCI database and the synthesised compounds towards recombinant DEN-2

NS2B-NS3 protease by using fluorogenic peptide substrate Boc-Gly-Arg-Arg-MCA.

The validity of selected pharmacophore models, S5T5H06 and S2T3H01 was

experimentally established by the identification of three new DEN-2 protease

inhibitors retrieved from NCI database with Ki values of 17 ± 6 μM (non-

competitive), 26 ± 4 μM (competitive) and 95 ± 35 μM (competitive). One

competitive inhibitor (Ki = 78 ± 41 μM) and two non-competitive inhibitors (Ki = 29

xxv

± 6 μM and Ki = 97 ± 49 μM) were found from the NADI guided compounds. This

interesting finding revealed that the natural products from Malaysian biodiversity,

which are available in NADI database, are valuable resources of active

compounds/active fragments for the development of anti-dengue. It was postulated

that the active site and the allosteric binding site on DEN-2 protease share common

binding mode to the ligand that carry the pharmacophores of the model S5T5H06.

From SAR study, a potential lead structure for DEN-2 NS2B-NS3 protease non-

competitive inhibitor was suggested. The structure should has a central nucleus made

up of diphenylmethane connected by imines at the position 1,1’ to two phenyl lateral

arms, R1 and R2. Additionally, the structure should have a hydrophobic and a

hydrogen bond acceptor (HBA) as side chains to the phenyl group R1 and a

hydrogen bond donor (HBD) as a side chain to the phenyl group R2. The findings

concluded that pharmacophoric models S5T5H06 and S2T3H01 can be useful for

pharmacophoric exploration of chemical databases in searching for the potential

DEN-2 NS2B-NS3 protease inhibitors as well as a guide to design new potential

inhibitors from the active hits.

1

1 CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 PROBLEM STATEMENT

Dengue is the most prevalent arthropod-borne viral infection in human. It is

estimated that dengue virus infection is 50 – 100 million per year globally in over

100 tropic and sub-tropic countries where over 2.5 billion people (over 40% of

world’s population) are at risk. Out of these 2.5 billion people at risk, 1.8 billion (>

70%) are in Asia Pacific countries (Kumar et al., 2012; WHO, 2009, 2013a, 2013b).

The trend of reported dengue cases in Western Asia Pacific region varies from region

to region for example from 2006 to 2012, Australia, Cambodia, Lao PDR, Malaysia,

Philippines and Vietnam have reported more cases in 2012 than in 2011. Malaysia in

particular reported 21,900 of Dengue cases with 35 deaths (CFR 0.2%) for the year

2012 compared to 19,884 cases (36 deaths, CFR 0.2%) in 2011 (WHO, 2013a). As at

December 2013, Australia, Lao PDR, Malaysia, New Caledonia and Singapore have

reported higher cumulative cases in 2013 compared to 2012 for the same time period

(WHO, 2013c). In Malaysia, the cases rapidly increased (43346 cases with 92 death)

by the end of 2013, which is two-fold increase relative to 2012 (WHO, 2014). Recent

statistics of dengue cases and fatalities in Malaysia are ringing the alarm bells across

the country. From 1st to 22

nd January 2014, there were 6,155 dengue cases reported,

compared to 1,792 cases within the same period in 2013 (six-fold increase). The

number of deaths has doubled from five deaths in 2013 to 10 deaths in 2014, all

within the same period (Ismail, 2014).

2

The continuous prevalent of dengue cases drive the researchers to search and

develop the vaccine and anti-dengue drugs for the treatment of dengue. The search

for lead anti-dengue compounds has been done from many resources. In traditional

medicines plants extracts, several species have been reported with anti-dengue

activity (Reis et al., 2008). A sulphated polysaccharide, named fucoidan, from

marine algae was found as a potential anti-dengue agent (Hidari et al., 2008) while

there have been many discoveries of potential anti-dengue from synthetic compounds

using many approaches such as in silico screening from compounds libraries

(Kampmanna et al., 2009; Wang et al., 2009), high throughput in vitro screening and

structure-based drug design (Tomlinson et al., 2009).

Figure 1.1: Several structures of potential anti-dengue compounds reported by

Kampmanna et al., 2009 ((a) and (b)), Wang et al., 2009 (c) and Tomlinson et al.,

2009 ((d) and (e)).

N

N N

S NH

NH

O

Cl

Cl

Cl

O

N

N

S

CH3

N NH

CH3

Cl

Cl

(a)

IC50 32 ± 17 μM

(b)

IC50 1.2 ± 0.7 μM

3

Figure 1.1: Continued.

From the understanding of the viral life cycle, the virus structural proteins

(capsid protein, C, membrane-associated protein, prM and envelope protein, E) as

well as non-structural proteins (NS1, NS2B, NS3 and NS5) have been identified as

potential targets for the development of vaccine and anti-dengue drug. The

development of imaging techniques, high resolution x-ray crystallography and

computer simulation technology enable the proteins structure models of the virus to

be available (Erbel et al., 2006) to assist researchers to understand the interactions of

the target proteins and the drugs. This availability helps the researchers to speed up

the finding of new drugs at lower cost.

Nevertheless, the development of vaccines and antiviral therapy has seen little

success where no licensed antiviral therapy is currently available (Sampath &

N

N NH N

S

Cl

OH O OH

NO2ONO2

N

O

CH3 CH3

O NH2

OH

CH3

OH

(c)

EC50 0.119 ± 0.03 μM

(e) (d)

EC50 35 ± 8 μM EC50 4.2 ± 9 μM

4

Padmanabhan, 2009). The control of disease is focused on the control of mosquito

vector, which is costly and often met with limited success (Gubler, 1998). Therefore,

the search for the lead compounds and derivatives with their information on the

structures and activities is needed in discovering novel inhibitor for the development

of anti-dengue.

1.2 DENGUE

Dengue is a disease caused by the infection of the dengue virus. There are

four antigenically related but genetically distinct serotypes of dengue viruses; DEN-

1, DEN-2, DEN-3 and DEN-4, which are transmitted to human by female Aedes

aegypti and Aedes albopictus mosquitoes. The symptom of a disease similar to

dengue was reported since the end of 18th

century. In 1780, Benjamin Rush, a

physician of Philadelphia, provided the first detailed description of what we know

now as dengue fever. However, the first extensively documented outbreak of dengue

haemorrhagic fever (DHF) occured in the Philippines in 1953 followed by Thailand

in 1958. Since 1960s dengue has spread to over 60 countries (Howard, 2005).

Dengue is most prevalent in tropical Asia, Latin America and Caribbean

(Deen et al., 2006; Kyle & Harris, 2008; Mukhopadhyay, et al., 2005). From the

estimated number of dengue virus infection of 50 – 100 million per year globally,

250,000 – 500,000 patients develop more severe diseases of dengue haemorrhagic

fever (DHF) and dengue shock syndrome (DSS) with 1-5% of cases result in fatality

(Rawlinson et al., 2006). The incidence of dengue has increased dramatically in the

past 50 years due to the population growth and uncontrolled urbanization in tropical

and subtropical countries that become breeding sites for the mosquitoes and

5

problematic vector control (Kyle & Harris, 2008). In addition, the increased global

transports networks such as air travel has enabled infected humans to spread viruses

(Gibbons & Vaughn, 2002).

Dengue symptoms appear after five to eight days following the bite by an

infected mosquito (Smith, 1995). The person infected by dengue virus may manifest

ranges of symptoms from silent infection (no symptom) to fatal dengue

heamorrhagic fever (DHF)/ dengue shock syndrome (DSS) (Gibbons & Vaughn,

2002). The WHO scheme has classified dengue virus infection into three categories;

dengue fever, DHF and DSS.

Dengue fever is clinically defined as an acute febrile illness with two or more

manifestations (headache, retro-orbital pain, myalgia, arthralgia, rash, haemorrhagic

manifestations, leucopenia) and the occurrence should be at the same location and

time as other cases of dengue fever. DHF is defined as a case that must meet all four

of the following criteria: fever or history of fever lasting 2-7 days, a haemorrhagic

tendency shown by a positive tourniquet test or spontaneous bleeding,

thrombocytopenia and evidence of plasma leakage. DHF is further classified into

four severity grades according to the presence or absence of spontaneous bleeding

and the severity of plasma leakage. The dengue shock syndrome (DSS) refers to

DHF grades III and IV, in which shock is present as well as all four DHF defining

criteria (Deen et al., 2006; Gubler, 1998). Schematic of WHO classification of

symptomatic dengue infection and WHO classification of dengue haemorrhagic fever

are shown in Figure 1.2 and Table 1.1, respectively.

6

Figure 1.2: WHO classification of symptomatic dengue infection (Source: Deen et

al., 2006).

Among the infected person, less than 1% developed DHF/DSS. The

development of DHF/DSS was influenced by many factors. The patience with

sequential infection of distinct dengue virus serotype from the primary infection

showed to have 15-80-fold likely to develop DHF. However, patience that has not

undergone a previous infection can also develop DHF. The trigger factors to the

development of DHF could be host related such as age, sex, race, nutritional status

and genetic predisposition. Children under 15 years old are more likely to develop

DHF. However, in the case of DEN-2 outbreak in Santiago De Cuba in 1997, all

DHF are in adults. Female are more likely to develop a more severe disease. In the

case of race factor, in Cuban epidemic in 1981, white Cubans were 3.5-fold more

hospitalised compared to the black Cubans (Howard, 2005; Guzman, et al., 2006;

Rawlinson et al., 2006).

7

Table 1.1: WHO classification of dengue haemorrhagic fever. Source: (Howard,

2005).

Grade Clinical description

I Fever with non-specific constitutional symptoms and the only

haemorrhagic manifestations being a positive tourniquet test

II As for Grade I, but accompanied by more extensive haemorrhagic

manifestations

III Sign of circulatory failure or hypertension

IV Profound shock with pulse and blood pressure being undetectable

Another trigger factor is the viral factor. The differences in genotype between

strains also influence the severity of the disease. This phenomenon is shown by the

capability of DEN-2 strain from Southeast Asia to cause DHF while the same strain

did not cause DHF in epidemic event in Peru in 1995 even though the population

had undergone an epidemic of DEN-1 five years before. This is due to the fact that

DEN-2 from Southeast Asia and Peru have differences in six amino acid in structural

and non-structural proteins as well as at the 5’ and 3’ un-translated region of virus

RNA. However, the exact factors for the minor cases of DHF/DSS development is

still not clear (Rawlinson et al., 2006).

1.3 DENGUE VIRUS

Dengue virus is a member of Flaviviridae family from Flavivirus genera. The

Flaviviridae family consists of three genera; Flavivirus, Pestivirus and Hepacivirus.

Genus Flavivirus contains more than 70 viruses including dengue virus (DENV),

Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile

virus (WNV) and yellow fever virus (YFV). Dengue viruses are further divided into

four serotypes 1, 2, 3 and 4 (DEN-1, DEN-2, DEN-3 and DEN-4).

8

1.3.1 The structure and genome of dengue virus

Dengue virion is a spherical enveloped virus with diameter approximately

500Å. It comprises a single, positive-strand RNA genome of ~10,700 nucleotides

that is packed by three structural proteins and a lipid bilayer. The structural proteins

are capsid, C; membrane-associated (M, which is expressed as prM, precursor to M)

and envelope, E (Kuhn et al., 2002; Mukhopadhyay et al., 2005; Rey, 2003). The

RNA of dengue virus consist of a 5’-untranslated region (UTR), a single open

reading frame (ORF) and 3’-UTR (Dong et al., 2008). The RNA has type 1 cap at the

5’-terminus (m7GpppAmp) and polyadenylate [Poly(A)] tail at 3’-terminus (Wahab

et al., 2007). The three structural proteins (C, prM and E) are encoded by 5’-

terminus of the ORF of the genome. The remainder of the ORF of the genome

encodes seven nonstructural proteins i.e. NS1, NS2A, NS2B, NS3, NS4A, NS4B and

NS5 (Figure 1.3).

Figure 1.3: Schematic representation of flavivirus genome organization and

polyprotein processing. Sites of polyprotein cleavage mediated by the viral NS2B-

NS3 and by host signalase are shown. The enzymatic activities of NS3 and NS5 are

also indicated. Source: (Sampath & Padmanabhan, 2009)

2K

Hydrophobic peptide

9

1.3.2 Life cycle of Flavivirus

The primary mammals’ cells infected by dengue virus are monocytes,

macrophages and dendritic cells (Rawlinson et al., 2006; Sampath & Padmanabhan,

2009). The life cycle of dengue virus and flavivirus in general, started with the

attachment and binding of the virions on the host cell surface, followed by the entry

into the cell by the process of endocytosis to form endosome. Endosome then

undergoes the process of fusion where the RNA is released into the cytoplasm. The

viral RNA is next translated into a polyprotein, which is cleaved into viral proteins

(structural and non-structural) by polyprotein processing. Then, the replication of

RNA and virus assembly occur. Finally, budding occurs where the new mature

virions are released from the host cell by exocytosis (Guzman, et al., 2006; Lescar et

al., 2008; Mukhopadhyay et al., 2005). The schematic diagram of the flavivirus life

cycle is illustrated in Figure 1.4.

10

Figure 1.4: Flavivirus life cycle. Numbers shown in boxes refer to the pH of the

respective compartments. Source: (Perera et al., 2008).

1.3.2.1 Entry to the cell

In the initial dengue virus infection process, the virion attaches to the host cell

by the binding of E protein onto the receptor on the host cell surface (Rawlinson et

al., 2006) . Then, the virion enters the cell by receptor-mediated endocytosis to form

endosome (Sampath & Padmanabhan, 2009). There are different receptors for

dengue virus on different cell types for the attachment. Fcɣ-R is the receptor for

macrophages and monocytes, while the C-type lectine DC-SIGN(CD209) is the

receptor for dendritic cell for the infection of all four dengue serotypes (Rawlinson et

al., 2006).

11

Following endocytosis, the RNA genome is released into the cytoplasm of the

host cell through the fusion of the viral membrane and the host-cell endosomal

membrane. Acidic environment (low pH) in endosome mediates the structural

reorganization of E protein (Sampath & Padmanabhan, 2009) from homodimers into

homotrimers (Rawlinson et al., 2006). The trimeric conformation of the E protein

forms more open structure, exposing the fusion peptide, thereby bringing the viral

and endosomal membrane into close proximity and allowing insertion of the virus

into the host cell membrane. As a result, fusion of the viral and host-cell endosomal

membranes occurs. Upon fusion, the nucleocapsid, which carries virus RNA is

released into the cytoplasm of the host cell (Lescar et al., 2008; Mukhopadhyay et

al., 2005; Rawlinson et al., 2006). The capsid protein and viral RNA dissociation is

probably a spontaneous process that occurred upon the release of nuclecapsid

(Rawlinson et al., 2006).

1.3.2.2 Translation and polyprotein processing

In cytoplasm, the viral RNA served as mRNA that is directly translated into a

long single polyprotein precursor of approximately 370 kDa. The polyprotein

precursor is arranged in the order of NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-

NS4B-NS5-COOH (Wahab et al., 2007). The polyprotein is then cleaved into the

three viral structural (C-prM-E) and seven non-structural (NS1-NS2A-NS2B-NS3-

NS4A-NS4B-NS5) proteins by viral-encoded serine protease (NS2B-NS3), host cell

proteases (signalase and furin) as well as other unknown protease (Lescar et al.,

2008; Mukhopadhyay et al., 2005; Rawlinson et al., 2006; Sampath & Padmanabhan,

2009).

12

In addition to the ten proteins, two small hydrophobic peptides are released

from the polyprotein (Figure 1.3). One is derived from the C-terminus of the

anchored capsid protein; after the cleavage of this fragment, the mature capsid is

released. The second one is a small fragment between the NS4A and the NS4B that

is called 2K (Guzman, et al., 2006).

The viral NS2B-NS3 protease is involved in the cleavage on the cytoplasmic

side of the endoplasmic reticulum (ER) membrane whilst host cellular protease takes

part in the cleavage in the ER lumen (Lescar et al., 2008). The viral NS2B-NS3

protease is responsible for the cleavage at the NS2A/2B, NS2B/NS3, NS3/NS4A,

NS4A/2K and NS4B/5. Host protease cleaves at the C/prM, prM/E, E/NS1 and

2K/NS4B junctions (Figure 1.3) that release prM, E and NS1 proteins in the ER

lumen (Guzman, et al., 2006; Rawlinson et al., 2006).

1.3.2.3 RNA replication

Genome replication occurs in intracellular membranes (Mukhopadhyay et al.,

2005) and Golgi-derived membranes called vesicle packed (Sampath &

Padmanabhan, 2009). Non-structural proteins mediate the transcription of viral RNA.

Thus, transcription started after the non-structural proteins have been synthesised and

released from the polyprotein precursor (Lescar et al., 2008). Starting from the

genomic plus-strand RNA, the complimentary negative-strand RNA is synthesised,

which then served as the template for the production of an additional genomic plus-

strand RNA (Guzman, et al., 2006).

13

1.3.2.4 Viral assembly and release of new progeny virion

Virus assembly occurs on the surface of the endoplasmic reticulum (ER)

(Mukhopadhyay et al., 2005). The newly synthesised plus stranded RNA is coated

with C protein to form nuclecapsid. The mechanism of interaction of the C protein in

nucleocapsid is still not clear (Sampath & Padmanabhan, 2009). Then, E and prM

envelop the nucleocapsid resulting in the formation of non-infectious, immature viral

and subviral particles in the lumen of ER. Immature viral particle contains E and prM

proteins, lipid membrane and nucleocapsid. On the other hand, subviral particle does

not contain nucleocapsid. These particles are non-infectious as they cannot induce

host-cell fusion. They are transported through the trans-Golgi network where the

maturation of the virus particles occurs. In the trans-Golgi network, the prM is

cleaved to M by the host protease furin and rearrangements of E protein occur,

resulting in the formation of mature, infectious particles (Perera et al., 2008).

Subviral particles are also cleaved by furin. Mature and subviral particles are

subsequently released from the cell by exocytosis (Mukhopadhyay et al., 2005;

Sampath & Padmanabhan, 2009).

1.4 PROTEINS OF DENGUE VIRUS

1.4.1 Structural proteins and functions

The capsid protein (~100 amino acids) is involved in the packaging of viral

genome/form nucleocapsid core (NC). The prM protein (~165 amino acids) might

function as a chaperone for the folding and assembly of E protein during particle

maturation. The E protein (~495 amino acids) constructs the envelope structure of

the virus.

14

1.4.1.1 Capsid protein

Dengue-2 virus capsid protein exists in dimer form and has been suggested to

be the building block for nucleocapsid assembly. Nuclecapsid core formed in the

early stage of assembly process, consists of one copy of genomic RNA and multiple

copies of the capsid protein. However, NCs are rarely found in flavivirus-infected

cells, which indicated that the particle formation is a coordinated process between the

membrane-associated capsid protein and the prM-E heterodimers in the ER

(Mukhopadhyay et al., 2005). The cryoEM reconstruction of mature and immature

dengue virus show that the nucleocapsid is not a well-formed protein shell. It has

been suggested that the capsid protein facilitates the binding of the nucleocapsid to

the lipid membrane (Ma et al., 2004).

1.4.1.2 E and M proteins

The E protein, the external structural protein of dengue virus is responsible for

the binding with cellular receptor and interacts with the host cell membrane for

fusion and penetration, as well as viral assembly and budding. E protein also

possesses antigenic determinants that elicit neutralizing antibodies (Huang et al.,

2008; Kuhn et al., 2002; Modis et al., 2004, 2005; Mukhopadhyay et al., 2005;

Zhang et al., 2004). The ability of monoclonal antibody to neutralise dengue virus

serotype 1, 2 and 3, is primarily by inhibiting the attachment to the host. Therefore,

the change in viral surface structure was assumed to be responsible for inhibiting

viral attachment to cell (Lok et al., 2008).

15

During the flavivirus life cycle, the virion exists in three main conformational

states: immature, mature and fusion-activated. The conformation of the E protein

differs in these three states. The conformational rearrangement occur from prM–E

heterodimers in immature particle to E homodimers in the mature particle, and

finally to E homotrimers in the fusion-activated particle (Perera et al., 2008).

Following the assembly process, an immature virus particle is produced in

ER, which have pre-membrane protein (prM) that must be proteolytically processed

during virion maturation. There are two types of immature virus; “spiky” and

“smooth” virus particles. Immature virus particles have a diameter of ∼600 Å

(Figure 1.5A and 1.5C) with a spiky surface protein shell consisting of prM and E

proteins, which form 180 heterodimers that are arranged as 60 trimeric spikes.

Within each spike there are three prM-E heterodimers (Zhang et al., 2003; Zhang et

al., 2004). The prM protein forms a cap-like structure that protects the fusion peptide

on E protein and prevents the fusion of the virus with the host membrane.

In Golgi apparatus, low pH environment trigger rotational and translational

movement of E protein resulting in the transition of E protein from prM heterodimer

(Figure 1.5C) to anti-parallel E homodimers (Figure 1.5D). The “smooth” viral

morphology is formed but is still considered immature due to the presence of prM

protecting the fusion peptide on E. Maturation process of flavivirus is directed by

proteolitic cleavage of the precursor membrane protein (prM) (Li et al., 2008). Then,

furin cleaves the prM into pr and M. The cleaved pr portion remains associated with

E and is only released from the virus particle following the exit into neutral pH

environment. Following maturation, the particle is released into a neutral

16

environment and pr peptide is released from the virion (Perera et al., 2008).

Figure 1.5: Structure of flaviviruses. (A) A surface shaded view of the cryo-EM

reconstruction of immature DENV-2 showing the spiky surface features. (B) A

surface shaded view of the cryo-EM reconstruction of mature DENV-2 showing the

relatively smooth surface features (C) Fit of the atomic coordinates of the E protein

into the immature virus and (D) Mature virus showing the arrangement of the E

proteins on the surface of the virion. Source: (Perera et al., 2008)

Mature flavivirus particles (diameter ∼500 Å) have a relatively smooth

surface with the lipid bilayer membrane completely covered by the envelope (E) and

membrane (M) protein shell (Figure1.5B and 1.5D). This shell consists of 180 copies

of the E protein arranged as 90 homodimers forming a herringbone pattern or so-

17

called protein rafts that lie flat on the viral surface. Following the attachment of

mature virus particle, specific cell-surface receptors mediate endocytosis of the virus.

In the endosome, in the low pH environment, the E homodimers within the mature

virion dissociate and re-arrange into fusion-active homotrimers. The fusion peptide

of the virus becomes exposed at the distal end of the E protein and is inserted into the

host membrane, which promotes fusion of the viral and host membranes (Modis et

al., 2004; Perera et al., 2008).

1.4.2 Non-structural proteins and functions

Nonstructural proteins are essential for viral replication. NS1 involves in early

steps of viral replication (Lindenbach & Rice, 1999), while NS3 shows enzymatic

activities, which is involved in the viral polyprotein processing and genome

replication. NS3 functions as a serine protease (NS2B-NS3 complex mediate

proteolytic processing of polyprotein), 5’-RNA triphosphatase (RTPase), nucleoside

triphosphatase (NTPase) and helicase (Luo et al., 2008). NS5 exhibits two enzymatic

activities which are involved in the methylation of 5’-cap structure of genomic RNA

(methyltransferase) and RNA-dependent RNA polymer (Dong et al., 2008; Geiss et

al., 2009; Zhou et al., 2007). Due to the improved knowledge on the structures and

functions of the proteins, the development of vaccine and therapeutic design

currently target E, NS1, NS3 and NS5 proteins (Wahab et al., 2007).

18

1.4.2.1 Dengue virus NS2B-NS3 protease

Proteins are made up of a long chains of amino acids that bonded by carbon-

nitrogen bonds called amide bonds or peptide bonds. Chemical hydrolysis of amide

bond requires activation energy of ~25 kcal/mol. Therefore, under normal

physiological conditions (pH~7, T = 37 °C), hydrolysis of amide bond would be very

slow. However, catalysis of amide bond hydrolysis under physiological condition is

accomplished at much faster rate by enzymes called proteases or peptidase.

The protease or proteolytic enzymes not only catalyze protein hydrolysis at a

fast rate but also specific and selective in order to prevent uncontrolled proteolysis of

the organism. Proteases can be divided into classes based on their mechanism of

action, which are serine, cysteine, aspartic, threonine and metallo proteases. As an

example, a serine protease is an enzyme in which one of the amino acids at the active

site is serine and the catalytic nucleophile in serine protease is hydroxyl group of the

active site serine.

Protease binds with the substrate in a specific manner that is represented by

Schechter and Berger system of nomenclature. According to this system, the amino

acid residues (or side chains) of substrate are labeled from the N to C terminus as

Pn,.......,P3,P2,P1,P1’,P2’,P3’,........Pn’ and the corresponding binding sub sites of the

enzyme are labelled as Sn,.......,S3,S2,S1,S1’,S2’,S3’,........Sn’. The peptide bond

hydrolysis is carried out between P1,P1’ and the bond is called the scissile bond

(Figure 1.6). The P1 residue of the substrate is called the primary specificity residue.

The specificity and selectivity of a protease depend on the nature of the P1 residue.

As an example trypsin only cleaves at lysine or arginine residues (positively charged

http://en.wikipedia.org/wiki/Amino_acids

19

residues/basic residues at P1). Serine proteases are classified as elastase-like (small

hydrophobic residues at P1), trypsin-like (positively charged residues/basic residues

at P1) or chymotripsin-like (large hydrophobic residues at P1).

Figure 1.6: Schehter and Berger System of Nomenclature.

NS2B-NS3 protease is a serine protease. Serine protease utilises a

combination of mechanisms that are common to enzyme catalysis. First, enzyme

binds to the substrate to form an enzyme-substrate Michaelis-Menten complex (E-S

complex) utilizing non-covalent bonding interaction such as ionic interactions,

dipole-dipole interactions, hydrophobic interactions, hydrogen bonding and Van der

Waal’s interactions. When the substrate is bound to the active site of the enzyme, the

carbonyl group of the scissile amide bond is exposed for catalysis by enzyme.

The active site of serine protease consists of three conserved amino acid

residue; serine, histidine and aspartic acid that together form the “catalytic triad”.

Serine protease utilises an active site serine residue in a covalent catalytic cleavage

of peptide bonds. Nucleophilic group of serine is hydroxyl, which is a poor

nucleophile. However, the serine hydroxyl is activated by general base catalysis

NHNHNH

O

P1'O

P1O

NH

P2'

O P3'

CH3NH

P2O

P3

CH3

S1 ' S2

S2 ' S

1 S3

scissile bond

20

through hydrogen bonding network involving aspartic acid, histidine and serine

residues nearby. This process converts serine to alkoxide (ionised serine) through a

mechanism called the charge relay system (Silverman, 2004). The active site of

serine (activated nucleophile) then attacks the carbonyl group of the scissile amide

bond (Figure 1.7).

Figure 1.7: Charge relay system for activation of an active site serine residue.

Dengue NS3 protein is made up of 618 amino acids. It consists of two

domains that possess enzymatic activity: a trypsin-like serine protease located in the

N-terminal domain of the protein and a C-terminal domain that possess RNA

helicase and NTPase activity (Murthy et al., 1999). The NS3 protease domain adopts

a chymotrypsin-like fold with two β-barrels, each formed by six β-strands

(Figure1.8), with the catalytic triad (His51-Asp75-Ser135) located at the cleft

between the two β-barrels.

NNH

His

O

Ser

H

Asp

O-

O

NHNH

P1

O P1'

O

NHPeptide2

Peptide1

O

21

Figure 1.8: Structures of NS2B–NS3 protease (Blue, NS3pro; Green, NS2B).

Residues of the catalytic triad Histidine 51 (HIS 51), Aspartate 75 (ASP 75) and

Serine 135 (SER 135) are shown as red-colored stick models. Source: (Wichapong et

al., 2009).

It has been reported that Dengue virus NS3 protease alone is not active for

proteolysis. The enzymatic activity of NS3 protease is enhanced by its interaction

with NS2B protein (130 amino acid) that functions as a co-factor. Both NS2B and

NS3 are required for proteolytic processing of dengue virus polyprotein (Falgout et

al., 1991). From the X-ray crystal structure of NS2B (PDB ID: 2FOM) (Erbel et al.,

2006), residues 51-57 of NS2B form a β-strand which associates with the NS3

protease. Erbel et al. (2006) also expressed a construct containing residues 49–66 of

NS2B in a soluble form, indicating that the N-terminal part of NS2B is sufficient to

stabilise the enzyme. However, the truncated NS2B-NS3 protein is catalytically

22

inactive and cannot bind a substrate-based inhibitor, which suggests that the C-

terminal part of NS2B directly interacts with the substrate-binding site. This is in

contrast to HCV NS3 protease, where a short fragment of NS4A, including residues

corresponding to β-strand 1, is sufficient to yield a fully active enzyme (Erbel et al.,

2006).

It is also suggested that NS2B functions as a chaperone in assisting the

folding of NS3 protease to an active conformation (Leung et al., 2001). A 47-

residues central hydrophobic region of NS2B is found to be essential for the

activation of NS3 protease where the absence of NS2B has adverse effects on both

the stability and the catalytic activity of NS3 protease (Murthy et al., 1999; Yusof et

al., 2000). Leung et al. (2001) also reported that the enzyme is inactive on its own or

after the addition of 13-residues cofactor but is active when fused to the 40-residues

cofactor. The first homology model with complete structure of the protease including

the complete cofactor NS2B in the productive form was presented by Wichapong et

al. (2009).

The active site of NS2B-NS3 protease consists of four pockets, which are S1,

S2, S3 and S4 that interact with the substrate residues P1, P2, P3 and P4,

respectively. The sequence alignment of Dengue 2 Virus (PDB ID: 2FOM) and West

Nile Virus (2PF7) of NS2B and NS3 domains (Erbel et al., 2006) showed that all

residues in the pockets are either identical or similar for these two viruses

(Wichapong et al., 2009) as shown in the Figure 1.9.

23

Figure 1.9: Sequence alignment of DENV and WNV NS2B and NS3 domains. Stars

indicate residues that are identical whereas dots and colons indicate similar residues.

Residues which are drawn italic and underlined are missing residues in the X-ray

structures. Residues located in the S1, S2, S3, and S4 pockets are colored cyan,

yellow, green, and magenta, respectively, and catalytic triads are displayed in bold

red. Source: (Wichapong et al., 2009).

Previous alignment studies indicated that NS3 protease has structurally

similar to trypsin-like serine protease (Murthy et al., 1999). However, unlike trypsin,

NS3 protease has a marked preference for a pair of basic residues (either Arg or Lys)

at P2 and P1 in its peptide substrate followed by a small, unbranch amino acid (Gly

or Ser) at P1’ (Chambers et al., 1991; Chambers et al., 1990)

Wichapong and co-workers (2009) had constructed homology model of DEN-

2 Virus NS2B-NS3 protease complexed with the peptidic inhibitor (Bz-Nle-Lys-Arg-

Arg-H). Based on the MD simulation of this homology model, the interactions

between the enzyme active sites and inhibitors were revealed. It was suggested that

His51 and Asp129 of NS3 protease play significant role in stabilizing the basic

inhibitor P1 residue Arg or Lys (Li et al., 2005; Wichapong et al., 2009). From the

simulation, the interaction of Arg-P2 and residues Asp75 (C-terminus NS2B),

Asn152 (NS3) and Gly182 (NS2B) were observed. This result suggested that C-

terminus of NS2B not only play an important role for stabilizing and reorganizing the

24

NS3 protease but is also essential for the interaction with P2 residue of the inhibitor.

All residues in S3 pocket are made up from NS2B domain. MD simulation

showed that the H-bond was only observed between Lys-P3 and Tyr161. The S4

pocket of DEN-2 protease is formed by Val154, Val155, Thr156 and Arg157

suggested that it is dominated by hydrophobic residues. Thus the preferred residue at

the P4 position of peptidic inhibitor is norleucine or leucine (Li et al., 2005).

Wichapong and co-workers (2009) also revealed that the hydrogen bond between

Nle-P4 of inhibitor and the S4 pocket is very low.

1.4.2.2 Dengue virus NS2B-NS3 protease as a potential drug target

The replication of flaviviruses requires the correct processing of their

polyprotein by the viral NS2B-NS3 protease. Therefore, NS2B-NS3 protease is a

potential target for the development of therapeutics against the dengue virus

(Tomlinson et al., 2009). Moreover, protease inhibitors have a successful history as

being developed into antiviral drugs (Hsu et al., 2006; Malcolm et al., 2006;

Wlodawer & Vondrasek, 1998). A few protease inhibitors have been approved by

US Food and Drug Administration (FDA) for HIV treatement such as saquinavir

mesylate (1995), ritonavir (1996) and indinavir (1996), amprenavir (1999) and

atazanavir sulphate (2003) (Hsu et al., 2006). For dengue virus, the potential peptides

and small molecules as protease inhibitors have been studied by a few reserchers

focusing on NS2B-NS3 protease (Chanprapaph et al., 2005; Ganesh et al., 2005; Kiat

et al., 2006; Yin et al., 2006a).

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