Molecular Pharmacology E-Book

John Dickenson

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1111

Veröffentlichungsjahr: 2012

Beschreibung

This textbook provides a fresh, comprehensive and accessible introduction to the rapidly expanding field of molecular pharmacology. Adopting a drug target-based, rather than the traditional organ/system based, approach this innovative guide reflects the current advances and research trend towards molecular based drug design, derived from a detailed understanding of chemical responses in the body. Drugs are then tailored to fit a treatment profile, rather than the traditional method of ‘trial and error’ drug discovery which focuses on testing chemicals on animals or cell cultures and matching their effects to treatments.

Providing an invaluable resource for advanced under-graduate and MSc/PhD students, new researchers to the field and practitioners for continuing professional development, Molecular Pharmacology explores; recent advances and developments in the four major human drug target families (G-protein coupled receptors, ion channels, nuclear receptors and transporters), cloning of drug targets, transgenic animal technology, gene therapy, pharmacogenomics and looks at the role of calcium in the cell.

• Current - focuses on cutting edge techniques and approaches, including new methods to quantify biological activities in different systems and ways to interpret and understand pharmacological data.
• Cutting Edge - highlights advances in pharmacogenomics and explores how an individual’s genetic makeup influences their response to therapeutic drugs and the potential for harmful side effects.
• Applied - includes numerous, real-world examples and a detailed case-study based chapter which looks at current and possible future treatment strategies for cystic fibrosis. This case study considers the relative merits of both drug therapy for specific classes of mutation and gene therapy to correct the underlying defect.
• Accessible - contains a comprehensive glossary, suggestions for further reading at the end of each chapter and an associated website that provides a complete set of figures from within the book.

Leseprobe

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

This edition first published 2013 © 2013 by John Wiley & Sons, Ltd

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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

[Ca

2+

]

i

intracellular free ionised calcium concentration

[Ca

2+

]

n

nuclear free ionised calcium concentration

[Ca

2+

]

o

extracellular free ionised calcium concentration

2-APB

2-aminoethoxydiphenyl borate

4EFmut DREAM

4

th

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 Ca

2+

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

BK

Ca

big conductance Ca

2+

-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

Ca

2+

-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

Ca

V

voltage-gated Ca

2+

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

c

ytosine-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 P

450

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

FEV

1

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

I

crac

calcium release activated Ca

2+

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

IP

3

inositol 1,4,5-triphosphate

IP

3

R

IP

3

receptor

iPLA

2

β

β isoform of Ca

2+

independent phospholipase A

2

IRT

immunoreactive trypsinogen

I

sc

short circuit current

ISE

introns splicing enhancer

ISS

introns splicing silencer

K

2P

two-pore potassium channels

K3K4 HMT

histone methyl transferase

K

ATP

ATP-sensitive K

+

channels

kb

kilobase

K

Ca

Ca

2+

-activated K

+

channels

KCC

K

+

-Cl

−

co-transporter

KChIP

K

+

channel interacting protein

KCO

K

+

channel openers

Kd

Ca

2+

dissociation constant

K

G

G-protein gated K

+

channels

KID

kinase-inducible domain

K

ir

inwardly rectifying K

+

channels

K

V

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 Ca

2+

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

Na

V

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

-log

10

of the Ca

2+

concentration

PCR

polymerase chain reaction

PD

potential difference

PDE

phosphodiesterase

PDZ

PSD

95

-Dlg1-zo-1 (protein motif)

PEPT

dipeptide transporters

PG

prostaglandins

PGC-1α

peroxisome proliferator-activated receptor α, co-activator 1α

PGE

2

prostaglandin E

2

P-gp

permeability glycoprotein (transporter)

Phe

phenylalanine (F)

Pi

inorganic phosphate

PI3

phosphatidylinositol 3-kinases

PIP

2

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 Ca

2+

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)

PSD

95

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 Ca

2+

ATPase

Shh

sonic hedgehog homolog gene/TF

siRNA

short interfering RNA

SK

Ca

small conductance Ca

2+

-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 Ca

2+

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

V

m

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

1.1 Introduction to molecular pharmacology

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 drug design. More recently researchers have used NMR spectroscopy to obtain a high-resolution structural information of the β-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 .