ii

Abstract

Alteration in dopaminergic and serotonergic neurotransmission influences

various neurological and mental disorders such as depression, anxiety, bipolar disorder,

schizophrenia and drug abuse. The naturally occurring aporphine alkaloids are well

known for their activity at D1, D2 and 5-HT1A receptors, but only a few have been

shown to bind to the 5-HT2A receptor. Aim of this study was to identify aporphines with

significant activity at dopamine and serotonin receptors using both in silico and in vitro

screening approaches. A 3D homology model of the rat 5-HT2A receptor was generated

using the crystal structure of the human β2-adrenergic receptor (PDB ID: 2RH1) and

validated with standard 5-HT2A receptor ligands. A filtered set of aporphines obtained

from the ZINC database using (S)-boldine as the backbone structure was docked into

the generated 5-HT2A receptor model. A set of 13 compounds were identified with

higher or comparable activity to (S)-boldine for experimental testing across the D1, D2,

5-HT1A and 5-HT2A receptors using a medium throughput radioligand receptor binding

assay. (R)-roemerine was found to have selective 5-HT2A binding affinity with 20–400-

fold higher affinity for the 5-HT2A receptor versus the D1, D2, and 5-HT1A receptors.

Investigation into the structures of the selected compounds revealed that substitution at

positions 1 and 2, particularly with a methylenedioxy group, non-substitution at

positions 10 and 11 and a protonated amino group at position 6 may be responsible for

the good affinity-selectivity profile of (R)-roemerine for the 5-HT2A receptor compared

to the other compounds. Further analysis of the binding modes of the selected

compounds also showed that the combination of an electrostatic interaction and the

hydrogen bonding between the protonated amino group of (R)-roemerine and Asp155

and a pi-cation interaction with Phe339 appears to explain its enhanced affinity and

selectivity as compared to the other compounds. The results illustrate the usefulness of a

iii

combined in silico and in vitro approach in the search for lead molecules for the

development of new selective drugs acting at dopamine and serotonin receptors.

iv

Abstrak

Perubahan pada neurotransmisi dopamin dan serotonin mempengaruhi pelbagai

gangguan neurologi dan mental seperti kemurungan, keresahan, gangguan bipolar,

skizofrenia dan penyalahgunaan dadah. Alkaloid aporphine semulajadi terkenal dengan

aktivitinya pada reseptor D1, D2 dan 5-HT1A, tetapi hanya sebilangan daripadanya

menunjukkan ikatan pada reseptor 5-HT2A. Tujuan kajian ini adalah untuk

mengenalpasti aporphine dengan aktiviti yang siknifikan pada reseptor dopamin dan

serotonin menggunakan kedua-dua pendekatan in silico dan in vitro. Satu model

homologi 3D bagi reseptor tikus 5-HT2A telah dijana dengan menggunakan struktur

kristal reseptor manusia β2-adrenergik (PDB ID: 2RH1) dan divalidasi dengan ligan

asas 5-HT2A. Satu set aporphines yang ditapis diperoleh dari pangkalan data ZINC

dengan menggunakan (S)-boldine sebagai struktur asas untuk didok dalam reseptor 5-

HT2A yang telah dijana. Sebanyak 13 kompaun telah dikenalpasti dengan aktiviti yang

lebih tinggi atau setanding dengan (S)-boldine untuk diuji secara experimental pada

reseptor D1, D2, 5-HT1A dan 5-HT2A dengan pengendalian asei reseptor radioligan. (R)-

roemerine menunjukkan ikatan afiniti yang selektif pada reseptor 5-HT2A dengan 20–

400-kali ganda lebih tinggi untuk reseptor 5-HT2A berbanding dengan reseptor D1, D2

dan 5-HT1A. Kajian menyeluruh bagi struktur kompaun yang terpilih menunjukkan

bahawa (R)-roemerine dengan penggantian pada kedudukan 1 dan 2, khususnya dengan

kumpulan metilenadioksi, tanpa penggantian pada kedudukan 10 dan 11 dan kumpulan

amino berproton pada kedudukan 6 bertanggungjawab kepada profil afiniti-selektiviti

yang baik oleh (R)-roemerine bagi reseptor 5-HT2A berbanding kompaun lain. Analisis

lanjutan bagi jenis interaksi oleh kompaun terpilih juga menunjukkan bahawa gabungan

interaksi elektrostatik dan ikatan hidrogen antara kumpulan amino berproton dalam (R)-

roemerine dan Asp155 dan interaksi pi-kation dengan Phe339 menjurus kepada

v

peningkatan afiniti dan selektiviti berbanding dengan kompaun lain. Keputusan ini

menunjukkan kepentingan gabungan kedua-dua kaedah in silico dan in vitro dalam

pencarian molekul baru bagi perkembangan ligan yang selektif yang bertindak pada

reseptor dopamin dan serotonin.

vi

Acknowledgements

First and foremost, I’m indebted to my supervisors, Prof. Dr. Chung Lip Yong

and Dr. Michael James Christopher Buckle for their support, guidance and

encouragement throughout this project. I’m thankful to Prof. Dr. Stephen Doughty for

his comments and guidance, especially in molecular modelling.

I wish to express my special and sincere thanks to Dr. Azura and all the staff

from the animal house for their assistance. I would also like to acknowledge the help of

all the staff from Department of Pharmacy and Department of Molecular Medicine. I

wish to thank my labmates and friends for their support and friendship.

I sincerely thank to Ministry of Science, Technology and Innovation, Malaysia

for the National Science Fellowship award (Ref No: M/0138/02/2008/LAIN2) and

University of Malaya for the research grants (PPP vote: PS192/2009C; UMRG:

RG009/09BIO).

At last but not the least I would like to thank my beloved family for their

undying support, love and blessings, and finally to God who made all the things

possible.

vii

Table of Contents

Title page i

Abstract ii

Abstrak iv

Acknowledgements vi

Table of Contents vii

List of Figures xii

List of Tables xviii

List of Symbols and Abbreviations xix

1.0 Introduction 1

1.1 Aim and Objectives of the Research 3

2.0 Literature Review

2.1 G-Protein Coupled Receptors (GPCRs) 4

2.1.1 G-Protein Coupled Receptor (GPCR) Overview 4

2.1.2 G-Protein Coupled Receptor (GPCR) Crystal Structures 6

2.2 Dopamine (D) and Serotonin (5-hydroxytryptamine; 5-HT) Receptors 8

2.2.1 Dopamine (D) Receptors 8

2.2.2 Serotonin (5-hydroxytryptamine; 5-HT) Receptors 8

2.2.3 Clinical Significance of Dopamine and Serotonin Receptors 9

2.3 Computer Aided Drug Design 11

2.3.1 Structure-Based Drug Design (SBDD) 11

2.3.2 Homology Modelling 11

2.3.3 Molecular Docking 14

2.4 Receptor Binding Assays 14

2.4.1 Fundamental of Receptor–ligand Interactions 14

viii

2.4.2 Radioligand Receptor Binding Assays 15

2.5 Aporphine Alkaloids 16

2.5.1 An Overview of Aporphines 16

2.5.2 Structural Diversity of Aporphines 17

2.5.3 Pharmacological Properties of Aporphines 17

3.0 Computational Investigation of the 5-HT2A Receptor Binding

Interactions of Standard Ligands and Aporphines

3.1 Introduction 20

3.1.1 5-HT2A Receptor (5-HT2AR) Structure 20

3.2 Materials 22

3.3 Methodology 22

3.3.1 Sequence Alignments 22

3.3.2 Model Construction 22

3.3.3 Docking Procedure 23

3.3.4 Compound Selection for Docking 23

3.4 Results and Discussion 25

3.4.1 Selection of Templates 25

3.4.2 Sequence Analysis 25

3.4.3 Model Construction 26

3.4.4 Model Evaluation 26

3.4.5 Docking 27

3.4.6 Compound Selection from Docking Studies 28

3.5 Conclusion 31

4.0 Optimisation and Evaluation of Filtration Based Receptor Binding

Assays for Dopamine (D1 and D2) and 5-Hydroxytryptamine (5-

HT1A and 5-HT2A)

ix

4.1 Introduction 32

4.1.1 Properties of Receptor Binding Assays 32

4.1.2 Selection of Receptor Source 33

4.1.3 Selection of Radioligand 34

4.2 Materials 37

4.2.1 Chemicals and Instrumentation 37

4.3 Methodology 38

4.3.1 Preparation of Dopamine (D1 and D2) and

5-Hydroxytryptamine (5-HT1A and 5-HT2A) Membrane

Receptors

38

4.3.2 Protein Determination 40

4.3.3 Receptor binding assays 40

4.3.3.1 Optimisation of Dopamine (D1 and D2) and 5-

Hydroxytryptamine (5-HT1A and 5-HT2A) Receptor

Binding Assays

40

4.3.3.2 Saturation experiments 41

4.3.3.3 Competition experiments 42

4.3.3.4 Zʹ factor 42

4.4 Data and Statistical Analysis 44

4.5 Results and Discussion 45

4.5.1 Protein determination 45

4.5.2 Effects of Different Physico-Chemical Properties on Dopamine

(D1 and D2) and 5-Hydroxytryptamine (5-HT1A and 5-HT2A)

Receptor Binding Assays

45

4.5.2.1 Selection of Membrane Concentration 46

4.5.2.2 Selection of Unlabelled Ligand and its Concentration 47

x

4.5.2.3 Selection of pH 47

4.5.2.4 Selection of Filter Plate Type 48

4.5.2.5 Selection of Incubation Temperature 49

4.5.2.6 Selection of Incubation Time 50

4.5.2.7 Selection of Washing Times 50

4.5.2.8 Solvent Interference 51

4.5.2.9 Saturation Experiments 56

4.5.2.10 Competition Experiments 57

4.5.2.11 Zʹ factor 60

4.6 Conclusion 61

5.0 Virtual and Biomolecular Screening of Potential Ligands for 5-HT2A

Receptor

5.1 Introduction 63

5.1.1 Structure Activity Relationship (SAR) of Aporphines 63

5.2 Materials 65

5.3 Methodology 65

5.3.1 Receptor Binding Assays 65

5.4 Results and Discussion 66

5.4.1 Receptor Binding Assays 66

5.5 Conclusion 76

6.0 General Conclusion, Limitations and Future Perspectives 77

7.0 References 79

8.0 Appendices 98

A Sequence Analysis 98

B Ramachandran Plot 105

C Binding Modes of Standard Ligands in 5-HT2A Receptor Model 106

xi

D ZINC IDs and the Corresponding Compound Names 112

E Binding Modes of the Selected Aporphines with the 5-HT2A

Receptor Model

113

F Abstracts of Research Presentations 119

xii

List of Figures

Figure 2.1 Schematic diagram of GPCRs with seven transmembrane

regions (TM1–TM7) connected by intracellular and

extracellular loop segments, as well as an extracellular N-

terminus and an intracellular C-terminus

6

Figure 2.2 Accuracy and application of protein structure models 13

Figure 2.3 General structure of aporphines 17

Figure 2.4 Structures of aporphines 19

Figure 3.1 General structure of naturally-occuring aporphines with

possible substitution sites indicated as R1–R11

24

Figure 3.2 Proposed binding mode of (S)-boldine in the 5-HT2A receptor 30

Figure 4.1 Diagrammatic representations of dissection procedure for rat

brain

39

Figure 4.2 Standard curve of BSA concentration against absorbance for

use in protein determination

45

Figure 4.3(a) Selection of membrane concentration for D1 receptor 52

Figure 4.4(a) Selection of membrane concentration for D2 receptor 53

Figure 4.5(a) Selection of membrane concentration for 5-HT1A receptor 54

Figure 4.6(a) Selection of membrane concentration for 5-HT2A receptor 55

Figure 4.3(b) Selection of unlabelled ligand concentration for D1 receptor 52

Figure 4.4(b) Selection of unlabelled ligand concentration for D2 receptor 53

Figure 4.5(b) Selection of unlabelled ligand concentration for 5-HT1A

receptor

54

Figure 4.6(b) Selection of unlabelled ligand concentration for 5-HT2A

receptor

55

xiii

Figure 4.3(c) Selection of pH for D1 receptor 52

Figure 4.4(c) Selection of pH for D2 receptor 53

Figure 4.5(c) Selection of pH for 5-HT1A receptor 54

Figure 4.6(c) Selection of pH for 5-HT2A receptor 55

Figure 4.3(d) Selection of filter plate type for D1 receptor 52

Figure 4.4(d) Selection of filter plate type for D2 receptor 53

Figure 4.5(d) Selection of filter plate type for 5-HT1A receptor 54

Figure 4.6(d) Selection of filter plate type for 5-HT2A receptor 55

Figure 4.3(e) Selection of incubation temperature for D1 receptor 52

Figure 4.4(e) Selection of incubation temperature for D2 receptor 53

Figure 4.5(e) Selection of incubation temperature for 5-HT1A receptor 54

Figure 4.6(e) Selection of incubation temperature for 5-HT2A receptor 55

Figure 4.3(f) Selection of incubation time for D1 receptor 52

Figure 4.4(f) Selection of incubation time for D2 receptor 53

Figure 4.5(f) Selection of incubation time for 5-HT1A receptor 54

Figure 4.6(f) Selection of incubation time for 5-HT2A receptor 55

Figure 4.3(g) Selection of washing times for D1 receptor 52

Figure 4.4(g) Selection of washing times for D2 receptor 53

Figure 4.5(g) Selection of washing times for 5-HT1A receptor 54

Figure 4.6(g) Selection of washing times for 5-HT2A receptor 55

Figure 4.3(h) Selection of DMSO effect for D1 receptor 52

Figure 4.4(h) Selection of DMSO effect for D2 receptor 53

Figure 4.5(h) Selection of DMSO effect for 5-HT1A receptor 54

Figure 4.6(h) Selection of DMSO effect for 5-HT2A receptor 55

Figure 4.7(a) Saturation binding curve of [3H] SCH23390 for D1 receptor 56

Figure 4.7(b) Saturation binding curve of [3H] spiperone for D2 receptor 56

xiv

Figure 4.7(c) Saturation binding curve of [3H] 8-OH-DPAT for 5-HT1A

receptor

56

Figure 4.7(d) Saturation binding curve of [3H] ketanserin for 5-HT2A

receptor

56

Figure 4.8(a) Competition binding curve of [3H] SCH23390 for D1 receptor 57

Figure 4.8(b) Competition binding curve of [3H] spiperone for D2 receptor 57

Figure 4.8(c) Competition binding curve of [3H] 8-OH-DPAT for 5-HT1A

receptor

57

Figure 4.8(d) Competition binding curve of [3H] ketanserin for 5-HT2A

receptor

57

Figure 4.9(a) Zʹ analysis for D1 receptor 60

Figure 4.9(b) Zʹ analysis for D2 receptor 60

Figure 4.9(c) Zʹ analysis for 5-HT1A receptor 60

Figure 4.9(d) Zʹ analysis for 5-HT2A receptor 60

Figure 5.1(a) Competition binding curves of (R)-roemerine towards [3H]

SCH23390 for D1 receptor, [3H] spiperone for D2 receptor,

[3H] 8-OH-DPAT for 5-HT1A receptor and [

3H] ketanserin for

5-HT2A receptor

68

Figure 5.1(b) Competition binding curves of (S)-bulbocapnine towards [3H]

SCH23390 for D1 receptor, [3H] spiperone for D2 receptor,

[3H] 8-OH-DPAT for 5-HT1A receptor and [

3H] ketanserin for

5-HT2A receptor

68

Figure 5.1(c) Competition binding curves of (S)-boldine towards [3H]

SCH23390 for D1 receptor, [3H] spiperone for D2 receptor,

[3H] 8-OH-DPAT for 5-HT1A receptor and [

3H] ketanserin for

5-HT2A receptor

68

xv

Figure 5.1(d) Competition binding curves of (R)-apomorphine towards [3H]

SCH23390 for D1 receptor, [3H] spiperone for D2 receptor,

[3H] 8-OH-DPAT for 5-HT1A receptor and [

3H] ketanserin for

5-HT2A receptor

68

Figure 5.1(e) Competition binding curves of (S)-apomorphine towards [3H]

SCH23390 for D1 receptor, [3H] spiperone for D2 receptor,

[3H] 8-OH-DPAT for 5-HT1A receptor and [

3H] ketanserin for

5-HT2A receptor

68

Figure 5.1(f) Competition binding curves of N-propylapomorphine towards

[3H] SCH23390 for D1 receptor, [

3H] spiperone for D2

receptor, [3H] 8-OH-DPAT for 5-HT1A receptor and [

3H]

ketanserin for 5-HT2A receptor

68

Figure 5.2 Proposed binding mode of (R)-roemerine in the 5-HT2A

receptor

71

Figure 8.1 Alignment of rat 5-HT family receptor 98

Figure 8.2 Sequence alignment of 5-HT2A multiple species 101

Figure 8.3 Sequence comparison of rat 5-HT2A against human 5-HT2A 103

Figure 8.4 Sequence comparison of rat 5-HT2A against rat 5-HT2B and 5-

HT2C sequences

104

Figure 8.5 Structure validation of the model using Ramachandran plot

computed with the PROCHECK program

105

Figure 8.6(a) The 5-HT2A receptor model in complex with endogenous

ligand, serotonin

106

Figure 8.6(b) The 5-HT2A receptor model in complex with tryptamine 106

Figure 8.6(c) The 5-HT2A receptor model in complex with 5-MeOT 107

Figure 8.6(d) The 5-HT2A receptor model in complex with (R)-(-)-DOI 107

xvi

Figure 8.6(e) The 5-HT2A receptor model in complex with (S)-(-)-DOI 108

Figure 8.7(a) The 5-HT2A receptor model in complex with spiperone 109

Figure 8.7(b) The 5-HT2A receptor model in complex with risperidone 109

Figure 8.7(c) The 5-HT2A receptor model in complex with haloperidol 110

Figure 8.7(d) The 5-HT2A receptor model in complex with ritanserin 110

Figure 8.7(e) The 5-HT2A receptor model in complex with ketanserin 111

Figure 8.8(a) Proposed binding mode of (S)-bulbocapnine in the 5-HT2A

receptor

113

Figure 8.8(b) Proposed binding mode of (S)-laureline in the 5-HT2A

receptor

113

Figure 8.8(c) Proposed binding mode of (S)-nuciferine in the 5-HT2A

receptor

114

Figure 8.8(d) Proposed binding mode (R)-apomorphine in the 5-HT2A

receptor

114

Figure 8.8(e) Proposed binding mode (S)-apomorphine in the 5-HT2A

receptor

115

Figure 8.8(f) Proposed binding mode of (R)-N-propylapomorphine in the 5-

HT2A receptor

115

Figure 8.8(g) Proposed binding mode of (R)-2-hydroxy-N-

propylapomorphine in the 5-HT2A receptor

116

Figure 8.8(h) Proposed binding mode of (S)-isocorydine in the 5-HT2A

receptor

116

Figure 8.8(i) Proposed binding mode of (S)-isothebaine in the 5-HT2A

receptor

117

Figure 8.8(j) Proposed binding mode of (S)-corydine in the 5-HT2A receptor 117

xvii

Figure 8.8(k) Proposed binding mode of (S)-corytuberine in the 5-HT2A

receptor

118

xviii

List of Tables

Table 2.1 Examples of some prescription drugs which target GPCRs for

the indicated condition or disease state

5

Table 2.2 List of three dimensional crystal structures of GPCRs 7

Table 3.1 Computed Ki values of the selected compounds 31

Table 4.1 Summary of the values used for the different parameters

involved in optimising the protocols for the radioligand

binding assays

43

Table 4.2 Ki values of the standard ligands for D1, D2, 5-HT1A and 5-

HT2A receptors

59

Table 4.3 Data from Zʹ factor analysis for the D1, D2, 5-HT1A and 5-

HT2A receptors

61

Table 4.4 Optimised assay conditions for D1, D2, 5-HT1A and 5-HT2A

receptors

62

Table 5.1 Binding affinities of the selected ligands towards rat D1, D2, 5-

HT1A and 5-HT2A receptors

69

Table 5.2 5-HT2A residues found from the docking studies to interact

with the selected aporphines

74

Table 8.1 The ZINC IDs of the compounds selected from virtual

screening

112

xix

List of Symbols and Abbreviations

2D 2 Dimensional

3D 3 Dimensional

5-HT 5-hydroxytryptamine, serotonin

5-HT1A 5-hydroxytryptamine1A

5-HT2A 5-hydroxytryptamine2A

5-MeOT 5-methoxytryptamine

8-OH-DPAT (±)-8-hydroxy-2-(di-n-propylamino)-tetralin

[3H] 8-OH-DPAT 8-Hydroxy-2-[2,3-

3H]di-n-(propylamino)tetralin

[3H] SCH 23390 [

3H] 7-chloro-8-hydroxy-3-methyl-5-phenyl-2,3,4,5-

tetrahydro-1-H-3-benzazepine

aa Amino acid

AMP Adenosine monophosphate

APDs Antipsychotic drugs

Bmax Maximal binding capacity

BSA Bovine serum albumin

cAMP Cyclic adenosine monophosphate

CNS Central nervous system

CV Coefficient of variation

D1 Dopamine1

D2 Dopamine2

DOI (±)-2, 5-dimethoxy-4-iodoamphetamine

DMSO Dimethyl sulphoxide

ECL Extracellular loop

EtOH Ethanol

xx

GF/B Glass fibre type B

GF/C Glass fibre type C

GPCR G-protein coupled receptor

HCl Hydrochloride

IC50 Concentration at 50% inhibition

Kd Equilibrium dissociation constant

Ki Inhibition constant

[L] concentration of radioligand

MDMA Methylenedioxymethamphetamine

MeOH Methanol

NSB Nonspecific binding

PDB Protein Data Bank

SAR Structure activity relationship

SB Specific binding

S/B Signal to background ratio

SCH 23390

R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-

tetrahydro-1H-3-benzazepine

SD Standard deviation

SEM Standard error mean

SBDD Structure-based drug design

TB Total binding

Tris Tris(hydroxymethyl)aminomethane

UV Ultraviolet

WHO World Health Organization

xxi

Units of Measurement

% Percent

Å Angstrom

°C Degree celsius

Ci Curie

ԑ Epsilon

× g Times gravity

µg Microgram

cpm Count per minute

M Molar

mg Milligram

min Minute

ml Milliliter

mM Millimolar

nM Nanomolar

min Minute

Rpm Revolutions per minute

v/v Volume per volume

w/v Weight per volume

xxii

List of Amino Acids

Ala (A) Alanine

Arg (R) Arginine

Asn (N) Asparagine

Asp (D) Aspartate

Cys (C) Cysteine

Gln (Q) Glutamine

Glu (E) Glutamate

Gly (G) Glycine

His (H) Histidine

Ile (I) Isoleucine

Leu (L) Leucine

Lys (K) Lysine

Met (M) Methionine

Phe (F) Phenylalanine

Pro (P) Proline

Ser (S) Serine

Thr (T) Threonine

Trp (W) Tryptophan

Tyr (Y) Tyrosine

Val (V) Valine