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