Table of Contents
Title Page
Copyright
Preface
Abbreviations
Chapter 1: Introduction to Drug Targets and Molecular Pharmacology
1.1 Introduction to molecular pharmacology
1.2 Scope of this textbook
1.3 The nature of drug targets
1.4 Future drug targets
1.5 Molecular pharmacology and drug discovery
References
Chapter 2: Molecular Cloning of Drug Targets
2.1 Introduction to molecular cloning—from DNA to drug discovery
2.2 ‘Traditional’ pharmacology
2.3 The relevance of recombinant DNA technology to pharmacology/drug discovery
2.4 The ‘cloning’ of drug targets
2.5 What information can DNA cloning provide?
2.6 Comparing the pharmacologic profile of the ‘cloned’ and the ‘native’ drug target
2.7 Reverse pharmacology illustrated on orphan GPCRs
2.8 Summary
References
Chapter 3: G Protein-coupled Receptors
3.1 Introduction to G protein-coupled receptors
3.2 Heterotrimeric G-proteins
3.3 Signal transduction pathways
3.4 Desensitisation and down-regulation of GPCR signalling
3.5 Constitutive GPCR activity
3.6 Promiscuous G-protein coupling
3.7 Agonist-directed signalling
3.8 Allosteric modulators of GPCR function
3.9 Pharmacological chaperones for GPCRs
3.10 GPCR dimerisation
3.11 GPCR splice variants
3.12 Summary
References
Useful Web sites
Chapter 4: Ion Channels
4.1 Introduction
4.2 Voltage-gated ion channels
4.3 Other types of voltage-gated ion channels
4.4 Ligand-gated ion channels
4.5 Summary
References
Chapter 5: Transporter Proteins
5.1 Introduction
5.2 Classification
5.3 Structural analysis of transporters
5.4 Transporter families of pharmacological interest
5.5 Transporters and cellular homeostasis
5.6 Summary
References
Chapter 6: Cystic Fibrosis: Alternative Approaches to the Treatment of a Genetic Disease
6.1 Introduction
6.2 Cystic fibrosis transmembrane conductance regulator
6.3 Mutations in CFTR
6.4 Why is cystic fibrosis so common?
6.5 Animal models of Cystic fibrosis
6.6 Pharmacotherapy
6.7 Gene therapy
6.8 Conclusion
References
Chapter 7: Pharmacogenomics
7.1 Types of genetic variation in the human genome
7.2 Thiopurine S-methyltransferase and K channel polymorphisms
7.3 Polymorphisms affecting drug metabolism
7.4 Methods for detecting genetic polymorphisms
7.5 Genetic variation in drug transporters
7.6 Genetic variation in G protein coupled receptors
7.7 Summary
References
Useful Web sites
Chapter 8: Transcription Factors and Gene Expression
8.1 Control of gene expression
8.2 Transcription factors
8.3 CREB
8.4 Nuclear receptors
8.5 Peroxisome proliferator-activated receptors
8.6 Growth factors
8.7 Alternative splicing
8.8 RNA editing
8.9 The importance of non-coding RNAs in gene expression
8.10 Summary
References
Chapter 9: Cellular Calcium
9.1 Introduction
9.2 Measurement of calcium
9.3 The exocrine pancreas
9.4 Calcium signalling in pancreatic acinar cells
9.5 Nuclear calcium signalling
9.6 Conclusions
References
Chapter 10: Genetic Engineering of Mice
10.1 Introduction to genetic engineering
10.2 Genomics and the accumulation of sequence data
10.3 The mouse as a model organism
10.4 Techniques for genetic engineering
10.5 Examples of genetically-engineered mice
10.6 Summary
References
Chapter 11: Signalling Complexes: Protein-protein Interactions and Lipid Rafts
11.1 Introduction to cell signalling complexes
11.2 Introduction to GPCR interacting proteins
11.3 Methods used to identify GPCR interacting proteins
11.4 Functional roles of GPCR interacting proteins
11.5 GPCR signalling complexes
11.6 GPCR and ion channel complexes
11.7 Ion channel signalling complexes
11.8 Development of pharmaceuticals that target GPCR interacting proteins
11.9 Development of pharmaceuticals that target protein-protein interactions
11.10 Lipid rafts
11.11 Receptor-mediated endocytosis
11.12 Summary
References
Chapter 12: Recombinant Proteins and Immunotherapeutics
12.1 Introduction to immunotherapeutics
12.2 Historical background of immunotherapeutics
12.3 Basis of immunotherapeutics
12.4 Types of immunotherapeutics
12.5 Humanisation of antibody therapy
12.6 Immunotherapeutics in clinical practice
12.7 Advantages and disadvantages of immunotherapy
12.8 The future
12.9 Summary
References
Glossary
Index
Companion website
This book is accompanied by a companion website:
www.wiley.com/go/dickenson/dnamolecular
The website includes:
Figures and Tables from the book for downloading
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Library of Congress Cataloging-in-Publication Data
Molecular pharmacology : from DNA to drug discovery / John Dickenson … [et al.].
p. ; cm.
Includes index.
ISBN 978-0-470-68444-3 (cloth)— ISBN 978-0-470-68443-6 (pbk.)
I. Dickenson, John.
[DNLM: 1. Molecular Targeted Therapy. 2. Pharmacogenetics– methods.
3. Drug Delivery Systems. 4. Drug Discovery. QV 38.5]
615.1′9– dc23
2012034772
A catalogue record for this book is available from the British Library.
Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.
First Impression 2013
Preface
Nottingham Trent University offers a suite of successful MSc courses in the Biosciences field that are delivered by full-time, part-time and distance (e-learning) teaching. The authors are members of the Pharmacology team at Nottingham Trent University and teach extensively on the MSc Pharmacology and Neuropharmacology courses. The content of this book was inspired by these courses as there is no comparable postgraduate textbook on molecular pharmacology and it is a rapidly expanding subject. The primary aim of this text was to provide a platform to complement our courses and enhance the student experience. Given the breadth and depth of this volume it will be of use to students from other institutions as a teaching aid as well as an invaluable source of background information for post-graduate researchers. The value of this book is enhanced by the research portfolio of the Bioscience Department and individual authors who have research careers spanning over 25 years.
This textbook illustrates how genes can influence our physiology and hence our pharmacological response to drugs used to treat pathological conditions. Tailoring of therapeutic drugs is the future of drug design as it enables physicians to prescribe personalised medical treatments based on an individual's genome. The book utilises a drug target-based approach rather than the traditional organ/system-based viewpoint and reflects the current advances and research trends towards in silico drug design based on gene and derived protein structure.
The authors would like to thank Prof Mark Darlison (Napier University, Edinburgh, UK) for providing the initial impetus, inspiration and belief that a book of such magnitude was possible. We would also like to acknowledge the unflagging encouragement and support of the Wiley-Blackwell team (Nicky, Fiona and Clara) during the preparation of this work. Finally thanks should also be given to the helpful, constructive and positive comments provided by the reviewers. We hope that you enjoy this book as much as we enjoyed writing it.
John Dickenson, Fiona Freeman, Chris Lloyd Mills, Shiva Sivasubramaniam and Christian Thode.
Abbreviations
[Ca2+]i | intracellular free ionised calcium concentration |
[Ca2+]n | nuclear free ionised calcium concentration |
[Ca2+]o | extracellular free ionised calcium concentration |
2-APB | 2-aminoethoxydiphenyl borate |
4EFmut DREAM | 4th EF hand mutant DREAM |
5F-BAPTA | 1,2-bis(2-amino-5,6-diflurophenoxy) ethane-N,N,N′,N′-tretracacetic acid |
5-HT | 5-hydroxytyrptamine / serotonin |
AAV | adeno-associated virus |
ABC | ATP-binding cassette (transporter) |
AC | adenylyl cyclase |
ACC | mitochondrial ADP/ATP carrier (transporter) |
ACh | acetylcholine |
ACS | anion-cation subfamily |
AD | Alzheimer's disease |
ADAR | adenosine deaminase acting on RNA (1, 2 or 3) |
ADCC | antibody-dependent cellular cytotoxicity |
ADEPT | antibody-directed enzyme pro-drug therapy |
ADHD | attention deficit hyperactivity disorder |
AF1/2 | transcriptional activating function (1 or 2) |
Ala | alanine (A) |
AM | acetoxylmethyl |
AMPA | α-amino-3-hydroxy-5-methylisoxazole 4-propionic acid |
Apo- | apolipoproteins (A, B or C) |
APP | amyloid precursor protein |
AQP | aquaporins |
ARC channels | arachidonic acid regulated Ca2+ channels |
Arg | arginine (R) |
ASIC | acid sensing ion channels |
ASL | airways surface liquid |
Asn | asparagine (N) |
Asp | aspartic acid (D) |
ATF1 | activation transcription factor 1 |
ATP | adenosine triphosphate |
AV | adenovirus |
Aβ | amyloid β peptide |
BAC | bacterial artificial chromosome |
BBB | blood brain barrier |
BCRP | breast cancer resistant protein |
BDNF | brain-derived neurotrophic factor |
BKCa | big conductance Ca2+-activated K+ channels |
BLAST | Basic Local Alignment Search Tool |
bp | base pairs |
BRET | bioluminescence resonance energy transfer |
Brm/brg1 | mammalian helicase like proteins |
BTF | basal transcription factors |
BZ | benzodiazepine |
Ca-CaM | Ca2+-calmodulin |
CaCC | calcium activated chloride channel |
cADPr | cyclic adenosine diphosphoribose |
CaM | calmodulin |
CaMK | calcium-dependent calmodulin kinase |
cAMP | cyclic adenosine 3′,5′ monophsophate |
CaRE | calcium responsive element |
catSper | cation channels in sperm |
CaV | voltage-gated Ca2+ channels |
CBAVD | congenital bilateral absence of the vas deferens |
CBP | CREB binding protein |
CCCP | carbonyl cyanide m-chlorophenylhydrazone |
CCK | cholecystokinin |
CDAR | cytosine deaminase acting on RNA |
cDNA | complementary DNA |
CDR | complementarily-determining region |
CF | cystic fibrosis |
CFP | cyan fluorescent protein |
CFS | colony stimulating factors |
CFTR | cystic fibrosis transmembrane conductance regulator |
cGMP | cyclic guanosine 3′,5′ monophosphate |
CHF | congestive heart failure |
CHO | Chinese hamster ovary cell line |
CICR | calcium induced calcium release |
CIF | calcium influx factor |
ClC | chloride channel |
CMV | cytomegalovirus |
CNG | cyclic nucleotide-gated channel |
CNS | central nervous system |
CNT | concentrative nucleoside transporter |
COS | CV-1 cell line from Simian kidney cells immortalised with SV40 viral genome |
COX | cyclooxygenases (1, 2 or 3) |
CPA | monovalent cation/proton antiporter super family |
CpG | cytosine-phosphate-guanine regions in DNA |
CPP | cell penetrating peptide (transporter) |
CRE | cAMP responsive element |
CREB | cAMP responsive element binding protein |
CREM | CRE modulator |
CRF | corticotropin-releasing factor |
CRM | chromatin remodelling complex |
CRTC | cAMP-regulated transcriptional co-activator family |
CSF | cerebral spinal fluid |
CTD | C terminal domain |
CTL | cytotoxic T lymphocyte |
CYP | cytochrome P450 |
Cys | cysteine (C) |
DAG | diacylglycerol |
DAX1 | dosage-sensitive sex reversal gene/TF |
DBD | DNA-binding domain |
DC | dicarboxylate |
DHA | drug:H+ antiporter family (transporter) |
Dlg1 | drosophila disc large tumour suppressor |
DNA | deoxyribonucleic acid |
DOPA | dihydroxyphenylalanine |
DPE | downstream promoter element |
DRE | downstream regulatory element |
DREAM | DRE antagonist modulator |
dsRNA | double-stranded RNA |
EBV | Epstein Barr virus |
EGF | epidermal growth factor |
EGFR | epidermal growth factor receptor |
EGTA | ethylene glycol tetraacetic acid |
ELISA | enzyme linked immunosorbent assay |
ENaC | epithelial sodium channel |
EPO | erythropoietin |
ER | endoplasmic reticulum |
ERK | extracellular-signal-regulated kinases |
eRNA | enhancer RNA |
ERTF | oestrogen receptor transcription factor |
ES cells | embryonic stem cells |
ESE | exon splicing enhancer |
ESS | exon splicing silencer |
EST | expressed sequence tag |
Fab | antibody binding domain |
FACS | fluorescent-activated cell sorting |
Fc | constant fragment of the monoclonal antibodies |
FEV1 | forced expiratory volume in 1 second |
FGF-9 | fibroblast growth factor |
FIH | factor inhibiting HIF |
FISH | fluorescence in situ hybridisation |
FOXL2 | fork-head box protein |
FRET | fluorescence resonance energy transfer |
FXS | fragile-X syndrome |
G3P | glucose-3-phosphate |
GABA | gamma-aminobutyric acid |
GAT | GABA transporters |
GC | guanylyl cyclase |
GFP | green fluorescent protein |
GIRK | G-protein-gated inwardly rectify K+ channel |
Gln | glutamine (Q) |
GlpT | sn-glycerol-3-phosphate/phosphate antiporter |
GltPh | Pyrococcus horikoshii glutamate transporters |
Glu | glutamic acid (E) |
GLUT | glucose transporters |
Gly | glycine (G) |
GLYT | glycine transporters |
GMP | guanosine monophosphate |
GPCR | G protein coupled receptor |
GPN | glycyl-L-phenylalanine-2-napthylamide |
GRK | G-protein coupled receptor kinase |
GST | Glutathione S-transferase |
H+ | hydrogen ion; proton |
HAD | histone deacetylases |
HAMA | human anti-murine antibodies |
HAT | histone acetyltransferases |
HCF | host cell factor |
HCN | hyperpolarisation-activated cyclic nucleotide-gated channels |
HDL | high density lipoprotein |
HIF | hypoxia inducible factor |
His | histidine (H) |
HMG | high mobility group |
HMIT | H+/myo-inositol transporter |
hnRNP | nuclear ribonucleoproteins |
HOX | homeobox |
HPLC | high-performance liquid chromatography |
HRE | hypoxia response elements |
Hsp70 | heat shock protein of the 70 kilodalton family |
HSV | herpes simplex virus |
HSV-tk | herpes simplex virus thymidine kinase |
HTS | high-throughput screening |
Htt | Huntingtin |
IBMX | 3-isobutyl-1-methylxanthine |
Icrac | calcium release activated Ca2+ channel |
ICSI | intra-cytoplasmic sperm injection |
Ifs | interferons |
Ig | immunoglobulins |
IGF-1 | insulin-like growth factor-I |
iGluR | ionotropic glutamate receptor |
IHD | ischaemic heart disease |
IL-10 | interleukin-10 |
Ile | isoleucine (I) |
INN | international non-proprietary names |
INR | initiator element |
INSL3 | insulin-like factor 3 |
IP3 | inositol 1,4,5-triphosphate |
IP3R | IP3 receptor |
iPLA2β | β isoform of Ca2+ independent phospholipase A2 |
IRT | immunoreactive trypsinogen |
Isc | short circuit current |
ISE | introns splicing enhancer |
ISS | introns splicing silencer |
K2P | two-pore potassium channels |
K3K4 HMT | histone methyl transferase |
KATP | ATP-sensitive K+ channels |
kb | kilobase |
KCa | Ca2+-activated K+ channels |
KCC | K+-Cl− co-transporter |
KChIP | K+ channel interacting protein |
KCO | K+ channel openers |
Kd | Ca2+ dissociation constant |
KG | G-protein gated K+ channels |
KID | kinase-inducible domain |
Kir | inwardly rectifying K+ channels |
KV | voltage-gated K+ channel |
LacY | lactose:H+ symporter |
LBD | ligand binding domains |
LDL | low density lipoprotein |
Leu | leucine (L) |
LeuTAa | Aquifex aeolicus leucine transporter |
LGIC | ligand-gated ion channel |
lncRNA | long non-coding RNA |
LPS | lipopolysaccharide |
lys | lysine (K) |
Mab | monoclonal antibodies |
MAC | membrane attack complex |
MAPK | mitogen-activated protein kinase |
MATE | multidrug and toxic compound extrusion superfamily (transporter) |
Mb | megabase |
MCT | mono carboxylate transporters |
MCU | mitochondrial Ca2+ uniporter |
MDR | multidrug resistance (transporter) |
MDR1 | multidrug resistant transporter 1 |
Met | methionine (M) |
MFP | periplasmic membrane fusion protein family (transporter) |
MFS | major facilitator superfamily (transporter) |
MHC | histocompatibility complex |
miRNA | microRNA |
mPTP | mitochondrial permeability transition pore |
mRNA | messenger RNA |
MSD | membrane spanning domain |
MTF | modulatory transcription factors |
Myc | myc oncogene |
NAADP | nicotinic acid adenine dinucleotide phosphate |
nAChR | nicotinic acetylcholine receptors |
NAD+ | nicotinamide adenine dinucleotide |
NADP+ | nicotinamide adenine dinucleotide phosphate |
NALCN | sodium leak channel non-selective protein channel |
NAT | natural antisense transcript |
NaV | voltage-gated Na+ channels |
NBD | nucleotide binding domain |
ncRNA | non-coding RNA |
neoR | neomycin resistance |
NES | nuclear endoplasmic space |
NFAT | nuclear factor of activated T cells |
NFκB | nuclear factor kappa of activated B cells |
NHA | Na+/H+ antiporters |
NhaA | Escherichia coli Na+/H+ antiporter |
NHE | Na+/H+ exchanger |
NKCC | sodium potassium 2 chloride cotransporter |
NM | nuclear membrane |
NMDA | N-methyl-D-aspartate |
NMR | nuclear magnetic reasonance |
NO | nitric oxide |
NPA | Asn-Pro-Ala motif |
NPC | nuclear pore complex |
NR | nucleoplasmic reticulum |
NR-HSP | nuclear receptor-heat shock protein complex |
NRSE | neuron restrictive silencer element |
NSS | neurotransmitter sodium symporter (transporter) |
nt | nucleotide |
NTD | N- terminal domain |
NVGDS | non viral gene delivery systems |
OA- | organic anion |
OAT | organic anion transporters |
OCT | organic cation transporters |
Oct/OAP | octomer/octomer associated proteins |
OMF | outer membrane factor family (transporter) |
ORCC | outwardly rectifying chloride channel |
ORF | open-reading frame |
OSN | olfactory sensory neurons |
OxlT | oxalate:formate antiporter |
Pax | paired box gene/TF |
pCa | -log10 of the Ca2+ concentration |
PCR | polymerase chain reaction |
PD | potential difference |
PDE | phosphodiesterase |
PDZ | PSD95-Dlg1-zo-1 (protein motif) |
PEPT | dipeptide transporters |
PG | prostaglandins |
PGC-1α | peroxisome proliferator-activated receptor α, co-activator 1α |
PGE2 | prostaglandin E2 |
P-gp | permeability glycoprotein (transporter) |
Phe | phenylalanine (F) |
Pi | inorganic phosphate |
PI3 | phosphatidylinositol 3-kinases |
PIP2 | phosphatidylinositol 4,5-bisphosphate |
PKA | protein kinase A |
PKC | protein kinase C |
PLC | phospholipase C |
PLCβ | β isoform of phospholipase C |
pLGICs | pentameric ligand-gated ion channels |
PM | plasma membrane |
PMCA | plasma membrane Ca2+ ATPase |
PP1 | protein phosphatase 1 |
PPAR | peroxisome proliferator-activated receptors (α, β, δ, or γ) |
PPRE | PPAR response element |
pRB | retinoblastoma protein |
Pro | proline (P) |
PSD95 | post synaptic density protein-95 |
Q1/Q2 | glutamine-rich domains (1 or 2) |
RaM | rapid mode uptake |
RAMP | receptor-activity modifying protein |
Ras | rat sarcoma (causing factor) |
RBC | red blood cell |
REST | repressor element-1 transcription factor |
RFLP | restriction fragment length polymorphism |
rhDNase | recombinant human DNase |
RICs | radio-immunoconjugates |
RIP | receptor-interacting protein |
RISC | RNA-induced silencing complex |
RLF | relaxin-like factor |
RNA pol | RNA polymerases |
RNA | ribonucleic acid |
RNAi | RNA interference |
RND | resistance-nodulation-cell division (transporter) |
ROS | reactive oxygen species |
rRNA | ribosomal RNA |
RSPO1 | R-spondin-1 |
RT-PCR | reverse-transcription polymerase chain reaction |
RXR | retinoic acid receptor |
RyR | ryanodine receptors |
SAM | intraluminal sterile α motif |
SBP | substrate binding protein |
Ser | serine (S) |
SERCA | sarco/endoplasmic reticulum Ca2+ ATPase |
Shh | sonic hedgehog homolog gene/TF |
siRNA | short interfering RNA |
SKCa | small conductance Ca2+-activated K+ channels |
SLC | solute carrier superfamily (transporter) |
SMN | survival of motor neurons protein |
SMR | small multidrug resistance superfamily (transporter) |
snoRNA | small nucleolar RNA |
SNP | single nucleotide polymorphism |
snRNA | spliceosomal small nuclear RNA |
SOC | store operated Ca2+ channel |
Sox9 | SRY-related HMG box-9 gene/factor |
SR | sarcoplasmic reticulum |
SRC-1 | steroid receptor co-activator-1. |
SREBP | sterol regulatory element-binding proteins |
SRY | sex-determining region Y |
SSS | solute sodium symporter (transporter) |
STAT | signal transducer and activator of transcription (1, 2 or 3) |
STIM | stromal interaction molecule |
SUG-1 | suppressor of gal4D lesions −1 |
SUMO | small ubiquitin like modifier |
SUR | sulfonylureas receptor |
SW1/SNF | switching mating type/sucrose non-fermenting proteins |
TAD | transactivation domain |
TAP | transporters associated with antigen processing |
TCA | tricarboxlyic acid |
TCR | T cell receptor |
TDF | testis-determining factor |
TEAD | TEA domain proteins |
TEF | transcription enhancer factor |
TESCO | testis-specific enhancer of Sox9 |
TGF | transforming growth factor |
TGN | trans-Golgi network |
TH | tyrosine hydroxylase |
Thr | threonine (T) |
TIF-1 | transcription intermediary factor |
TIRF | total internal reflection fluorescence imaging |
TMAO | trimethylamine N-oxide |
TMD | transmembrane domain |
TMS | transmembrane segments |
TNFs | tumour necrosis factors |
TPC | two pore calcium channels |
TPEN | N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine |
Trk | tyrosine kinase receptor (A, B or C) |
tRNA | transfer RNA |
TRP | transient receptor potential channels |
Trp | tryptophan (W) |
TTX | tetrodotoxin |
Tyr | tyrosine (Y) |
TZD | thiazolidinedione |
Ubi | ubiquitination |
UTR | untranslated region |
Val | valine (V) |
VDAC | voltage dependent anion channel |
VEGF | vasculoendothelial growth facto |
VFT | venus flytrap |
vGLUT | vesicular glutamate transporter |
VHL | von Hippel-Lindau protein |
VIP | vasoactive intestinal peptide |
VLDL | very low density lipoprotein |
Vm | membrane potential |
VOCC | voltage-operated calcium channels |
WNT4 | wingless-type mouse mammary tumour virus integration site |
YAC | yeast artificial chromosome |
YFP | yellow fluorescent protein |
YORK | yeast outward rectifying K+ channel |
ZAC | zinc-activated channel |
Zo-1 | zonula occludens-1 protein |
POST-FIXes
Chimeric antibodies—xiMabs
Human antibodies—muMbs
Humanised antibodies—zumab
Monoclonal antibodies—oMabs
Chapter 1
Introduction to Drug Targets and Molecular Pharmacology
1.1 Introduction to molecular pharmacology
1.2 Scope of this textbook
1.3 The nature of drug targets
1.4 Future drug targets
1.5 Molecular pharmacology and drug discovery
References
During the past 30 years there have been significant advances and developments in the discipline of molecular pharmacology—an area of pharmacology that is concerned with the study of drugs and their targets at the molecular or chemical level. Major landmarks during this time include the cloning of the first G-protein coupled receptor (GPCR) namely the β2-adrenergic receptor in 1986 (Dixon et al., 1986). This was quickly followed by the cloning of additional adrenergic receptor family genes and ultimately other GPCRs. The molecular biology explosion during the 1980s also resulted in the cloning of genes encoding ion channel subunits (e.g. the nicotinic acetylcholine receptor and voltage-gated Na+ channel) and nuclear receptors. The cloning of numerous drug targets continued at a pace during the 1990s but it was not until the completion of the human genome project in 2001 that the numbers of genes for each major drug target family could be determined and fully appreciated. As would be expected, the cloning of the human genome also resulted in the identification of many potentially new drug targets. The completion of genome projects for widely used model organisms such as mouse (2002) and rat (2004) has also been of great benefit to the drug discovery process.
The capacity to clone and express genes opened up access to a wealth of information that was simply not available from traditional pharmacology-based approaches using isolated animal tissue preparations. In the case of GPCRs detailed expression pattern analysis could be performed using a range of molecular biology techniques such as in situ hybridisation, RT-PCR (reverse transcriptase-polymerase chain reaction) and Northern blotting. Furthermore having a cloned GPCR gene in a simple DNA plasmid made it possible for the first time to transfect and express GPCRs in cultured cell lines. This permitted detailed pharmacological and functional analysis (e.g. second messenger pathways) of specific receptor subtypes in cells not expressing related subtypes, which was often a problem when using tissue preparations. Techniques such as site-directed mutagenesis enable pharmacologists to investigate complex structure-function relationships aimed at understanding, for example, which amino acid residues are crucial for ligand binding to the receptor. As cloning and expression techniques developed further it became possible to manipulate gene expression in vivo. It is now common practice to explore the consequences of deleting a specific gene either from an entire genome (knockout) or from a specific tissue/organ (conditional knockout). It is also possible to insert mutated forms of genes into an organism's genome using knockin technology. These transgenic approaches allow molecular pharmacologists to study developmental and physiological aspects of gene function in vivo and in the case of gene knockin techniques to develop disease models.
The molecular biology revolution also enabled the development of novel approaches for studying the complex signal transduction characteristics of pharmacologically important proteins such as receptors and ion channels. These include reporter gene assays, green fluorescent protein (GFP) based techniques for visualising proteins in living cells and yeast two hybrid-based assays for exploring protein-protein interactions. You will find detailed explanations of these and other current molecular-based techniques throughout this textbook. Another major breakthrough in the 2000s was the development of methods that allowed high resolution structural images of membrane-associated proteins to be obtained from X-ray crystallography. During this time the first X-ray structures of GPCRs and ion channels were reported enabling scientists to understand how such proteins function at the molecular level. Indeed crystallography is an important tool in the drug discovery process since crystal structures can be used for in silico drug design. More recently researchers have used NMR spectroscopy to obtain a high-resolution structural information of the β2-adrenergic receptor (Bokoch et al., 2010). A distinct advantage of NMR-based structural studies, which are already used for structural studies of other drug targets such as kinases, would be the ability to obtain GPCR dynamics and ligand activation data which is not possible using X-ray based methods. Some of the molecular pharmacology based approaches used to interrogate drug targets are outlined in Figure 1.1.
Despite this increased knowledge of drug targets obtained during the molecular biology revolution, there has been a clear slowdown in the number of new drugs reaching the market (Betz, 2005). However, since it takes approximately 15 years to bring a new drug to market it may be too early to assess the impact of the human genome project on drug discovery. In 2009 the global pharmaceutical market was worth an estimated $815 billion. However during the next few years a major problem facing the pharmaceutical industry is the loss of drug patents on key blockbusters. The hope for the future is that the advances in molecular pharmacology witnessed during the last decade or so will start to deliver new blockbuster therapeutics for the twenty-first century.
As briefly detailed above there have been numerous exciting developments in the field of molecular pharmacology. The scope of this textbook is to explore aspects of molecular pharmacology in greater depth than covered in traditional pharmacology textbooks (summarised in Figure 1.2). Recent advances and developments in the four major human drug target families (GPCRs, ion channels, nuclear receptors and transporters) are covered in separate chapters (Chapters 3–5 and 8). The molecular targets of anti-infective drugs (anti-bacterial and anti-viral) whilst of great importance are not covered in this book. Other chapters deal with the cloning of drug targets (Chapter 2) and transgenic animal technology (Chapter 10). The concept of gene therapy is explored in a case study-based chapter which looks at current and possible future treatment strategies for cystic fibrosis, the commonest lethal genetic disease of Caucasians (Chapter 6). Another major development in molecular pharmacology has been the discipline of pharmacogenomics: the study of how an individual's genetic makeup influences their response to therapeutic drugs (Chapter 7). These naturally occurring variations in the human genome are caused predominantly by single nucleotide polymorphisms (DNA variation involving a change in a single nucleotide) and there is a major research consortium aimed at documenting all the common variants of the human genome (The International HapMap project). The information from the project, which is freely available on the internet, will enable scientists to understand how genetic variations contribute to risk of disease and drug response. Finally, we take an in depth look at the role of calcium in the cell, looking at techniques used to measure this important second messenger (Chapter 9).
How many potential drug targets are there in the human genome? This is an important question often asked by the pharmaceutical industry since they are faced with the task of developing novel therapeutics for the future. When the draft sequence of the human genome was completed in 2001 it was estimated to contain approximately 31,000 protein-coding genes. However since its completion the number of human protein-coding genes has been continually revised with current estimates ranging between 20,000 and 25,000. Of these it is predicted that about 3000 are feasible protein drug targets. In 2005 it was calculated that about 100 drug targets account for all prescription drugs. On this basis there is obviously considerable scope for the development and discovery of novel drug targets to treat disease. At present the classical drug targets include GPCRs (Chapter 3), ion channels (Chapter 4), nuclear receptors (Chapter 8), transporters (Chapter 5) and enzymes. These important classical drug targets, whilst briefly covered in this Introduction, are extensively covered in later chapters. The distribution of drug targets expressed as a percentage of total products approved by the Food and Drug Administration (FDA; agency in the USA responsible for approving drugs for therapeutic use) is illustrated in Figure 1.3.
GPCRs represent the largest single family of pharmaceutical drug target accounting for approximately 30% of the current market. Their primary function is to detect extracellular signals and through heterotrimeric G-protein activation trigger intracellular signal transduction cascades that promote cellular responses (Figure 1.4). Whilst their share of the overall drug market is likely to fall in the future they still represent ‘hot’ targets for drug discovery programmes. GPCRs are conventionally targeted using small molecules (typically less than 500 Da) that are classified as agonists (receptor activating) or antagonists (inhibit receptor function by blocking the effect of an agonist). Some key examples of drugs that target GPCRs are listed in Table 1.1. Chapter 3 will explain in detail many of the recent developments in GPCR structure, function, pharmacology and signal transduction including GPCR dimerisation. Many of these exciting advances have revealed new pharmaceutical approaches for targeting GPCRs such as inverse agonists, allosteric modulators, biased agonists and bivalent ligands that target GPCR heterodimers. Since the completion of the human genome project it has emerged that the total number of human GPCRs may be as high as 865, which would account for approximately 3.4% of total predicted protein-coding genes (assuming a total of 25,000). For many cloned GPCRs the endogenous ligand(s) are unknown (so called ‘orphan’ GPCRs) and the identification of these orphan receptor ligands is the focus of drug discovery programmes within the pharmaceutical industry. The process of GPCR de-orphanisation is addressed in Chapter 2. In Chapter 11 the concept that GPCRs interact with a host of accessory proteins that are important in modulating many aspects in the life of a GPCR including the formation of signalling complexes will be explored. Indeed, targeting such GPCR signalling complexes with drugs that disrupt protein-protein interactions is another exciting avenue for future drug development not only in the field of GPCRs but also in other areas of signal transduction.
Ion channels represent important drug targets since they are involved in regulating a wide range of fundamental physiological processes. Indeed, at present they are the second largest class of drug target after GPCRs. They operate the rapid transport of ions across membranes (down their electrochemical gradients) and in doing so trigger plasma and organelle membrane hyperpolarisation or depolarisation. They are also potential drug targets for the treatment of rare monogenic hereditary disorders caused by mutations in genes that encode ion channel subunits. Such conditions termed ‘ion channelopathies’ include mutations in sodium, chloride and calcium channels that cause alterations in skeletal muscle excitability. The understanding of ion channel diversity and complexity increased significantly following the completion of the human genome project which identified over 400 genes encoding ion channel subunits. Given this number of genes it has been suggested that ion channels may rival GPCRs as drug targets in the future (Jiang et al., 2008). Other major developments include the first 3D resolution of ion channel structure by X-ray crystallography, which was reported for the voltage-gated potassium channel in 2003 (MacKinnon et al., 2003). Despite these important advances in the understanding of ion channel diversity and structure very few new ion channel drugs have reached the market during the last decade. Some key examples of ion channels as drug targets are shown in Table 1.2.
Ion channel | Drug (brand name) | Condition/use |
Voltage-gated Ca2+ channel | Amlodipine (Norvasc) | Hypertension and angina |
Voltage-gated Na+ channel | Phenytoin (Dilantin) | Epilepsy |
ATP-sensitive K+ channel | Glibenclamide (Glimepride) | Type II diabetes |
GABAA receptor | Benzodiazepines (Diazepam) | Anxiety |
5-HT3 receptor | Ondansetron (Zofran) | Nausea and vomiting |
Ion channels are broadly classified into two main groups (Figure 1.5). Firstly there are ligand-gated ion channels or ionotropic receptors which open when activated by an agonist binding to a specific ion channel subunit. Examples of this class include the nicotinic acetylcholine receptor, GABAA receptor, glycine receptor, 5-HT3 receptor, ionotropic glutamate receptors, and ATP-gated channels. The second group which includes voltage-gated or voltage-operated ion channels are opened by other mechanisms including changes in plasma membrane potential. Examples include voltage-gated Ca2+, Na+, and K+ channels. The molecular structure and classification of ion channels together with their use as drug targets will be explored in detail in Chapter 4.
Nuclear receptors are a large family of transcription factors that play a pivotal role in endocrine function. In contrast to other families of transcription factor the activity of nuclear receptors (as their name suggests) is specifically regulated by the binding of ligands (Figure 1.6). Such ligands, which are small and lipophilic, include steroid hormones (glucocorticoids, mineralocorticoids, androgens, oestrogens and progestogens), thyroid hormones (T3 and T4), fat soluble vitamins D and A (retinoic acid) and various fatty acid derivatives. Since the completion of the human genome sequencing project 48 members of the human nuclear receptor family have been identified. However, for many nuclear receptors the identity of the ligand is unknown. These ‘orphan’ nuclear receptors are of significant interest to the pharmaceutical industry since they may lead to the discovery of novel endocrine systems with potential therapeutic use. Whilst the total number of nuclear receptors is small in comparison to GPCRs they are the target of approximately 13% of all prescribed drugs. For example, the chronic inflammation associated with asthma can be suppressed by inhaled glucocorticoids and oestrogen-sensitive breast cancer responds to treatment with the oestrogen receptor antagonist tamoxifen. The structure, classification, signal transduction mechanisms and therapeutic uses of nuclear receptor targeting drugs will be explored in detail in Chapter 8.
The concentration of some neurotransmitters within the synaptic cleft is tightly regulated by specific plasma membrane-bound transporter proteins. These transporters, which belong to the solute carrier (SLC) transporter family, facilitate the movement of neurotransmitter either back into the pre-synaptic neuron or in some cases into surrounding glial cells. There are two major subclasses of plasma-membrane bound neurotransmitter transporter: the SLC1 family which transports glutamate and the larger SLC6 family which transports dopamine, 5-HT, noradrenaline, GABA and glycine (Figure 1.7). Both SLC1 and SLC6 families facilitate neurotransmitter movement across the plasma membrane by secondary active transport using extracellular Na+ ion concentration as the driving force. As might be expected drugs that target neurotransmitter transporters have a wide range of therapeutic applications such as treatment for depression, anxiety and epilepsy. Indeed, neurotransmitter transporters are the target for approximately one-third of all psychoactive drugs (see Table 1.3). The molecular structure and classification of neurotransmitter transporters and their value as important current and future drug targets will be discussed in detail in Chapter 5.
Transporter | Drug (brand name) | Condition/ use |
5-HT transporter (SERT) | Sertraline (Zoloft) | Antidepressant |
Dopamine transporter (DAT) | Cocaine | Drug of abuse |
Noradrenaline transporter (NET) | Bupropiona (Welbrutin) | Antidepressant |
GAT-1 (GABA) | Tiagabine | Epilepsy |
Affinity for DAT as well
At present more than 50% of drugs target only four major gene families, namely GPCRs, nuclear receptors, ligand-gated ion channels and voltage-gated ion channels (Figure 1.3). It is likely that the market share of these classical drug targets will shrink as new drug targets and approaches are developed in the future.
It is predicted that protein kinases (and lipid kinases), one of the largest gene families in eukaryotes, will become major drug targets of the twenty-first century. Protein phosphorylation is reversible and is one of the most common ways of post-translationally modifying protein function. It regulates numerous cellular functions including cell proliferation, cell death, cell survival, cell cycle progression, and cell differentiation. The enzymes that catalyse protein phosphorylation are known as protein kinases, whereas the enzymes that carry out the reverse dephosphorylation reaction are referred to as phosphatases (Figure 1.8a). The human genome encodes for 518 protein kinases and approximately 20 lipid kinases. The predominant sites of protein phosphorylation are the hydroxyl groups (−OH) in the side chains of the amino acids serine, threonine and tyrosine (Figure 1.8b). When a phosphate group is attached to a protein it introduces a strong negative charge which can alter protein conformation and thus function.
Enzymes are the drug target for approximately 50% of all prescribed drugs. Some key examples are listed in Table 1.4. However, because of their diverse nature they will not be the focus of a specific chapter in this book. It is also important to remember that many prescribed drugs target bacterial and viral enzymes for the treatment of infectious disease and HIV. Also many enzymes, whilst not direct drug targets, play important roles in drug metabolism for example cytochrome P450 enzymes.
Enzyme | Drug (brand name) | Condition/use |
HMG-CoA reductase | Statins | Used to lower blood cholesterol levels |
Phosphodiesterase type V | Sildenafil (Viagra) | Erectile dysfunction and hypertension |
Cyclo-oxygenease | Aspirin | Analgesic and anti-inflammatory |
Angiotensin-converting enzyme | Captopril (Capoten) | Hypertension |
Dihydrofolate reductase | Methotrexate | Cancer |
Protein kinases are classified according to the amino acid they phosphorylate and are grouped into two main types: serine/threonine kinases and tyrosine kinases. In both cases ATP supplies the phosphate group with the third phosphoryl group (γ; gamma phosphate) being transferred to the hydroxyl group of the acceptor amino acid. Examples of serine/threonine kinases include protein kinase A (PKA; activated by the second messenger cyclic AMP) and protein kinase C (PKC; activated by the second messenger diacylglycerol). Examples of tyrosine kinases include tyrosine kinase linked receptors for insulin and epidermal growth factor and non-receptor tyrosine kinases such as Src and JAK (Janus-associated kinase). Given the prominent role of protein phosphorylation in regulating many aspects of cell physiology it is not surprising that dysfunction in the control of protein kinase signalling is associated with major diseases such as cancer, diabetes and rheumatoid arthritis. These alterations in protein kinase and in some cases lipid kinase function arise from over-activity either due to genetic mutations or over-expression of the protein. It is estimated that up to 30% of all protein targets currently under investigation by the pharmaceutical industry are protein or lipid kinases. Indeed, there are approximately 150 protein kinase inhibitors in various stages of clinical development, some of which are highlighted in Table 1.5. Whilst protein kinases are important new human drug targets they are also present in bacteria and viruses and thus represent potential targets for infectious disease treatment.
Drug | Protein kinase target | Use |
AZD 1152 | Aurora B Kinase | Various cancers |
NP-12 | Glycogen synthase kinase 3 (GSK3) | Alzheimer's disease |
Bay 613606 | Spleen tyrosine kinase (Syk) | Asthma |
INCB-28050 | Janus-associated kinase 1/2 (JAK1/2) | Rheumatoid arthritis |
BMS-582949 | p38 mitogen-activated protein kinase (p38 MAPK) | Rheumatoid arthritis |
Since the launch of imatinib in 2001 several other small-molecule protein kinase inhibitors have successfully made it to the market place as novel anti-cancer treatments (Table 1.6). The majority of these drugs are tyrosine kinase inhibitors and in some cases function as multi-kinase inhibitors (e.g. sunitinib) targeting PDGFR (proliferation) and VEGFR (angiogenesis) dependent signalling responses. Monoclonal antibodies are also used to block the increased tyrosine kinase linked receptor activity that is associated with many forms of cancer and these will be discussed in Chapter 12.
Drug (brand name) | Targets | Use |
Imatinib (Gleevec®) | c-Abl-kinase, c-Kit | Chronic myeloid leukaemia |
Gefitinib (Iressa®) | EGFR | Various cancers |
Sunitinib (Sutent®) | PDGFR, VEGFR | Renal cell carcinoma |
Dasatinib (Sprycel®) | c-Abl-kinase, Src | Various cancers |
Everolimus (Afinitor®) | mTORa | Various cancers |
Serine/threonine kinase. Abbreviations: EGFR, epidermal growth factor receptor; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor.
A useful approach for assessing the therapeutic potential of novel drug targets is the number of approved patents for each target (Zheng et al., 2006). The level of patents gives an indication of the degree of interest in that particular target and hence likelihood of successful drugs being developed. Future targets with a high number of US-based patents include matrix metalloproteinases (MMPs) as a target for cancer treatment. MMPs are proteases which break down the extracellular matrix thus facilitating cancer cell invasion and metastasis. Other targets include phosphodiesterase 4 (PDE4), caspases and integrin receptors. Only time will tell whether any of these novel targets result in the development of effective therapeutics. For further reading on the identification and characteristics of future drug targets see the review by Zheng et al. (2006).