i

α-conotoxins targeting neuronal nAChRs:

Understanding molecular pharmacology and potential therapeutics

This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Shiva Nag Kompella B.Sc., MS (The University of Queensland)

School of Medical Sciences, Health Innovations Research Institute

RMIT University October 2013

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Declaration by the Candidate

I, Shiva Nag Kompella, declare that:

a) except where due acknowledgement has been made, this work is that of myself alone;

b) this work has not been submitted previously, in whole or part, to qualify for any other

academic award;

c) the content of the thesis is the result of work that has been carried out since the official

commencement date of the approved research program;

d) any editorial work, paid or unpaid, carried out by a third party is acknowledged.

Signed: Date:

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Statement of contributions by others to the thesis as a whole The collaborators for the work in this thesis include Dr. Richard Clark (School of

Biomedical Sciences, The University of Queensland ) for peptide synthesis and Dr. Norelle

Daly (Institute for Molecular Biosciences, The University of Queensland) for NMR

structural analysis in Chapter 3. Dr. Andrew Hung (RMIT University) for molecular

modelling and docking simulations.

Statement of parts of the thesis submitted to qualify for the award of another degree None Published works by the candidate incorporated into the thesis

1. Chapter 3 Section 3.3.1:

Franco A, Kompella SN, Akondi KB, Melaun C, Daly NL, Luetje CW, Alewood PF, Craik DJ, Adams DJ, Marí F. (2012) RegIIA: an α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs. Biochem Pharmacol. 83(3):419-26

2. Chapter 4

Inserra MC †, Kompella SN †, Vetter I, Brust A, Daly NL, Cuny H, Craik DJ, Alewood PF, Adams DJ, Lewis RJ. (2013) Isolation and characterisation of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors. Biochem Pharmacol. 86(6):791-9. (†co-first author).

3. Chapter 6

van Lierop BJ †, Robinson SD †, Kompella SN †, McArthur JR, Hung A, MacRaild C, Adams DJ, Norton RS, Robinson AJ. (2013) Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors. ACS Chem Biol. 8(8):1815-21. (†co-first author).

Additional published works by the candidate during PhD but not incorporated into the thesis

1. Yu R, Kompella SN, Adams DJ, Craik D, Kaas Q. (2013) Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic acetylcholine receptor. J Med Chem. 56(9):3557-67.

2. Safavi-Hemami H, Siero WA, Kuang Z, Williamson NA, Karas JA, Page LR, MacMillan D, Callaghan B, Kompella SN, Adams DJ, Norton RS, Purcell AW. (2011) Embryonic toxin expression in the cone snail Conus victoriae: primed to kill or divergent function? J Biol Chem. 286(25):22546-57.

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Talks at Conferences/Meetings:

1. Kompella SN, Hung A, Clark RJ, Adams DJ. (2012) α-Conotoxins targeting nAChRs: mutagenesis studies improving selectivity and potency. Biomed Link Conference, St. Vincent’s Hospital, University of Melbourne.

2. Kompella SN, Marco Inserra, Irina Vetter, Andreas Brust, Paul Alewood, Richard Lewis, David J. Adams. (2012) α-Conotoxin LsIA targeting nAChRs: structure – function relationship studies for future design and development of selective ligands. Higher Degree Research Student Conference – ‘From Inception to Excellence’, RMIT University.

3. Kompella SN, Nevin S, Hung A, Adams DJ. (2010) α-conotoxins targeting neuronal nicotinic acetylcholine receptors (nAChRs) – potential therapeutic drug development. Higher Degree Research Student Conference– ‘Presenting Tomorrow's Knowledge’, RMIT University.

Poster presentation at Conferences/Meetings:

1. Kompella SN, Shaoqiong Xu, Tianlong Zhang, Mengdi Yan, Xiaoxia Shao, Chengwu Chi, Jianping Ding, and Chunguang Wang, David J. Adams (2013) Novel Strategy of Blocking nAChR Revealed by Dissecting a Dimeric Conotoxin αD-GeXXA. ‘Nicotinic Acetylcholine Receptors as Therapeutic Targets: Emerging Frontiers in Basic Research & Clinical Science, Society for neuroscience satellite symposium, San Diego, CA.

2. Kompella SN, Hung A, Clark RJ, Adams DJ. (2013) α-Conotoxins targeting nAChRs: mutagenesis studies improving selectivity and potency. Gage Ion Channels and Transporters Conference, Canberra Boys Grammar School, ACT, Australia.

3. Kompella SN, Hung A, Clark RJ, Adams DJ. (2013) α-Conotoxins targeting nAChRs: mutagenesis studies improving selectivity and potency. Biophysical Society 57th Annual Meeting, Philadelphia, Pennsylvania, USA.

4. Kompella SN, Nevin S, Hung A, Adams DJ. (2010) α-conotoxins targeting neuronal nicotinic acetylcholine receptors (nAChRs) – potential therapeutic drug development. Higher Degree Research Student Conference– ‘Presenting Tomorrow's Knowledge’, RMIT University.

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Acknowledgements I am deeply thankful to my supervisor Prof. David J. Adams for the opportunity to do this

PhD and his constant support towards experimental designs and publications. I would also

like to thank my supervisor for the opportunity to attend various national and international

conferences to understand the scope of my research work and network with other leading

scientists. I like to thank my co-supervisor Dr. Brett A. Cromer and all my lab members

during the period of my PhD for their support and feedback for publications, conference

talks and poster presentations. I am thankful for Dr. Richard Clark and Dr. Norelle Daly for

providing the opportunity to visit their labs to learn peptide synthesis and NMR structural

analysis respectively. I wish to thank all the members and groups of my publications (listed

above) with whom I have collaborated, for the opportunity to be part of their various

projects. Thanks to our collaborator Dr. Frank Marí (Department of Chemistry &

Biochemistry, Florida Atlantic University, USA) for providing the α-conotoxin RegIIA for

structural and functional characterisation (Chapter 3). My thanks to Chunguang Wang

(Institute of Protein Research, Tongji University, China), Jianping Ding (State Key

Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai

Institutes for Biological Sciences, China) and their lab members for providing the

opportunity to collaborate with respective to the αD-GeXXA project (Chapter 5).

Last, but not the least I would like to thank my family for their phenomenal support in

undertaking and completion of my PhD.

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Table of Contents

List of Figures…………………………………………………………………………………..….ix

List of Tables…………………………………………………………………………………..…...xi

Abbreviations…………………………………………………………………………………..…..xii

Abstract…………………………………………………………………………………………..….1

Chapter 1: General Introduction……………………………………………………………..…5

1.1 Nicotinic acetylcholine receptors

1.1.1 Discovery…………………………………………………………………………......6

1.1.2 Subunit diversity……………………………………………………………………...8

1.1.3 Receptor structure………………………………………………………………………8

1.3.1 Extracellular domain (ECD) or Ligand-binding domain (LBD)………….......9

1.3.2 Transmembrane domain (TMD) and Intracellular domain…………………….12

1.1.4 Subunit stoichiometry of nAChRs: structural and functional implications…………...14

1.1.5 Function and Biophysical properties……………………………………………15

1.1.6 Calcium signaling and modulation of neuronal growth……………………………….18

1.1.7 Nicotinic acetylcholine receptors and diseases.…………………………………....23

1.1.7.1 nAChRs and pain……………………………………………………………..23

1.1.7.2 nAChR signalling and lung cancer……….…………………………………..25

1.1.8.1.1 Role of α3 nAChR subunit………………………………………...25

1.1.8.1.2 Physiological to biochemical translation: cellular difference mediated by α7 and α3 subunits………………………………….28

1.1.8.1.3 Targeting α3β4 nAChR…………………………………………....29

1.2. Conotoxins……………………………………………………………………………..31

1.2.1 Venom apparatus and biosynthesis……………………………………………………...31

1.2.2 Classification and nomenclature……………………………………………………...33

1.2.3 Conotoxin structural diversity…………………………………………………………….35

1.2.4 Conotoxin structure–activity relationship……………………………………………36

1.2.4.1 Use of alanine scanning mutagenesis…………………………….. . . . . .38

1.2.5 Conotoxin re-engineering………………………………………………………...39

1.2.5.1 Dicarba modification…………………………………………………….....39

1.2.6 Conotoxins: potential therapeutics…………………………………………………40

1.3 References……………………………………………………………………………..41

Chapter 2: Materials and Methods……………………………………………………………..67

2.1 Materials………………………………………………………………………………..68

2.2 Peptide Synthesis……………………………………………………………………….68

2.2.1 Solid Phase Peptide Synthesis (SPPS) ……………………………………………68

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2.2.2 Fmoc SPPS…………………………….……………………...……………….69

2.2.2.1 Peptide assembly…………………………….……………………...69

2.2.2.2 Peptide cleavage…………………………….…………….………...71

2.2.2.3 Directed disulphide bond formation………………………………..72 2.2.3 NMR Spectroscopy…………………………….……………………………..74

2.3 Electrophysiological recordings in Xenopus oocyctes……………………………….74

2.4 Data analysis…………………………….…………………………….………………76

2.5 References…………………………….…………………………….…………………77 Chapter 3: α-RegIIA targeting α3β4 nAChR: unlocking novel therapeutics towards lung

cancer 3.1 Introduction………………………………………………………………………..80

3.1.1 Nicotinic acetylcholine receptors (nAChRs)……………………………......80

3.1.2 Pathophysiological role of α3β4 nAChR subtype…………………………...80

3.1.3 α-Conotoxins targeting neuronal nAChRs……………………………….....81

3.2 Aims………………………………………………………………………………..83

3.3 Results

3.3.1 Selective α-conotoxin RegIIA inhibition of recombinant nAChR subtypes…84

3.3.2 Directed Peptide Synthesis of α-conotoxin RegIIA analogues and NMR……86

3.3.3 Inhibition of nAChR subtypes by α-conotoxin RegIIA analogues……………87

3.3.4 Double mutant [N11A,N12A]RegIIA selectively inhibits α3β4 nAChR

subtype……………………………………………………………………….91

3.4 Discussion

3.4.1 Conus regius: characterization of novel α-conotoxin RegIIA………………..92

3.4.2 Alanine mutagenesis reveals pharmacological role of –NNP– motif in RegIIA………………………………………………………………………..93

3.4.3 Molecular modelling and MD reveal structural topology of α3β2 and α3β4

subtypes and residues interacting with RegIIA………………………………93

3.5 Summary and conclusion ……………………………………………………………….95

3.6 References…………………………………………………………………………….…97

Chapter 4: Isolation and characterisation of α-conotoxin LsIA

4.1 Introduction………………………………………………………………………….…106

4.2 Aims……………………………………………………………………………………107

4.3 Results…………………………………………………………………………….……108

4.3.1 LsIA inhibition of recombinant nAChR subtypes…………………….……..108

4.3.2 Influence of C-terminal carboxylation and N-terminal truncation of LsIA….113

4.4 Discussion……………………………………………………………………..….……115

4.5 Summary and Conclusion …………………………………………………….….……118

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4.6 References……………………………………………………………………..….……119

Chapter 5: Characterization of Dimeric αD-Conotoxin GeXXA Reveals a Novel Strategy of Blocking nAChRs

5.1 Introduction………………………………………………………………………….…125

5.2 Aims ……………………………………………………………………………………127

5.3 Results…………………………………………………………………………….……128

5.3.1 Concentration-dependent inhibition of α3-containing receptors by α-D

conotoxin GeXXA……………………………………………..……............128

5.3.2 Monomeric α-D conotoxin GeXXA selectively inhibits the α9α10 nAChR……………………………………………………………..….…….130

5.3.3 α9α10 hybrid nAChR studies reveal the site of monomeric α-D conotoxin

GeXXA binding……………………………………………………………..131

5.4 Discussion and conclusion…………………………………………………..….……...132

5.5 References……………………………………………………………………..….……136

Chapter 6: Dicarba modification of α-conotoxins exhibits differential selectivity for nAChRs and GABAB receptors

6.1 Introduction………………………………………………………………………….…139

6.1.1 Conotoxins - Conus victoriae and Conus regius………………………….…139

6.1.2 The molecular mechanism of analgesia………………………….…………..139

6.1.3 The α9α10 nAChR subtype: expression and function…………………….…140

6.1.4 α-Conotoxin drug development: limitations and strategies……………….…141

6.2 Aims ……………………………………………………………………………………143

6.3 Results…………………………………………………………………………….……144

6.3.1 Regioselective dicarba Vc1.1 analogues exhibit differential activity at α9α10

nAChR subtypes……………………………………………………….……144

6.3.2 Dicarba modification of RgIA confers similar pharmacological effects to those of Vc1.1……………………………………………………….………….…145

6.4 Discussion ……………………………………………………………………..….……148

6.5 Summary and conclusion …………………………………………………….…..……150

6.6 References……………………………………………………………………..….……151

Chapter 7: Conclusion & future directions……………………………………………………...156

References………………………………………………………………………….…...160

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List of Figures

Figure 1.1: Structural features of Torpedo nAChR……………………………………………………7

Figure 1.2: nAChR subunits forming (A) homomeric and (B) heteromeric receptors………………..9

Figure 1.3: Cartoon representation of the ligand binding domain between α and δ subunit of Torpedo

nAChR……………………………………………………………………………………..11

Figure 1.4: Surface overlayed-cartoon representation of transmembrane domains of Torpedo

nAChR……………………………………………………………………………………..13

Figure 1.5: Various stoichiometric combinations of a heteromeric receptor………………………….14

Figure 1.6: nAChR-mediated Ca2+ signalling and modulation of neuronal growth…………………...20

Figure 1.7: nAChR mediated signalling pathway involved in lung cancer and their cells of origin….27

Figure 1.8: Predatory marine cone snails (A) Conus regius and (B) Conus victoriae………………...31

Figure 1.9: Schematic representation of cone snail’s venom apparatus representing the main

parts………………………………………………………………………………………...32

Figure 1.10: Classification of conotoxins……………………………………………………………...34

Figure 1.11: Structural diversity among conotoxins…………………………………………………...36

Figure 1.12: AChBP and [A10L,D14K]PnIA co-crystal structures…………………………………...38

Figure 2.1: The basic apparatus setup required for solid phase peptide synthesis…………………….69

Figure 2.2: Directed disulphide formation of α-conotoxins using orthogonal cysteine protecting

groups.……………………………………………………………………………………73

Figure 2.3: Schematic representation of the various steps involved in two-electrode voltage clamp

studies using Xenopus laevis oocytes. …………………………………………………….75

Figure 3.1: Concentration-dependent RegIIA inhibition of ACh-evoked current amplitude mediated by

(A) α3β2, (B) α3β4 and (C) α7 nAChRs expressed in oocytes……………………………84

Figure 3.2: Selectivity of α-conotoxin RegIIA inhibition of nAChR subtypes………………………..85

Figure 3.3: RegIIA and alanine analogues secondary αH shifts……………………………………….87

Figure 3.4: RegIIA and alanine analogue (300 nM) inhibition of various nAChR subtypes expressed in

Xenopus oocytes…………………………………………………………………………...88

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Figure 3.5: [N11A]RegIIA and [N12A]RegIIA exhibiting improved selectivity at α3β4 nAChR

subtypes……………………………………………………………………………………89

Figure 3.6: [N11A, N12A]RegIIA inhibition of α3β2 and α3β4 nAChR compared with that of wild-

type RegIIA………………………………………………………………………………..91

Figure 4.1: α-Conotoxin LsIA selectivity for various nAChR subunit combinations expressed in

Xenopus oocytes………………………………………………………………………….108

Figure 4.2: Kinetics of LsIA inhibition of peak ACh-evoked current amplitude as a function of

time……………………………………………………………………………………….110

Figure 4.3: Kinetics of α-conotoxin LsIA block and recovery at the α7 nAChR subtype…………...111

Figure 4.4: Influence of N-terminus truncation and C-terminus carboxylation of LsIA on ACh-evoked

current inhibition at the α7 (A) and α3β2 (B) nAChR subtypes………………………….113

Figure 5.1: Dimeric αD-conotoxin GeXXA inhibition of various nAChR subtypes………………...129

Figure 5.2: α9α10 hybrid nAChR inhibition by monomeric αD-conotoxin GeXXA………………...130

Figure 5.3: The sequence and disulphide linkage of αD-conotoxin GeXXA………………………...132

Figure 5.4: The crystal structure of αD-conotoxin GeXXA………………………………………….133

Figure 5.5: Pairwise sequence alignment of the ACh-binding site of the mature α10 subunit

peptide.……………………………………..……………………………………………..135

Figure 6.1: Dicarba Vc1.1 analogues (3 µM) percentage inhibition of ACh-evoked currents at (A)

α9α10 and (B) α3β4 nAChR subtypes……………………………………………………144

Figure 6.2: Dicarba Vc1.1 analogues concentration-dependent inhibition of ACh-evoked currents at

the α9α10 nAChR subtype………………………………………………………………..145

Figure 6.3: [3,12]-Dicarba RgIA analogue inhibition of ACh-evoked currents at rat α9α10 and human

α7 nAChRs expressed in Xenopus oocytes……………………………………………….147

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List of Tables

Table 1.1: Sequence alignment of α-conotoxins targeting neuronal nAChR subtypes………………..37

Table 3.1: α-Conotoxin RegIIA inhibition of recombinant nAChR subunits expressed in Xenopus

oocytes……………………………………………………………………………………..85

Table 3.2: RegIIA and analogues inhibition of nAChR subtypes……………………………………..90

Table 3.3: Sequence alignment of α-conotoxins targeting various nAChR subtypes…………………96

Table 4.1: α-Conotoxin LsIA’s kinetic constants for blocking nAChR subtypes……………………112

Table 4.2: Half-maximal inhibitory concentrations (IC50) and Hill slope (nH) values from

concentration–response curves for LsIA and its analogues at the α3β2 and α7 nAChR

subtypes…………………………………………………………………………………..114

Table 4.3: Sequence alignment of LsIA and α-conotoxins…………………………………………...115

Table 5.1: Sequence alignment of αD-conotoxin GeXXA with other, previously discovered αD-

conotoxins………………………………………………………………………………...126

Table 5.2: Pharmacological profile of dimeric αD-conotoxin GeXXA inhibition of various

nAChRs…………………………………………………………………………………...128

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Abbreviations

ACh Acetylcholine AChBP Acetylcholine binding protein ACN Acetonitrile Acm Acetamidomethyl Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid BiP Immunoglobulin-binding protein Boc tert-butyloxycarbonyl BSA Bovine serum albumin Ca2+ Channel Voltage gated calcium channel CaMPK Ca2+ -calmodulin-dependent protein kinase

CCI Chronic constriction nerve injury CI Confidence interval CICR calcium induced calcium release Cys Cysteine CNS Central nervous system CTD C-terminal domain

CREB cAMP response element-binding protein DCM Dichloromethane DIPEA N,N-diisopropylethylamine DMF Dimethylformamide DNA Deoxyribonucleic acid DRG Dorsal root ganglion ECD Extracellular domain

ERK/MARK Extracellular signal-regulated mitogen-activated protein kinase ES-MS Electrospray mass spectrometer Fmoc 9-Fluorenylmethyloxycarbonyl g Grams Gln Glutamine Glu Glutamic acid Gly Glycine GWAS Genome-wide association studies HBTU O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium- hexafluoro-phosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HF Hydrogen fluoride His Histidine HPLC High performance liquid chromatography HVA High voltage-activated Hyp Hydroxyproline IAP Inhibitors of Apoptosis ICD Intracellular domain Ile Isoleucine IC50 Half-maximal inhibitory concentration ICK Inhibitory cysteine knot IP3R inositol (1,4,5)-triphosphate receptors KCN Potassium cyanide Leu Leucine Lys Lysine MBHA 4-Methylbenzhydrylamine MD Molecular dynamics

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Mec Mecamylamine Met Methionine mg milligrams min minutes ml milliliters MS Mass spectroscopy MS-222 Tricaine methanesulphonate nAChR Nicotinic acetylcholine receptor NFκB Nuclear factor kappa beta nH Hill slope NHBE normal human airway epithelial cells

NNK 4-(methylnitrosamino)-1-(3-pyridyl)-butanone NNN N-nitrosonornicotine

NMR Nuclear magnetic resonance NOE Nuclear Overhauser Effect NOESY Nuclear Overhauser Effect Spectroscopy NSCLC Non-small cell lung carcinoma NTD N-terminal domain P13K phosphatidyl-inositol 3-kinase PAC Peripheral adenocarcinoma PCR Polymerase chain reaction PDI Protein disulfide bond isomerase Phe Phenylalanine PKC Protein kinase C PNEC Pulmonary neuroendocrine cells PNS Peripheral nervous system PPI Peptidyl-prolyl cis-trans isomerase Pro Proline PSNL Partial sciatic nerve ligation RP-HPLC Reversed-phase high performance liquid chromatography RNA Ribonucleic acid RyR Ryanodine receptors

RT-PCR Reverse transcription polymerase chain reaction s Seconds SCLC Small cell lung carcinoma SEM Standard error of the mean Ser Serine SNP Single nucleotide polymorphisms SPPS Solid phase peptide synthesis TFA Trifluoroacetic acid TH Tyrosine hydroxylase TIPS Triisopropylsilane TOCSY Total Correlated Spectroscopy Thr Threonine Trp Tryptophan Trt Trityl or triphenylmethyl Tyr Tyrosine Val Valine VDCC voltage-dependent calcium channels XIAP X-liked inhibitor of apoptosis

1

ABSTRACT:

INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion

channels expressed in both central nervous system (CNS) and peripheral nervous system

(PNS) and are involved in fast ACh-mediated synaptic transmission. Neuronal nAChRs are

pentamers composed of a combination of alpha subunits (α2-10) and beta subunits (β2-4)

exhibiting diverse structural and functional heterogeneity. nAChRs are shown to contribute to

the physiological roles of nAChRs in neurotransmitter release and synaptic plasticity. Further,

they are also implicated in various pathophysiological conditions including Alzheimer’s

disease, schizophrenia, tobacco addiction and lung cancer.

α-Conotoxins, a new class of peptides that act as nAChR antagonists have been identified

from the venom of predatory marine cone snails. α-Conotoxins are a class of short di-sulphide

rich peptides which specifically target various nAChRs subtypes and are excellent molecular

probes for identifying the physiological role of nAChR subtypes in both normal and disease

states. They are defined by their characteristic cysteine framework, CCXmCXnC (Xm and Xn

represent the number of non-cysteine residues), classifying them into various subclasses.

CHAPTER 3: The α3β4 subtype is shown to be involved in lung cancer, nicotine addiction

and drug-abusive behaviour. Despite this, the knowledge of the pathophysiological role of

α3β4 subtypes is limited by the lack of adequate subtype specific probes. To-date only five α-

conotoxins inhibiting α3β4 nAChR subtype are known. Of these, α-conotoxin AuIB

belonging to a unique 4/6 subclass is the only peptide shown to selectively inhibit α3β4

nAChR subtype with an IC50 of 2.5µM.

We report the discovery of new α4/7-conotoxin RegIIA which was isolated from Conus

regius. RegIIA has a classical ω-shaped globular structure with balanced distribution of

shape, charges and polarity. RegIIA specifically inhibits ACh-evoked currents of α3β2, α3β4,

and α7 nAChRs isoforms. The implication of α3β4 nAChRs in various diseases such as lung

2

cancer and nicotine addiction along with RegIIA being the most-potent α3β4 nAChR

antagonist to date led us to investigate and improve RegIIA’s selectivity profile at the α3β4

nAChR subtype. Using alanine scanning mutagenesis and modelling studies, we identified

critical residues of α-conotoxin RegIIA that interact with the ACh-binding site of α3β2, α3β4

and α7 nAChRs.

CHAPTER 4: α3β2 and α7 nAChR subtypes play vital roles in various functions, such as

neuronal plasticity, angiogenesis and gene regulation. α-Conotoxins targeting these receptors

form excellent probes to understand their physiological roles in normal and diseases

conditions. Here I describe the pharmacological properties of the novel α-conotoxin LsIA, the

first peptide isolated from Conus limpusi, a species of worm-hunting cone snail commonly

found on the south east coast of Queensland, Australia. LsIA is an α4/7-conotoxin with the

characteristic I–III and II–IV disulfide connectivity. LsIA exhibited selective and potent α7

and α3β2 nAChR subtype antagonism. In this report, I also examined the structure–function

relationship of the presence of a unique N-terminal serine at position 2 and C-terminal

carboxylation. Furthermore, I also investigated the pharmacological implications, involving

incorporation of the α5 subunit towards the inhibition of LsIA at α3β2 nAChR subtypes.

CHAPTER 5: Novel α-conotoxins of the D-superfamily have only recently been discovered

and functionally characterized. These were found to be non-competitive nAChR antagonist

and are naturally dimeric, which makes their blocking mechanism more intriguing. From the

venom of Conus generalis, we identified a novel αD-conotoxin GeXXA. This toxin is a

disulfide-linked homodimer of a 10-Cys-containing peptide with each peptide chain made of

50 amino acid residues. Each polypeptide chain is composed of an N-terminal domain (NTD,

residues 1-20) involved in dimerization and a C-terminal domain (CTD, residues 21-50). αD-

GeXXA is a non-selective inhibitor of nAChR subtypes exhibiting most potency at the α7

3

subtype with an IC50 of 210 nM. However, for rat and human α9α10 nAChRs, the inhibition

by αD-GeXXA is irreversible, indicating the tight binding of αD-GeXXA to α9α10 nAChR.

To get further insight about its inhibition mechanism, the CTD of each chain was isolated and

tested against various nAChR subtypes. Contrary to native peptide, GeXXA-CTD showed

selective and reversible inhibition of α9α10 nAChR subtype. This specificity was further

investigated using hybrid receptors of rat and human α9 and α10 subunits. GeXXA-CTD was

10-fold less potent on human α9α10 than on rat α9α10 nAChR while similar potency was

observed with hα9rα10 hybrid receptor indicating significant role of α10 subunit in inhibition.

These results provide more insight into the novel blocking mechanism of α-D conotoxins.

CHAPTER 6: Conotoxins have emerged as useful leads for the development of novel

therapeutic analgesics. However, the disulfide connectivity of α-conotoxins can change under

oxidative and reduced conditions leading to changes in structural conformation. Exploitation

of these and other peptides in research and clinical settings has been hampered by the lability

of the disulfide bridges that are essential for toxin structure and activity. One solution to this

problem is replacement of cysteine bridges with nonreducible dicarba linkages. We explore

this approach by determining the functional characteristics of dicarba analogues of a novel

analgesic α-conotoxin Vc1.1 and RgIA which is known to inhibit high voltage-activated

(HVA) calcium channel currents via GABAB receptors and α9α10 nAChR subtypes. When

tested, dicarba Vc1.1 and dicarba RgIA analogues showed differential activity wherein the

[2,8]-dicarba analogues of both Vc1.1 and RgIA were active at HVA calcium channel current

via GABAB receptors whereas the [3,16]-dicarba analogues retained its activity at α9α10

nAChR subtypes. These results provide new leads towards the elucidation of the biological

target responsible for the peptide’s potent analgesic activity.

4

SUMMARY: In the past-decade, nAChRs have been identified as potential drug targets for

various diseases. However, knowledge about the role of various nAChR subtypes in both

physiological and pathophysiological conditions is scarce. My research describes the

discovery, characterization and development of a novel α3β4 antagonist as potential tool

towards understanding its role in lung cancer. Further, dicarba modification of peptides and

characterization of new class of α- and αD-conotoxins provide future insights towards drug

development.

5

CHAPTER 1

INTRODUCTION

6

1.1 Nicotinic acetylcholine receptors

1.1.1 The discovery

After the initial discovery of nicotinic acetylcholine receptors (nAChR) in 1914 by Henry H

Dale [1] and Otto Loewi [2], substantial progress has been made to improving our

understanding of the nicotinic mechanisms brought us a step closer to ever growing

knowledge hill. Numerous reviews documenting this progress have been published and

focused on nAChR structure, function, physiology, pharmacological tools and potential

therapeutic use [3-8]. Ironically, these advances only raised more questions about the

complexity of the nervous system and neurotransmission, and the contribution of these

receptors in various diseases, such as Parkinson’s disease, Alzheimer’s disease,

schizophrenia, epilepsy, dementia and cancer [9, 10]. Today, the major breakthroughs in

understanding nAChRs seem but a fascinating history tale that starts with the initial discovery

of the chemical nature of neurotransmission by Claude Bernard, a French physiologist, in

1857. The complexity of the cholinergic transmission or role of acetylcholine (ACh) was as

yet unanticipated.

In the mid-1980s, advancements in molecular biology enabled the identification and cloning

of the genes that encoded the first nAChR from Torpedo marmorata [11]. Since then, more

nAChR family members have been identified, with 16 genes encoding structurally

homologous yet distinct nAChR subunits now known [12]. The muscle nAChR was primarily

isolated, purified and sequenced by Jean-Pierre Changeux from the Torpedo electric organ

[13]. Consequently, the neuromuscular junction (endplate) for the analysis of nAChR-

mediated neurotransmission became widely used. This enabled physiologists to conduct

various experiments to improve our understanding of the biochemical nature and physiology

of neurotransmission.

7

nAChRs are ionotropic channels belonging to the cys-loop superfamily of the ligand-gated

ion channels [3]. Other ion channels belonging to this superfamily include GABAA, glycine

(GlyR), 5-HT3 serotonin and zinc-activated receptors, which have similar structural features

to nAChRs [12]. These features include an extracellular NH2-terminal domain (ECD), four

transmembrane domains (M1-M4), and a intracellular COOH-terminal sequence (ICD)

[Figure 1.1] [14]. Structural homology of human nAChRs was found to be evolutionarily

linked to the ionotropic channels, dating back as far as nematodes and molluscs and to simple

life forms, such as prokaryotes. [15, 16]. Along with these features, various nAChR subunits

have high sequence homology with each other.

Figure 1.1: Structural features of Torpedo nAChR (PDB 2BG9 [14]). Schematic

representation of (A) the pentameric structure of a Torpedo nAChR (B) a nAChR subunit

showing various structural features (C) Cartoon representation of the three dimensional of the

α-subunit of Torpedo nAChR generated using PyMol Molecular Graphics System (DeLano

Scientific LLC.).

8

1.1.2 Subunit diversity

The nAChR is one of the most well-studied ligand-gated ion channel family members. This is

due to various multidisciplinary studies, including genetic, protein, microscopic and structural

studies, that stemmed from earlier studies on the Torpedo electric organ nAChR structure.

Original studies made two major significant findings: 1) nAChRs from Torpedo have

remarkable density, enabling the pseudo-crystalline studies of the receptor at 4Ǻ resolution

[14]; and 2) studies of the crystal structure of a water soluble protein, ACh binding-protein

(AChBP) [17].

The 16 subunits that have been discovered are broadly classified as muscle or neuronal

subtypes, based on their tissue expression. Muscle subtypes include α1, β1, δ, γ and ε

subunits, and neuronal subtypes include α2–α10 and β2–β4 subunits. However, although this

nomenclature is widely used and provides a simple way to class receptor subtypes, it is being

discouraged by the International Union of Pharmacology [12]. This is because various studies

have indicated that more than one neuronal subtype receptor is expressed in various non-

neuronal tissues [18-20].

1.1.3 Receptor structure

nAChRs are pentameric structures made from various transmembrane subunit combinations.

The above-mentioned subunits assemble in numerous combinations to form two distinct

receptors classes, namely homomeric receptors (composed of only α subunits, such as α7) and

heteromeric receptors (composed of α and β subunits, such as α4β2) [Figure 1.2]. Up until

recently, only α7, α9 and α10 subunits were believed to form only homomeric receptors [21].

However, recent studies have provided evidence of the formation of α7-containing

heteromeric receptors [22, 23]. The pentameric structure of these receptors was initially

identified during earlier studies into the muscle nAChRs isolated from the Torpedo electric

9

organ. Only two known muscle nAChR combinations have been characterised, α1β1δγ and

α1β1δε. These were identified as fetal and adult muscle subtypes, respectively, based on their

expression levels during the developmental stages [24, 25].

Figure 1.2: nAChR subunits forming (A) homomeric and (B) heteromeric receptors. The

ACh binding site in each receptor is indicated in red triangles [26].

1.1.3.1 Extracellular domain/Ligand-binding domain

As mentioned earlier, nAChRs are ligand-gated ion channels and activated or opened in the

presence of an endogenous agonist or ligand ACh. The ligand- or agonist-binding site exists

on the extracellular domain, within the interface of two α subunits (in homomeric receptors)

or between an α and β subunit (in heteromeric receptors) in the receptor. A nomenclature of

‘agonist-binding subunit’ for α subunits and ‘structural subunits’ for non-α subunits (β1–β4,

δ, γ and ε subunits), was used to indicate the presence of the cysteine loop in α subunits. The

cysteine loop is needed to aid the formation of the functional ligand-binding domain [12].

However, this nomenclature was discontinued after it was discovered how vital the non-α

subunits are in the binding pocket. Today, the α-subunit interface is called the ‘principal’ or

‘+’ face and the β-subunit interface is called the ‘complementary’ or ‘-’ face [Figure 1.3] [27,

28].

Our knowledge of the N-terminal domain has developed through various binding and

functional assays in combination with chemical modification and mutagenesis experiments

[29-31]. Initial studies of photo affinity labelling (photolysis of covalently bound chemical

10

tags to the active sites of protein molecules) on muscle nAChRs using the Torpedo receptor

identified key amino acid residues, such as α-Tyr 93, which constitute the agonist-binding site

[32]. These studies provided an early indication of the hydrophobic nature of the agonist-

binding pocket. In muscle nAChRs, this site was located between the interface of α1-δ and

α1-γ subunits in fetal form, and α1-δ and α1-ε subunits in adult form [33].

More recently, major improvements in understanding the three-dimensional structure of these

receptors came through X-ray crystallographic and high-resolution electron microscopy

studies of the ACh binding protein (AChBP) and Torpedo receptor [17, 34]. AChBP, isolated

from glial cells of molluscs (Lymnaea), is a small, water-soluble protein. Its features are

structurally similar to those of the N-terminal extracellular domain of nAChRs, but it lacks

any of the other characteristics of the cys-loop receptor superfamily. The binding interaction

of ACh to AChBP is similar to nAChR receptor activation. As such, it has received significant

attention as it may help improve our understanding of the ligand-binding domain of these

proteins [35].

AChBP crystal structures revealed that the α subunit plays a major role in the agonist-binding

site. The Cys-Cys pair present only within the α subunits that form the ‘principal’ face of the

binding site is essential for ligand binding [Figure 1.3]. Mutation of these residues, Cys191–

Cys192 (Torpedo α subunit numbering), significantly affected receptor function and assembly

[36]. Furthermore, structural and simulation models revealed conformational changes in the

Cys-Cys pair of up to ~ 11Ǻ, interlocking the agonist/ligand deep within the binding pocket

[37].

Along with the mobility of the Cys-Cys pair, a series of conserved aromatic amino acid

residues in the ‘principal’ face contribute to the agonist-binding site. These residues are

11

labelled and grouped into various loops (indicated in parenthesis): Tyr93 (loop A), Trp149

and Tyr 151 (loop B), Tyr190 and Tyr198 (loop C). As the numbering indicates, the Cys-Cys

pair belongs to the C-loop of the ‘positive’ interface [Figure 1.3] [38-40]. It is worth noting at

this stage that among all of the identified α subunits, α5 and α10 subunits do not form the

‘principal’ face of the agonist-binding site within the receptor complex. Therefore, despite the

presence of the Cys-Cys pair, these subunits do not form homomeric or heteromeric

functional receptors. This may be due to the lack of conservation within the key residues. For

example, substitution of Asp for Tyr at position 198 of the α5 subunit makes it inactive to the

nicotinic agonist [41, 42].

Figure 1.3: Cartoon representation of the ligand binding domain between α and δ

subunit of Torpedo nAChR (PDB 2BG9 [14]). Amino acids contributing by the α-subunit

to the ‘principal’ or ‘+’ interface of the ACh-binding pocket (Y93, W149, Y151, Y190 and

Y198) and those contributed by the β-subunit interface to the ‘complementary’ or ‘-’ interface

(W54 and L108) are represented in stick, overlayed with sphere representation. The Cys191–

Cys192 of α-subunit is coloured yellow. Image generated using PyMol Molecular Graphics

System (DeLano Scientific LLC.).

12

1.1.3.2 The transmembrane domain and intracellular domain

Agonist-bound AChBP crystals significantly contributed to our understanding of the various

conformational changes within the N-terminal extracellular domain and are a very valuable

scientific tool [43, 44]. However, the lack of other components, such as a transmembrane

domain and channel gating, hindered the extrapolation of the information to complete

nAChRs.

The transmembrane domain of each nAChR subunit is made of four segments, M1–M4

[Figure 1.1]. These segments are important for channel gating and anchoring the receptor in

the lipid bilayer. The four transmembrane segments are arranged inside the pentameric

structure of the receptor with the M2 of all of the five subunits lining the channel pore and the

M4 of all of the five subunits forming the outer surface of the receptor [Figure 1.4]. This

arrangement, originally hypothesised after the initial bilayer membrane experiments with

isolated M2 segments of the Torpedo nAChR, was authenticated only after the electron

microscopic study of Torpedo nAChR by the Unwin group [14]. While the M2 segment is

primarily involved with channel gating, M1 and M4 segments modulate receptor assembly,

function and localisation. Early studies using chimeric M1 and M2 constructs reveal that M1–

M2 coupling is involved in the pentameric assembly of α7 nAChR [45]. This was evaluated

through simultaneous studies on muscle nAChR, and indicated that the M1 contributes to the

heterodimer structure of the receptor [46].

The orientation of the M4 to the outer surface of the receptors [Figure 1.4] suggests it has a

role in receptor localisation through its interactions with lipids and cholesterol in the

membrane bilayer [47, 48]. Furthermore, scanning mutagenesis studies within this Torpedo

nAChR segment altered its function and assembly [49, 50].

13

Figure 1.4: Surface overlayed-cartoon representation of transmembrane domains of

Torpedo nAChR (PDB 2BG9) [14]. The M2 segment (red) of all five subunits forms the ion

pore of the channel. The M1, M2 and M4 segments are represented in cyan, blue and green

colour respectively. α-V255 and α-L251 residues involved in channel gating are represented

in yellow spheres. Image generated using PyMol Molecular Graphics System (DeLano

Scientific LLC.).

Although the ligand-binding domain and transmembrane domain are vital for nAChR function

and assembly, the intracellular domain also significantly contributes to it. However, the

cytoplasmic loops of M1–M2 and M3–M4 have the distinct role of signal transduction to

these receptors via phosphorylation. While further validation of this function is required,

recent studies suggested that potential intracellular proteins, such as Rapsyn, interact with the

α9 nAChR and modulate its surface distribution [51, 52]. Mutations within the large M3–M4

intracellular loop of the α7 nAChR significantly reduced receptor function and assembly, and

mutations in the α3 subunit disrupted the distribution of α3-containing receptors in the

interneuronal synapse [53, 54].

1.1.4 Subunit stoichiometry of nAChRs: structural and functional implications

14

Sixteen nAChR subunits that assemble into homomeric or heteromeric pentamers have been

identified. Heteromeric receptors exhibit a huge diversity in structure, function and receptor

localisation, which can be attributed to the various possible subunit stoichiometric

combinations. To date, about 30 known subunit combinations arranged in various

stoichiometries have been identified. Thus, homomeric structure of a receptor provides a key

advantage in structural and functional understanding of the receptor. [55].

Stoichiometry of a heteromeric receptor is defined by the identity of the 5th subunit. For

example, for the α3β2 receptor, the pentameric structure can assemble the 5th subunit with α3

or β2, producing a simple stoichiometry of this receptor of either (α4)3(β2)2 or (α4)2(β2)3

[Figure 1.5]. Complex stoichiometries evolve when other subunits such as α5, α6 and β3 are

introduced as the 5th subunit, to form (α4)2(β2)2α5, (α4)2(β2)2α6 and α4α6(β2)2β3 receptor

combinations [Figure 1.5]. In the case of muscle nAChRs, only one subunit stoichiometry

exist: (α1)2β1δε/γ [14]. While recent studies have highlighted some of the roles played by

different stoichiometric combinations [5], much is to be learned about their contribution to

receptor function, pharmacology and physiology.

Figure 1.5: Various stoichiometric combinations of a heteromeric receptor [26].

While the pure mathematical combinations of different subunits represent a complex issue,

native nAChRs assemble predominantly with specific subunit stoichiometry. For example,

15

single-channel recording and direct biochemical studies of α4β2 nAChR, a major subtype

expressed throughout central nervous system (CNS), showed assembly primarily in the

(α4)2(β2)3 stoichiometry [56, 57]. However, this composition can be changed in model

expression systems, such as Xenopus oocytes, via RNA injection of different subunit ratios.

Recent studies on recombinant receptors using this technique showed differential agonist

sensitivity based on the subunit ratio of α4 and β2 subunits used (1:10 or 10:1), along with

changes in calcium permeability [58, 59]. Similar differences in affinity for both agonist

(ACh) and antagonist (α-conotoxin AuIB) were observed with α3β4 nAChR stoichiometry

[60]. Furthermore, recent electrophysiological studies with α5, α6 and β3 subunits revealed

complex subunit stoichiometries with a unique functional role in the receptors such as

receptor desensitization and gating (these concepts will be discussed in the next section). The

β3 subunit was reported to extensively co-localise with the α6 subunit, while forming

complex stoichiometric combinations [61].

Different α6-containing receptor stoichiometries have been implicated in the pathophysiology

of neurological disorders, such as Parkinson’s disease [62]. In addition, when the α5 subunit

is co-assembled with the α3β4 nAChR, calcium permeability increases [63] and nicotine-

evoked dopamine release in synaptosomal preparations containing α4β2 nAChRs is affected

[64]. Therefore, subunit stoichiometry plays a vital role in the pharmacological and

physiological role of various nAChR subtypes.

1.1.5 Receptor function, biophysical properties and gating

Mammalian nAChRs are activated by the natural endogenous agonist ACh binding to the N-

terminal extracellular domain. Upon ligand binding, the receptor undergoes rapid

conformational changes which transition into channel opening. These receptors are cation

16

selective and modulate mono- and divalent cation influx, depolarising cell membranes and

creating neuronal excitability.

Various residues within the extracellular, transmembrane and intracellular domains of the

receptor contribute to the microsecond conformation changes from ligand binding to channel

gating [3]. M1 and M2 are involved in the gating process, and help determine the ionic

selectivity of these receptors. Mutagenesis study has shown the presence of charged glutamate

and valine within M2 and a lack of proline in the intracellular domain of M1–M2 correspond

with the cation permeability of nAChRs [65].

While there is no direct experimental proof that the receptor changes, the various transitions

within the protein structure of the receptor are based on computational simulations from

various mutational and structural studies. The current model [34] describes a rotation or

torque produced by significant C-loop movement (~ 11Ǻ) when the ligand interacts with the

agonist-binding site. This torque is transitioned through the receptor, influencing the

orientation of the M2 that lines the channel pore. These transitions place the receptor in three

structural states: closed, open and desensitised, which determine the receptor’s functional and

pharmacological properties and influence various intracellular signalling cascades.

The channel-gating process involves switching from a closed or non-ligand-bound receptor

state to an open state, with a change in channel pore size from ~ 4Ǻ to ~ 8Ǻ respectively [66].

This is accomplished via the transitional torque produced by the extracellular domain and

transitory shift of hydrophobic residues within the M2 helices. It leads to concurrent influx of

Na+, K+ and Ca2+ ions, which activates an array of signalling pathways.

17

The pore’s ion-gating function is attributed to two special properties: the near perfect

arrangement of M2 helices in a radially inward tilt towards the pore axis, and symmetric

orientation of hydrophobic residues α-Leu 251 and α-Val 255 that constrict to form a narrow

energetic barrier during the closed state of the receptor [Figure 1.4] [67, 68]. This model was

evaluated when mutational studies of these residues led to increased channel conductivity

[69].

The above-mentioned studies provided the first rationale linking the physiological function of

the nAChR with its biophysical properties. There are two major nAChR biophysical

properties that modulate its physiological functions. Firstly, the rapid transition of the receptor

between the closed and open state governs its primary role of fast synaptic transmission. The

release of ACh and subsequent activation of the nAChR in presynaptic junctions modulates

the release of other neurotransmitters, such as dopamine and GABA [70, 71]. Secondly, the

ionic permeability of nAChRs to Ca2+ mediates various downstream signalling cascades [72].

The relative permeability ratio (Ca2+/Na+) of the nAChR is calculated by two methods:

measuring the shift in reversal potential of nAChR-mediated current under varying calcium

concentrations [73]; and calcium fluorescence imaging [74]. These techniques indicate the

highest calcium permeability for homomeric α7 and heteromeric α9/α10 with a ratio of ≥10.

This ratio was the least for muscle nAChRs at ~ 0.1, while all of the other heteromeric

receptors exhibited a ratio of ~ 2.0 [75, 76]. While the residues that contribute to the barrier

within the pore are highly conserved in various nAChR subunits, the relative permeability of

calcium to sodium ions is affected by the arrangement of residues within the ion conduction

path of the pore. These residues include α-Glu 262, α-Ser 266 (top end of the pore) and α-Glu

241 (at the intracellular mouth of the pore), and were identified through mutational and

electrophysiological studies [77, 78]. This variance in calcium permeability changes the

18

physiological roles played by different nAChR subtypes [79]. The physiological implication

of nAChR-mediated Ca2+ signalling will be discussed in detail in Section 1.6.

While nAChRs were initially identified in neuromuscular and synaptic junctions, and their

primary role in fast cholinergic transmission is long established, recent discoveries showing

non-synaptic nAChR expression significantly expanded the functional scope of these

receptors [80]. Unlike synaptic junctions, where neurotransmitter levels are highly regulated

via release and active reuptake producing the instantaneous open and closed transition of

nAChR, non-neuronal nAChRs are prone to prolonged agonist application. This sustained

exposure led to the identification of a third state of the receptor: desensitised.

Desensitisation is a process in which the receptor transitions from an open state to an agonist-

bound non-conducting state. The transition rates between these states depend directly on the

dissociation rates of the agonist. Therefore, this is dependent on the nature of the agonist

bound and the subunit composition of that receptor. As α and β subunits contribute

differentially to the pharmacological properties of the receptor, the same is applicable to the

rate of receptor desensitisation. For example, α7 nAChRs desensitise more quickly than other

subtypes. Also, incorporating the α5 subunit has been shown to change the biophysical

properties of all three functional states of a receptor [63, 81].

1.1.6 nAChR-mediated Ca2+ signalling and modulation of neuronal growth

nAChRs exhibit strong calcium permeability, which is dependent on the receptor composition

and stoichiometry [59, 76]. Subtypes such as the α7 nAChR can directly raise the cytoplasmic

calcium levels due to their high permeability; however, indirect calcium influx has also been

shown to occur due to nAChR-mediated depolarisation. Indirect calcium influx occurs in two

19

ways: through voltage-dependent calcium channels (VDCC), and being released from calcium

intracellular stores, such as the endoplasmic reticulum [Figure 1.6] [82, 83].

VDCC-mediated calcium influx occurs at depolarising potentials of > –40 mV, which are

generated by nAChR activation. Under these conditions, the initial Ca2+ entry via nAChRs is

augmented and may be functionally complementary, an association that is also observed with

NMDA receptors [84].

The third process contributing to increased cytoplasmic Ca2+ signal is through calcium

induced calcium release (CICR) from intracellular stores [83]. While calcium signalling via

VDCCs is significantly mediated by α7-, α3- and/or β2-containing receptors in neuronal

ganglia, α7 also increases transient Ca2+ levels independently of VDCCs via CICR. Calcium-

quenching experiments in neurons of substantia nigra pars compacta showed reduced

cytoplasmic calcium levels when α7 nAChR was activated with choline [85]. Receptor

antagonists were later used to show that CICR involved ryanodine (RyR) receptors and

inositol (1,4,5)-triphosphate receptors (IP3Rs) expressed in the endoplasmic reticulum

[Figure 1.6] [82, 86]. Although the molecular mechanism behind nAChR-activated IP3R-

calcium release is still unclear, secondary signalling molecules, such as Ca2+-dependent

phospholipase C and Ca2+ -sensor proteins, have been implicated [87, 88]. The various

mechanisms of cytoplasmic calcium influx reflect a complex and intricate functional coupling

of nAChRs with RyR and IP3 receptors, which leads to a sustained calcium signal. These

calcium signals play a vital role in the activation of various downstream signalling processes.

20

Figure 1.6: nAChR-mediated Ca2+ signalling and modulation of neuronal growth.

Downstream signalling events upon nAChR activation are classed as immediate, interim and

long-term effects. The calcium ion influx occurs through three ways: nAChR activation,

VDCC activation and CICR mechanism from calcium internal stores (mediated by RyR and

IP3 receptors). The Ca2+ influx leads to immediate cell depolarisation and activation of

various kinases and proteins such as PKC, PKA and Calmodulin. This is followed by

subsequent activation of downstream signalling molecules, leading to various physiological

responses such as neuronal plasticity, memory, neuroprotection.

The above-discussed calcium influx is a sequential process occurring in the following order:

nAChR > VDCCs > ryanodine receptor > IP3 receptors. Therefore, it is only logical for the

various downstream signalling events upon nAChR activation to be sequential and classed as

immediate, interim and long-term effects based on their duration and timing.

21

1.1.6.1 Immediate effects

nAChRs expressed in presynaptic junctions of the peripheral nervous system (PNS) and CNS

mediate their primary role in fast synaptic transmission. This process not only depolarises

cells, but also initiates exocytosis either directly or via VDCC activation [Figure 1.6] [89]. In

addition, nAChRs and VDCCs play physiologically complimentary roles in the regulation of

neurotransmitter release. In striatal synaptosomes, VDCC mediates nAChR-evoked dopamine

release [90]. However [3H]noradrenaline release from hippocampal synaptosomes, triggered

by α3β4 nAChR activation, is VDCC-independent [91].

1.1.6.2 Interim effects

The two major short-term implications of nAChR activation include regulation of gene

expression and nAChR desensitisation. CICR-mediated calcium influx activates the IP3

second messenger system involving various Ca2+-sensor proteins and kinases, such as protein

kinase C (PKC), is classed as an immediate effect [Figure 1.6]. However, concurrent

activation of downstream molecules, such as extracellular signal-regulated mitogen-activated

protein kinase (ERK/MAPK) [Figure 1.6], that has been shown to modulate the gene

expression of the tyrosine hydroxylase (TH), is defined as a short-term Ca2+-signalling effect.

TH is a known modulator of neurotransmitter release in catecholamine-containing neurons.

Prolonged nicotine exposure has been shown to promote TH mRNA expression [92]. In

addition, the role of nAChRs in the regulation of c-Fos and c-Jun gene expression has long

been apparent [Figure 1.6] [93].

In synaptic and non-synaptic regions, prolonged exposure of agonist leads to nAChR

desensitisation. Furthermore, high intracellular calcium levels have been shown to affect

various desensitisation properties, such as recovery of α7-containing nAChRs in chromaffin

cells and rat hippocampal neurons [94, 95]. These changes to the nAChR’s biophysical

22

properties are implicated in synaptic efficacy, and corresponding changes in pathological

conditions suggest a complex reciprocal relationship [81].

1.1.6.3 Long-term effects

In a complex scenario, nAChR activation and intracellular calcium influx translates into vital

physiological functions, such as neuronal plasticity, memory mechanisms, neuroprotection

and regulation of cell death [79]. Recent studies showed activation of other kinases, such as

Ca2+ -calmodulin-dependent protein kinase (CaMPK) and cAMP response element-binding

protein (CREB), was mediated via nAChR and CICR calcium influx [Figure 1.6] [96].

ERK/MAPK is an important molecule, central to various signalling cascades. Activation of

the ERK/MAPK signalling cascade via nAChR activation is critical for regulating gene

expression. Disrupting these kinase signalling cascades and consequent modulation of α7

nAChR expression and function has been implicated in Alzheimer’s disease [97]. Today, α7-

evoked activation of phosphatidyl-inositol 3-kinase (PI3K) and ERK/MAPK is a known

pathway for mediating neuroprotection and stimulating angiogenesis [Figure 1.6]. This anti-

apoptotic property of nAChR signalling is implicated in various cancers and is currently a

major target for novel therapeutics [98]. The nAChRs role in cancer is detailed in section

1.7.3.

The many signalling pathways mediated by direct and indirect calcium influx via nAChR-

activation reflect the complexity, yet specificity, of intracellular mechanisms. While recent

studies have shed light on these dynamic processes, much is yet to be proven and learnt about

the exact cellular pathways mediated by various specific nAChR subtypes, to improve our

understanding of their pathological implications.

23

1.1.8 nAChRs and diseases

Since their discovery, nAChRs have been extensively researched to build knowledge about

their structure and function. However, it was only in the past few decades that their true

nature and complexity has been understood. nAChR subunits have various different

compositions and stoichiometries, which correspond with specific biophysical and functional

properties, including tissue distribution. This suggests nAChRs may be involved in a huge

range of identified and as yet unidentified health conditions. nAChRs are known to be

implicated in numerous pathophysiological conditions, such as epilepsy, schizophrenia,

Alzheimer’s disease, Parkinson’s disease, pain, auto-immune disease, lung cancer and

nicotinic dependence, with other conditions being investigated and identified [3, 7, 99]. Each

of these conditions involves dynamic cellular interactions with a specific receptor subtype.

However, only three conditions will be discussed within the scope of this thesis: pain, nicotine

dependence and lung cancer.

1.1.8.1 nAChRs and pain

Pain is one of the most common health conditions and affects millions of people throughout

the world. It is a sensory response to nociception (a neuronal process involving noxious

stimuli), can be classified as either acute (sudden onset) or chronic (prolonged persistent

pain), and involves various pain pathways.

Nicotinic acetylcholine receptors have long been implicated in pain mediation, and various

nicotinic compounds that produce analgesic effects have been identified [100]. The initial

breakthrough came with the discovery of epibatidine which is a potent analgesic and a

nAChR agonist, although this drug was discontinued due to its severe side effects [101]. This

knowledge highlighted an alternative pain target to opioid receptors, which were targeted by

conventional analgesics. Epibatidine is a potent agonist of α4β2 nAChRs, one of the most

24

abundant subtypes in the CNS, and inhibits the nociceptive signals transmitted through the

dorsal horn of spinal cord [102]. The α4β2 nAChR’s role as a target for analgesics was further

established with the discovery of the promising therapeutic candidate ABT-594, a potent α4β2

nAChR inhibitor that lacks the side effects of opioid-targeting drugs [103, 104]. In

conjunction with α4 and β2 knockout mice studies, this paved the way for nAChRs to be

novel therapeutic target in pharmaceutical industry [105] and various compounds that target

α4β2 nAChRs are currently in clinical trials [5].

While, α4β2 nAChR subtype is a prominent candidate, recent study showed various other

nAChR subtypes with α7, α3, α9, α10, β2 and β4 subunits, to be involved in pain pathways

[106]. This study re-established the role of the α4β2 nAChR subtype in the analgesic efficacy

of various compounds. However, it also suggested that the α3β2 and α3β4 subtypes have a

significant role in the analgesia by these compounds. While this study excludes the role of the

α7 nAChR subtype in pain alleviation, other studies contradict this idea [107, 108]. These

other studies showed that α7 nAChR subtype activation induces anti-nociceptive in acute pain

model and anti-inflammatory effects in peritoneal macrophages. This was collaborated by

further studies in which α7 knockout mice exhibited increased hyperalgesia (an exaggerated

response to pain stimulus) and pain inflammation [109].

A new mechanism of nAChR-mediated pain modulation via receptor desensitisation has also

been proposed, and involves direct activation of these receptors. Incorporating auxiliary α5

subunits in α4β2 and α3β4, increases receptor desensitisation [110].

25

Finally, α9-containing receptor inhibition by a novel class of conotoxins showed significant

analgesic effects in peripheral neuropathy pain models [111, 112], and as a result, α9 has also

been proposed as novel target for analgesic drugs. Conotoxins and their analgesic properties

will be discussed in detail in the Chapters 6.

1.1.8.2 nAChR signalling and lung cancer

The pathophysiology of lung cancer has studied for almost half a century. Divided into two

major types: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC),

it is the second most frequent and leading cause of cancer-related deaths worldwide. NSCLC

accounts for 80% of all lung cancer cases [113] and smoking, specifically tobacco intake, has

been a documented risk factor for lung cancer since the 1950s [114, 115]. Nicotine, polycyclic

aromatic hydrocarbons and nicotine-derived metabolites, such as 4-(methylnitrosamino)-1-(3-

pyridyl)-butanone (NNK) and N-nitrosonornicotine (NNN), are among the carcinogens in

tobacco smoke that are primary agents that initiate cancer [116]. Tobacco-related

tumorigenesis is caused either via the genocentric model, in which genomic interaction of

nicotine and carcinogenic nitrosamines leads to DNA adduct formation and mutation [117], or

the biochemical model, in which tobacco-derived nicotine and nitrosamines act as potent

nAChR agonists [118] and mediate the cell signalling pathways of growth, proliferation [119]

and apoptosis [120]. The biochemical model also leads to various biological effects, including

nicotine addiction and dependence [121].

1.1.8.2.1 Role of α3 subunit

Following the Schuller group’s first report in 1989, which implicated nAChRs in the

regulation of cancer cell growth [119], many studies started exploring the role of nAChRs in

cancer development and progression. In 1998, Schuller’s group conducted radiolabelled

binding assay studies using [125I]α-BTX and [3H]EB. These studies conclusively showed that

26

NNK and NNN, tobacco-specific carcinogenic nitrosamines, were potent nAChR agonists

[118]. At this point, concentrations of these nitrosamines were known to be 5000–10,000

times greater than nicotine in tobacco smoke, with the NNN concentration two–three times

that of NNK [117].

The binding assay experiments in this study also revealed the selective binding profile of

these nitrosamines. NNK was found to be a high-affinity ligand of the α-BTX-sensitive α7

nAChR subtype, while NNN is selective to the α4/α3 nAChR subtype, classified as an EB-

sensitive neuronal nAChR [Figure 1.7]. Also, the proliferation assay, which used the

incorporation of [3H]thymidine, identified that NNK’s binding to the α7 nAChR subtype is a

potent mitogenic stimulus. However, NNN and nicotine showed no effect in the proliferation

assay. These results, along with previous studies showing apoptotic inhibition of lung cancer

cells by nicotine, provided strong evidence that nAChRs are involved in tumour progression

[120].

Consecutively, NNK and nicotine were also shown to have a direct biochemical effect on

rapid Akt activation in normal human airway epithelial cells (NHBE) [122]. Serine/Threonine

kinase Akt is a key regulator of cell cycle process. Its activation leads to various signalling

cascades involved in cell proliferation and apoptosis [123]. This was confirmed by Dennis’

group, which demonstrated constitutive activation of Akt/protein kinase B in 90% of NSCLC

cell lines [124] and increased phosphorylation of downstream Akt substrates, such as MDM2,

mTOR and NFκB [Figure 1.7] [125]. This is known to promote cellular survival, resistance

to chemotherapy and radiation, and NFκB-dependent apoptotic inhibition.

27

Figure 1.7: nAChR mediated signalling pathway involved in lung cancer and their cells

of origin. A summarised model of the various cellular signalling molecules involved in SCLC

and NSCLC, mediated by the activation of α7 and heteromeric α3-containing receptor such as

αβ34 nAChRs. Activation of α7 nAChR by NNK or nicotine has shown to enhance cell

proliferation via β-Arrestin-SRC pathways involving the activation of Raf1 and ERK family

of kinases. Whereas, α7 and α3-containing receptor mediated activation of Akt, contribute to

the anti-apoptotic properties of NNK, NNN and Nicotine.

- kinases; - transcription factors; - IAP proteins

In 2003, Dennis’ group provided the first evidence that nAChR stimulation causes Akt

overexpression in NSCLCs [122]. It also conclusively provided the first indication that α3

antagonists are potential therapeutic drugs towards NSCLC. Treatment with NNK and

nicotine, led to rapid activation of Akt in NHBEs. This action was attenuated in the presence

28

of the α3/α4 antagonist DHβE, but not the α7 nAChR antagonists α-BTX and MLA, which

showed no effect. Further, Akt phosphorylation increases in NHBEs were observed in the

presence of an α3-specific agonist, α-ATX [126]. This supports the idea that nicotine causes

α3-mediated Akt activation. This was later confirmed by Arredondo’s study, in which

mecamylamine (Mec), a nAChR antagonist effectively abolished NNN’s pathobiologic

effects in BEP2D cells [127].

1.1.8.2.2 Physiological to biochemical translation: cellular difference mediated by α7 and

α3 subunits

SCLC and NSCLC exhibit different physiological profiles as well as different major

histological types. NSCLCs show constitutive Akt activation, increased NFκB-dependent

cellular survival and inhibition of apoptosis, mediated by α3/α4 nAChR subunits and due

primarily to the agonist effects of tobacco carcinogens NNN and nicotine. Concurrent studies

show that NNK’s tumorigenic effects are mediated by the α7 nAChR subtype predominantly

in SCLC, but also in NSCLC [128, 129]. Molecular studies show that α7 nAChR expression

in SCLC and pulmonary neuroendocrine cells (PNEC) is high [128], which is consistent with

α7 nAChR expression being upregulated as a result of chronic NNK exposure [130]. These

results, in conjunction with previous binding studies, show tumorigenesis in SCLC and

PNECs is mediated via the α7 nAChR subtype, while both α7 and heteromeric α3 nAChR

subtypes are involved in NSCLC and peripheral adenocarcinoma (PAC) [118, 129].

An in vitro proliferation assay of PNECs or SCLC in the presence of nicotine or NNK

induced a serotonergic autocrine loop, which was abolished when an α7 nAChR antagonist or

serotonin uptake inhibitor was added [131, 132]. This autocrine response involved the

activation of cellular regulators the serine/threonine kinase RAF1, protein kinase C (PKC),

mitogen-activated kinases ERK1 and ERK2, and transcription factors FOS, JUN and MYC

29

[Figure 1.7] [129, 131, 132]. Along with studies that show pharmacological inhibition of

these regulators block the proliferation and apoptosis response identified α7 as the key

regulator of cell proliferation [133, 134]. However in NSCLC, NNK-induced cell

proliferation activated signal transducers, transcription 1 (STAT1), NFκB, GATA3, β-

arrestin-SRC, and ERK1 and ERK2 [Figure 1.7] [127, 135]. While, NNK’s anti-apoptotic

activity in SCLC was modulated by activating Bcl-2, in NSCLC, E2F1-mediated upregulation

of survivin and X-liked inhibitor of apoptosis (XIAP) contributed to nicotine-induced

apoptotic inhibition [Figure 1.7] [134, 136]. However, these results are yet to identify the role

of IAP (Inhibitors of Apoptosis) proteins (survivin and XIAP) as characteristic signalling

proteins underlying the anti-apoptotic effect in NSCLC.

1.1.8.2.3 Targeting the α3β4 nAChR: unlocking novel therapeutics for NSCLC

Previous studies have identified nicotine and its derivatives as potent nAChR agonists and

highlighted their regulatory role in cancer cell apoptosis [118, 120]. Recent candidate–gene

analysis and genome-wide association studies (GWAS) have also identified gene clusters and

single nucleotide polymorphisms (SNP) encoding α3, α5 and β4 nAChR subunits associated

with lung cancer [118, 137]. These studies showed the existence of CHRNA5/A3/B4 gene

cluster overexpression and other specific nAChR subunits in lung cancer tissue [138, 139]. An

important transcription factor, ASCL1, was shown to play a key regulatory role in the

initiation and development of SCLC, and the overexpression of the CHRNA5/A3/B4 gene

cluster [140, 141]. These studies suggest a strong correlation between the overexpression α3,

α5 and β4 subunits and lung cancer.

NNK and NNN are leading carcinogens involved in the development of lung cancer in

smokers. Along with nicotine, these nitrosamines have also been shown to be strong nAChR

agonists and easily displace the endogenous nAChR agonist, ACh [118]. Binding studies have

30

shown that NNK is a selective α7 nAChR agonist, and NNN is a selective agonist of α3/α4-

containing nAChRs [118]. Subsequent studies also showed that expression and activation of

signalling molecules involved in cell proliferation and the apoptotic inhibition of NSCLC

increased in the presence of NNN and NNK. This response was abolished when an α7 or α3

nAChR antagonist was added [124, 136]. More recent GWAS also suggest α3β4 nAChRs are

key modulators of cell proliferation and apoptotic inhibition of NSCLC [137]. However, a

lack of adequate molecular α3β4-specific probes has limited the study of the physiological

role of this receptor subtype in disease progression.

31

1.2. Conotoxins

The venom of various animals, such as snakes, spiders and molluscs, has long attracted the

attention of scientists for their ability to kill or paralyse their prey, almost instantly. Most

venomous animals target invertebrates, with few targeting vertebrates. The venom of animals

that do target vertebrates has been found to be dangerous – even fatal – to humans. Various

bioactive peptides, called conotoxins, in the venom bind selectively and potently to different

ion channel receptors. This makes them biomedically and pharmacologically significant

[142].

Figure 1.8: Predatory marine cone snails (A) Conus regius [143] and (B) Conus victoriae.

Predatory marine snails of the genus Conus from the Conoidae superfamily, is one of the

largest and most diverse genera, with around 500 snail species [Figure 1.8]. Few snails in this

genus prey on vertebrates like fish. Their venom, which includes thousands of conotoxins,

also targets different ion channels in humans. α-Conotoxins, a conotoxin subclass found in

this venom, are short, disulphide-rich peptides that selectively target different nAChR subtype

isoforms [144].

1.2.1 Venom apparatus and biosynthesis

The vast diversity in conotoxin structure and sequence corresponds to the complex venom

apparatus and biosynthesis process in cone snails. The cone snails’ general venom apparatus

32

includes a long, convoluted tubular gland that synthesises venom, a muscular bulb that moves

the venom, and the proboscis, which helps deliver the venom and consists of various hallow,

harpoon-shaped radula teeth [Figure 1.9] [143, 145].

Figure 1.9: Schematic representation of cone snail’s venom apparatus representing the

main parts [146].

The conotoxin diversity in venom extends from cone snail gene and proteome level to post-

translational modifications. Interestingly, thousands of peptides in the venom are generated

from a relatively small number of gene families [147]. The genes encoding conotoxins

translate into a precursor protein molecule consisting of three distinct segments: N-terminal

signal sequence, a pro-peptide region and the mature toxin sequence. The N-terminal signal

sequence and pro-peptide region are highly conserved, however, hypermutation is observed in

the mature toxin sequence [147, 148].

After the gene is translated, various enzymes are responsible for the production of the mature

toxin. These include protein disulphide isomerase (PDI), peptidyl-prolyl cis-trans isomerase

(PPI), cysteine-rich protease Tex31 and immunoglobulin-binding protein (BiP). Recent

studies have identified various PDI isoforms that contribute to the conotoxins diversity at

proteomic level [149]. In addition to this variability in the mature toxin sequence, mature

peptides can undergo post-translational modifications, such as C-terminal amidation and

33

proline hydroxylation, which confers a unique functional activity compared with unmodified

sequences [150, 151].

Due to their potential pharmacological significance, it is vital to find a way to effectively and

accurately synthesise conotoxins in vitro and characterise their structure and function.

Understanding the diverse post-transcriptional and post-translational regulation of cone snail

venom biosynthesis will provide important information that will help to improve in vitro

synthesis of complex conotoxins.

1.2.2 Classification and nomenclature

The venom repertoire of cone snails is mainly constituted of disulphide-rich conopeptides and

are categorised into various superfamilies and classified by their structural and functional

features, such as cysteine pattern and pharmacology. Figure 1.10 illustrates some of these

superfamilies. This classification is also used to name conotoxins.

The first nomenclature guidelines were introduced in 1985 and updated several years later

[152]. They outline that conotoxins that have been functionally characterised should be

labelled with (in the order listed below):

• a Greek letter indicating their pharmacological target

• one (uppercase) or two letters (second letter is lowercase) from the Conus species from

which the peptide was isolated

• a Roman numeral indicating the cysteine framework pattern

• an uppercase letter representing the peptide variant discovered in respective cone species.

For example, α-RgIA represents a conotoxin isolated from Conus regius with cysteine

framework I that targets ACh receptors.

34

Figure 1.10: Classification of conotoxins. The classification of conotoxins into various

superfamilies is based on their cysteine pattern and pharmacology. Subclass α- and αD-

conotoxins discussed in the following chapter of the thesis are boxed in red. Na+ – voltage

gated sodium channel; K+ – voltage gated potassium channel; Ca+2 – voltage gated calcium

channel; nAChR – nicotinic acetyl choline receptors [153].

There has been a significant increase in the number of conotoxins discovered from complex

venom repertoire due to recent advances in protein and molecular techniques such as high-

performance liquid chromatography (HPLC) and reverse transcription polymerase chain

reaction (RT-PCR) [154]. The pharmacology of some of these peptides is unknown, so they

are labelled differently from those that have been functionally characterised. They are labelled

with:

35

• one or two letters (both lowercase) from the Conus species from which the peptide was

isolated

• an Arabic numeral representing the cysteine framework

• a lowercase letter representing the peptide variant.

For example, before its pharmacological characterisation, α-RgIA was represented as rg1a.

1.2.3 Conotoxin structural diversity

Along with high sequence hypervariability, conotoxins show structural diversity. The

disulphide framework of different conotoxin classes translates these peptides into specific,

rigid, three-dimensional structures [155]. These structures play an important role in

determining pharmacological properties of a peptide, including its potency and selectivity

[156]. In the past decade, nuclear magnetic resonance (NMR) has enabled to determine the

three-dimensional structures of many conotoxins with characteristic cysteine frameworks

[157]. For example, α-conotoxins of the A superfamily of conopeptides, fold into a ω-shaped

structure with a characteristic α-helix secondary structure [Figure 1.11(A)], whereas, χ-

conotoxin MrIA of the T superfamily is dominated by β-helix secondary structures [Figure

1.11(B)] [157, 158]. Conotoxins with three or more disulphide bonds, such as ω-conotoxin

MVIIA, have complex cysteine knot structures [Figure 1.11(C)] [159]. α-Conotoxins of the

D superfamily are composed of five disulphide bonds and exhibit dimerization that yields

complex three-dimensional structures [160]. These characteristic folds reveal critical residues

that interact with the ion channel and determine the pharmacological activity of conotoxins.

36

Figure 1.11: Structural diversity among conotoxins. Cartoon representation of the three

dimensional structures of conotoxins (A) α-Vc1.1 (PDB 2H8S) [161] (B) χ-MrIA (PDB

2EW4) [162] (C) ω-MVIIA (PDB 1MVI) [163]. α-helics are shown in blue, β-sheets in red

and disulphide bonds are shown in yellow. Images generated using PyMol Molecular

Graphics System (DeLano Scientific LLC.).

Though rich in cysteines, these residues are buried within the conotoxin’s three-dimensional

structure, exposing a hydrophobic patch at the surface [164]. The surface-exposed residues

contribute to overall net charge of the conotoxin. This charge distribution influences the

peptide’s selectivity and potency for its pharmacological target. For example, α-conotoxins

with net positive charge target muscle nAChR subtypes, and those with neutral or negative net

charge target neuronal nAChR subtypes [165].

1.2.4 Conotoxin structure–activity relationship

Conotoxin diversity enables them to identify various isoforms of ion channels, making them

excellent probes to study different receptors. α-Conotoxins are excellent examples of this,

because they specifically and potently target nAChR subtypes [166]. This has increased our

understanding of the physiological role each receptor subtype plays in normal and disease

states. In this thesis, I will limit my discussion to α- and αD-conotoxins.

α-Conotoxins range from 12–25 amino acids in length and belong to the A superfamily of

conopeptides. They are characterised with the CCXnCXmC cysteine framework, where n and

37

m represent the number of amino acids and indicate the subclass of a peptide. Most native α-

conotoxins exhibit I–III and II–IV disulphide connectivity, yielding a ω-shaped, three-

dimensional globular conformation [155]. Although, α-conotoxins exhibit sequence

hypervariability, conserved residues are observed among various α-conotoxins. Table 1.1 lists

some of the α-conotoxins that target neuronal nAChR subtypes. Residues in loop1 (residues

between Cys II and III) are highly conserved, with most peptides showing a –SXPA– motif.

Table 1.1: Sequence alignment of α-conotoxins targeting neuronal nAChR subtypes.

α-Conotoxin Sequence nAChR selectivity Reference

Ls1a SGCCSNPACRVNNPNIC* α3β2≈α7>α3α5β2 [167] GID IRDγCCSNPACRVNNOHVC# α7≈α3β2>α4β2 [164] ArIB DECCSNPACRVNNPHVCRRR# α7≈α6β2^>α3β2 [168] RegIIA GCCSHPACNVNNPHIC* α3β2>α3β4> α7 [169] OmIA GCCSHPACNVNNPHICG* α3β2>α7>α6β2^ [170] GIC GCCSHPACAGNNQHIC* α3β2≈α6β2*>α7 [171] AuIB GCCSYPPCFATNPD-C* α3β4 [172] RgIA GCCSDPRCRYR----CR# α9α10>>α7 [173] * amidated C-terminus; # carboxylated C-terminus; ^ receptors containing the subunits; conserved cysteine framework is indicated in yellow.

Significant interaction between these loop1 residues and the principal face of the receptor was

identified using X-ray studies of AChBP, ImI, [A10L,D14K]PnIA [Figure 1.12] and

[A10L]TxIA co-crystal structures [174, 175]. These studies identified that loop1 has a role in

the peptide’s secondary structure and affinity, and loop2 residues interact with the

complementary side of the receptor, contributing to the α-conotoxin’s subtype selectivity

[Figure 1.12] [156].

38

Figure 1.12: AChBP and [A10L,D14K]PnIA co-crystal structures (PDB 2BR8) [176]

shown in (A) surface representation and (B) cartoon representation. Mutated residues

Leu10 and Lys14 of loop2 (green) interact with the (-) interface (orange) while Leu5, Pro6

and Pro7 residue side chains of loop1 (cyan) have shown to interact with the ‘principal’/(+)

interface (blue) of the ACh binding pocket. Disulphide bonds of the conotoxin and Cys-Cys

pair of (+) interface are indicated in yellow. Images generated using PyMol Molecular

Graphics System (DeLano Scientific LLC.).

1.2.4.1 Use of alanine scanning mutagenesis

To date, most mutational studies have involved replacing various non-cysteine residues with

the inert residue alanine. These experiments provided vital information about the various α-

conotoxin residues that interact with receptors, and in combination with modelling simulation

studies, they have helped to determine α-conotoxin pharmacological selectivity [166]. For

example, loop2 residues (11–15) in Vc1.1 have been shown to be important for its activity at

α9α10 nAChR subtypes. Alanine mutation of these residues causes significant loss in activity

at this subtype [177]. In RgIA, similar experiments showed an approximate 1500-fold loss in

activity for [R9A]RgIA at the α9α10 subtype. Surprisingly, the opposite effect was seen at the

39

α7 subtype, with an approximate 5-fold increase in activity [178]. This mutation was

homologous with that of α4/3-conotoxin ImI, which selectively inhibits α7 and α3β2 nAChR

subtypes [179].

Alanine scanning mutagenesis not only contributed to the molecular basis for various

conotoxins’ antagonism of their respective nAChR subtypes, but also significantly improved

our understanding of these peptides’ subtype selectivity. Furthermore, alanine mutation

improved α-conotoxin potency and selectivity. For example, E11A mutation in α-conotoxin

MII improved its selectivity and potency towards α6-containing nAChRs [180]. In Vc1.1,

N9A mutation increased potency at α9α10 nAChR subtypes about 10-fold [177].

1.2.5 Conotoxin re-engineering

α-Conotoxin use in research and clinical settings has been hampered because of their peptidic

nature, which makes them susceptible to natural degradation, such as proteolytic attack, or

disruptions of their disulphide connectivity. These problems drastically affect their

bioavailability and half-life. In vitro chemical synthesis of conotoxins allows them to be

chemically modified to improve their structure and ability to be used in research and clinical

settings.

1.2.5.1 Dicarba modification

Various strategies to improve α-conotoxin stability have been implemented, such as

cyclisation and selenocysteine modification [181, 182]. Recent studies have shown that

replacing the cysteine bridges (S–S motif) with non-reducible dicarba links (CH2–CH2,

CH=CH groups) [183] is a novel way to improve stability and resistance to peptide

degradation and scrambling [184]. In addition, this modification maintains the

pharmacological activity of α-conotoxins [185, 186].

40

1.2.6 Conotoxins: potential therapeutics

That conotoxins selectively and potently inhibit various ion channels has put them at the

forefront of novel drug development. Several conotoxins are, and have been, tested clinically

to treat a range of health conditions, including neuropathic pain and benign prostatic

hyperplasia [187]. For example, ω-Conotoxin MVIIA, isolated from Conus magus, inhibits N-

type calcium channels [188] and is an FDA-approved drug for pain relief. Peptides

undergoing clinical trials include χ-MrIA (a non-competitive neuronal noradrenaline receptor

inhibitor) to treat benign prostatic hyperplasia symptoms [189], and conantokin-G (a selective

NMDA receptor inhibitor) to manage epilepsy [190].

Recent studies have also identified nAChR subtypes that are involved in various health

conditions, such as schizophrenia (α7 and α4β2), Parkinson’s disease (α6-containing

receptors), Alzheimer’s disease (α7 and α4β2), lung cancer (α3β4) and pain (α4β2). These

discoveries have provided new therapeutic applications for α-conotoxins that target neuronal

nAChRs [40, 99, 144, 191].

41

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164. Nicke, A., Loughnan, M. L., Millard, E. L., Alewood, P. F., Adams, D. J., Daly, N. L.,

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172. Luo, S., Kulak, J. M., Cartier, G. E., Jacobsen, R. B., Yoshikami, D., Olivera, B. M.,

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173. Ellison, M., Haberlandt, C., Gomez-Casati, M. E., Watkins, M., Elgoyhen, A. B.,

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174. Ulens, C., Hogg, R. C., Celie, P. H., Bertrand, D., Tsetlin, V., Smit, A. B., and Sixma,

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176. Celie, P. H., Kasheverov, I. E., Mordvintsev, D. Y., Hogg, R. C., van Nierop, P., van

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K., and Smit, A. B. (2005) Crystal structure of nicotinic acetylcholine receptor

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homolog AChBP in complex with an alpha-conotoxin PnIA variant, Nature structural

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177. Halai, R., Clark, R. J., Nevin, S. T., Jensen, J. E., Adams, D. J., and Craik, D. J. (2009)

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178. Ellison, M., Feng, Z. P., Park, A. J., Zhang, X., Olivera, B. M., McIntosh, J. M., and

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179. Johnson, D. S., Martinez, J., Elgoyhen, A. B., Heinemann, S. F., and McIntosh, J. M.

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CHAPTER 2

MATERIALS AND METHODS

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This chapter describes the protocols followed for the synthesis, and structural and functional

characterisation of conotoxins.

2.1 Materials

Rink amide methylbenzhydrylamine (MBHA) resin (Novabiochem), O-benzotriazole-

N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU), N-(9-fluorenyl)

methoxycarbonyl (Fmoc), dimethylformamide (DMF), N,N′-diisopropylethylamine (DIPEA),

trifluoroacetic acid (TFA), triisopropylsilane (TIPS), ether, HPLC grade acetonitrile (ACN),

potassium cyanide (KCN), ninhydrin, tricaine methanesulphonate (MS-222), acetylcholine

(ACh), hydrogen fluoride (HF).

2.2 Peptide synthesis

2.2.1 Solid-phase peptide synthesis (SPPS)

Chemical synthesis using SPPS involves building a peptide chain that is covalently attached

to a solid resin support. This concept was introduced by Merrifield in 1963 [1]. Stepwise

assembly of the amino acids on the resin uses one of two types of chemistry, depending on the

N-terminal protecting group of amino acids - 9-fluorenylmethoxycarbonyl (Fmoc) and tert-

butyloxycarbonyl (t-Boc). Along with one of these orthogonal protecting groups, side chain

protecting groups are also present on amino acids which prevent any intermediate reactions

during peptide elongation.

Peptide elongation involves two basic steps: N-terminal deprotection and a coupling reaction.

These steps are where the major differences between Fmoc and Boc peptide synthesis are

seen. In Boc chemistry, the deprotection step uses an acid, such as TFA, whereas Fmoc

chemistry uses a base, such as piperidine. The two also require different reactions to cleave

the peptide from the solid resin. Boc needs HF, while Fmoc uses TFA [1, 2]. Because Fmoc

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uses low-hazard reagents, it is safer than Boc, which is why many scientists prefer it.

However, both methods have specific advantages, and the type of SPPS method used depends

on the final peptide needed.

2.2.2 Fmoc SPPS

All peptide analogues discussed in Chapter 3 were synthesised using Fmoc chemistry. The

various steps involved in this method are detailed below. Fmoc peptide synthesis was carried

out in collaboration with Dr Richard Clark (The University of Queensland, Brisbane).

Our collaborators provided the peptides described in other chapters.

2.2.2.1 Peptide assembly

The basic apparatus for SPPS includes a reaction vessel with a sintered glass filter and tap at

the bottom, which is connected to a vacuum line via a solvent trap [Figure 2.1]. The filter

supports the resin upon which the peptides are assembled. The tap allows solvents to be

removed from the vessel.

Figure 2.1: The basic apparatus set up needed for solid-phase peptide synthesis.

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All peptide analogues were assembled on rink amide MBHA resin (Novabiochem;

0.7 mmol·g−1) using HBTU-mediated manual SPSS with an in situ neutralisation procedure

for Fmoc chemistry [3]. The amount of resin needed for each reaction was calculated based

on the scale of peptide synthesised in mmoles and the resin’s substitution value (provided

with resin).

Mass of resin = scale in mmoles/substitution value

All peptide analogues were synthesised at 0.25 mmoles scale, where 460 mg of rink amide

MBHA resin (substitution value of 0.54) was used, calculated using the above formula. The

resin was allowed to swell overnight, soaked in DMF, and then transferred into the reaction

vessel.

Each amino acid assembly cycle consisted of Fmoc deprotection with 20% piperidine

in DMF, followed by Fmoc amino acid coupling using HBTU and DIPEA in DMF. A two-

fold excess of Fmoc amino acids was used in the coupling reactions. Each coupling reaction

used 2 mmoles of each amino acid. The amino acids are activated by dissolving them in 2 mL

of 0.5 M of HBTU/DMF solution followed by the addition of 174 µL of DIPEA. When

DIPEA was added, the amino acid mixture was immediately added to the resin and left for

10–15 min to allow for efficient coupling.

A Kaiser Ninhydrin test was carried out to determine the coupling percentage [4]. The Kaiser

Ninhydrin test detects the amount of free amine present on the resin, indicated by the solution

turning purple. In this test, 3–5 mg of the resin was removed, washed with 50%

DCM/methanol and then air dried. To this dried resin, 2 drops of 76% w/w phenol in ethanol,

4 drops of 0.2 mM KCN in pyridine and 2 drops of 0.28 M ninhydrin in ethanol was added.

71

The sample was incubated at 100 oC for 5 min, and then 2.8 mL of 60% ethanol in water was

added to it before it was briefly centrifuged to settle the resin at the bottom. The solution’s

absorbance at 570 nm, was then calculated against the reagent blank using ultraviolet-visible

spectrophotometry. The coupling % was calculated using the following equation:

% coupling = 100 x (1-(A570 x 200/SV x mass of resin))

where, A570 is the absorbance value obtained at 570 nm and SV is the substitution value of the

resin.

Values > 99% indicated a successful coupling reaction, and peptide assembly continued with

the addition of the next amino acid [4]. However, the ninhydrin test cannot be used to

determine the coupling efficiency of an amino acid after proline. This is due to lack of

primary amines at the N-termini of proline. Under these conditions, the coupling reaction is

generally either repeated or an Isatin test can be performed to check for coupling.

In the Isatin test, 5 drops of Isatin were dissolved in 3% n-butanol and 2 drops of 10% acetic

acid were added to the dry resin, before the solution was incubated for 5 min at 100 oC.

Yellow or colourless resin indicated efficient coupling, whereas a blue or grey-green colour

represented poor coupling [5].

2.2.2.2 Peptide cleavage

When the last amino acid was coupled, the N-terminal Fmoc group was removed using the

usual deprotection procedure and washed with DMF. Peptide cleavage from the dried resin

(0.4 g) was achieved by treating it with 100 mL of a chemical cocktail composed of TFA,

TIPS and water (95:2.5:2.5 TFA:TIPS:water v/v/v). TIPS and water within the cocktail are

scavengers that prevent the modification of unprotected side-chains. The reaction proceeded

at room temperature (20–23 °C) for 2.5 h. The TFA was then evaporated (not completely

72

dried) and the peptide was precipitated with 30 mL of ice-cold ether. The peptide was then

extracted using 50% buffer A/B (Buffer A: H2O/0.05% TFA; Buffer B: 90%

CH3CN/10%H2O/0.045% TFA) using a separating funnel. Any residual ether was removed

and the peptide was lyophilised [6].

2.2.2.3 Directed disulphide formation

All peptides were synthesised in globular conformation (I–III; II–IV disulphide connectivity)

through Fmoc – Cys(Acm) – OH coupling at positions 2 and 8 of the amino acid sequence (I–

III disulphide bond). The scavengers in the chemical cocktail used for cleaving the peptide do

not deprotect the Acm group on I–III Cys residues.

Crude peptides were purified by reversed-phase high-performance liquid chromatography

(RP-HPLC) on a Phenomenex C18 column using a gradient of 0–80% B in 80 min, with the

eluent monitored at 215/280 nm. These conditions were used in subsequent purification steps

unless stated otherwise. Electrospray mass spectroscopy (ES-MS) confirmed the molecular

mass of the fractions collected. Those fractions displaying the correct molecular mass of

linear peptide were pooled and lyophilised for oxidation.

Linear peptides were oxidised in two steps [Figure 2.2]. First, they were dissolved in

0.1 m NH4HCO3 (pH 8.2) at a concentration of 0.3 mg/mL, and stirred overnight at room

temperature. This created the II–IV disulphide bond. The Acm protecting groups were stable

under these conditions. The [II–IV]-disulphide peptides were purified using RP-HPLC,

confirmed with ES-MS and then lyophilised.

Second, the second disulphide bond (I–III) was formed using the oxidation by iodine method.

The peptides were dissolved (1 mg/mL) in buffer A (H2O/0.05% TFA) before iodine in

73

acetonitrile was added until the solution turned orange/yellow. The reaction was incubated for

5 min at 37 oC. Excess iodine was destroyed by adding sodium ascorbate. The oxidised

peptides were then purified by RP-HPLC using a gradient of 0–80% buffer B over 160 min.

Analytical RP-HPLC and ES-MS confirmed the synthesised peptides’ purity and molecular

mass [7].

Figure 2.2: Directed disulphide formation of α-conotoxins using orthogonal cysteine

protecting groups.

74

2.2.3 NMR spectroscopy

Dr Richard Clark (School of Biomedical Sciences, The University of Queensland) carried out

all NMR spectroscopy experiments.

1H NMR was done to determine the successful folding of all peptide analogues outlined in

Chapter 3. NMR data for all peptides were recorded on Bruker Avance 500- and 600-MHz

spectrometers, with samples of > 95% purity dissolved in 90% H2O and 10% D2O

(Cambridge Isotope laboratories, Massachusetts, USA).

Two-dimensional NMR experiments included total correlation spectroscopy (TOCSY) and

nuclear overhauser effect spectroscopy (NOESY) recorded at 280 K. Spectra were analysed

using Topspin 1.3 (Bruker) and Sparky software. Most spectra were recorded at pH 3.5.

Sequence-specific resonance assignment was carried out for each peptide’s spectra in

collaboration with Dr Norelle Daly (Institute for Molecular Biosciences, The University of

Queensland) [8].

2.3 Electrophysiological recordings in Xenopus oocytes

Xenopus laevis frogs were anesthetised using MS-222 (1.3 g/L) solution before oocytes were

surgically extracted. The oocytes were then incubated with collagenase (3 mg/mL) dissolved

in OR2 buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2 and 5 mM HEPES, pH 7.4) for 1–2

h (until the vitelline membrane surrounding the oocytes was digested). The collagenase was

then removed by washing the oocytes with OR2 buffer.

Oocytes at stages V–VI (larger in size) with distinct animal (dark) and vegetal pole (white)

were selected for mRNA injection [Figure 2.3]. cDNAs encoding the rat α3, α4, α9, α10, β2

and β4 nAChR subunits and human α7 nAChR subunit, subcloned into the oocyte expression

75

vector pT7TS, were used for the mRNA preparation using the mMESSAGE mMACHINE Kit

(Ambion Inc, USA). All oocytes were injected with 25 ng of cRNA for α9 and α10, and 5 ng

of cRNA for all other subunits. They were then kept at 18 °C in ND96 buffer (96 mM NaCl, 2

mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.4) supplemented with 50

mg/L gentamicin and 100 µg/units/mL penicillin streptomycin for 2–5 days before recording

[Figure 2.3].

Membrane currents from Xenopus oocytes were recorded using two-electrode voltage clamp

(virtual ground circuit) with either a GeneClamp 500B amplifier (Molecular Devices) or an

automated workstation with eight channels in parallel, including drug delivery and online

analysis (OpusXpressTM 6000A workstation, Axon Instruments Inc.) [Figure 2.3]. Voltage-

recording and current-injecting electrodes were pulled from borosilicate glass (GC150T-7.5,

Harvard Apparatus Ltd) and had resistances of 0.3–1.5 MΩ when filled with 3 M KCl [9].

Figure 2.3: Schematic representation of the various steps involved in two-electrode

voltage-clamp studies using Xenopus laevis oocytes.

76

All recordings were made at room temperature using a bath solution of ND96, as described

above. During recordings, oocytes were perfused continuously at a rate of 2 mL/min, and

peptides were incubated for 300 s before the ACh was added. ACh (200 µM for α7 and 50

µM for all other nAChR subtypes) was applied for 2 s at 2 mL/min, with 180–240 s washout

periods between applications. Cells were voltage-clamped at a holding potential of −80 mV.

Data were filtered at 10 Hz and sampled at 500 Hz [10].

Desensitisation experiments were carried out using 50 µM ACh applied for 30 s, followed by

a 300 s washout. The onset (kon) and recovery (koff) from block by the peptide was measured

by bath applying the peptide at 2 mL/min for 5 min, followed by washout. On-rate kinetics

were carried out with ACh + peptide pulse applied at the indicated time intervals. ACh pulses

during recovery from block contained no peptide. The percentage response or percentage

inhibition was obtained by averaging the peak amplitude of three control responses directly

before exposure to the peptide.

2.4 Data analysis

Concentration–response curves for antagonists were fitted by unweighted nonlinear regression

to the logistic equation:

Ex = Emax XnH/(XnH + IC50

nH)

where Ex is the response, X is the antagonist concentration, Emax is the maximal response, nH

is the slope factor and IC50 is the antagonist concentration that gives 50% inhibition of the

maximal response.

All electrophysiological data were pooled (n = 4–8 for each data point) and represent

arithmetic means ± standard error of the fit.

77

The rates of onset and recovery from block during peptide washout were obtained from

exponential fits to the data using GraphPad Prism 5. The ki for the peptide was then

calculated using the equation:

ki = koff / kon.

Computation was done using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA,

USA).

2.5 References

1. Merrifield, R. B. (1963) Solid Phase Peptide Synthesis .1. Synthesis of a Tetrapeptide,

J Am Chem Soc 85, 2149-&.

2. Meienhofer, J., Waki, M., Heimer, E. P., Lambros, T. J., Makofske, R. C., and Chang,

C. D. (1979) Solid phase synthesis without repetitive acidolysis. Preparation of leucyl-

alanyl-glycyl-valine using 9-fluorenylmethyloxycarbonylamino acids, International

journal of peptide and protein research 13, 35-42.

3. Alewood, P., Alewood, D., Miranda, L., Love, S., Meutermans, W., and Wilson, D.

(1997) Rapid in situ neutralization protocols for Boc and Fmoc solid-phase

chemistries, Methods in enzymology 289, 14-29.

4. Sarin, V. K., Kent, S. B., Tam, J. P., and Merrifield, R. B. (1981) Quantitative

monitoring of solid-phase peptide synthesis by the ninhydrin reaction, Analytical

biochemistry 117, 147-157.

5. Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for

detection of free terminal amino groups in the solid-phase synthesis of peptides,

Analytical biochemistry 34, 595-598.

6. Lloyd-Williams, P., Albericio, F., and Giralt, E. (1997) Chemical approaches to the

synthesis of peptides and proteins, CRC Press, Boca Raton.

78

7. Banerjee, J., Gyanda, R., Chang, Y. P., and Armishaw, C. J. (2013) The chemical

synthesis of alpha-conotoxins and structurally modified analogs with enhanced

biological stability, Methods in molecular biology 1081, 13-34.

8. Wuthrich, K. (2003) NMR studies of structure and function of biological

macromolecules (Nobel lecture), Angewandte Chemie 42, 3340-3363.

9. Hogg, R. C., Hopping, G., Alewood, P. F., Adams, D. J., and Bertrand, D. (2003)

Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-

L247T nicotinic acetylcholine receptor, The Journal of biological chemistry 278,

26908-26914.

10. Nevin, S. T., Clark, R. J., Klimis, H., Christie, M. J., Craik, D. J., and Adams, D. J.

(2007) Are {alpha}9{alpha}10 nicotinic acetylcholine receptors a pain target for

{alpha}-conotoxins?, Mol Pharmacol.

79

CHAPTER 3

α-Conotoxin RegIIA targeting α3β4 nAChR:

unlocking novel therapeutics towards lung cancer

Conus regius

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3.1 INTRODUCTION

3.1.1 Nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels expressed in the

central nervous system (CNS) and peripheral nervous system (PNS) [1, 2]. They are

pentameric receptors composed of a combination of alpha subunits (α2–10) and beta subunits

(β2–4). nAChRs exhibit a diverse structural and functional heterogeneity through formation

of various heteromeric (e.g. α3β2 and α4β2) and homomeric isoforms (formed by only α7 and

α9 subunits) [3]. Their physiological role is well understood and they are known to modulate

pre- and post-synaptic transmission in the CNS, and visceral and somatic sensory

transmission in the PNS [4-6].

nAChRs have been implicated in various pathophysiological conditions, including

Alzheimer’s disease, schizophrenia, tobacco addiction and lung cancer [2]. Despite

considerable progress in understanding these pathological conditions, knowledge of the

distribution and neurophysiological role of individual receptor subtypes is limited by the lack

of adequate isoform-specific probes [7].

3.1.2 The pathophysiological role of the α3β4 nAChR subtype

Following the first report by Schuller’s group in 1989, which suggested that nAChRs may

have a role in regulating cancer cell growth [8], many studies were initiated to investigate the

role of nAChRs in cancer development and progression [9]. Two reports identified nicotine

and its derivatives as potent nAChR agonists and their regulatory role in cancer cell apoptosis

[10, 11]. Genome-wide association studies (GWAS) led to the identification of various single

81

nucleotide polymorphisms (SNP) within a gene cluster encoding α3, α5 and β4 nAChR

subunits, and these SNPs are associated with lung cancer [9, 12]. In addition, the α3β4

subtype has been shown to be involved in nicotine addiction and drug-abuse [13]. However,

knowledge of the distribution and physiological role of the α3β4 subtype is still limited. The

development of a α3β4 subtype-selective inhibitor, which could help develop drugs to treat

cancer and nicotine addiction, is very desirable.

3.1.3 α-Conotoxins targeting neuronal nAChRs

Conotoxins are bioactive peptides isolated from the venom of cone snails belonging to the

genus Conus [14, 15]. α-Conotoxins, a class of short disulphide-rich peptides from this venom

[16], specifically target various nAChR subtypes and are excellent molecular probes for

identifying the physiological role of nAChR subtypes in normal and disease states [17]. To

date, many α-conotoxins have been characterised, and their structural and functional

properties are well documented in various reviews and online databases

(http://www.conoserver.org) [14, 18-22]. While it is evident that α-conotoxins, such as ImII

and MII, exhibit selective inhibitory activity at α7 [23] and α3β2 [24] nAChR subtypes

respectively, most known peptides target multiple subtypes [25]. Mutagenesis experiments

have therefore become an important tool to improve the selectivity and potency of these

peptides [26]. Furthermore, α-conotoxin AuIB is the only peptide known to selectively target

the α3β4 nAChR subtype with an IC50 of 2.5 µM [27].

Here, I describe the discovery, and biochemical, biophysical and functional characterisation,

of RegIIA. This α4/7-conotoxin was isolated from the venom of Conus regius, a worm-

hunting cone snail species that inhabits the Western Atlantic Ocean. It is one of the most

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potent α3β4 nAChR antagonists, but does not inhibit the α4β2 subtype. This selectivity profile

makes RegIIA a prospective probe for studying nicotine addiction processes.

RegIIA has a classical α-conotoxin globular structure (ω-shaped fold) indicating that it has an

exquisite balance of shape, charges, and polarity exposed on its surface to enable it to potently

block the α3β4 nAChR. The pathophysiological association of α3β4 nAChRs in various

diseases such as lung cancer and nicotine addiction, along with RegIIA being one of the most

potent known α3β4 nAChR antagonists, led us to investigate and improve RegIIA’s

selectivity profile at the α3β4 nAChR subtype. Using alanine scanning mutagenesis and

modelling studies, we identified critical residues of α-conotoxin RegIIA that interact with the

ACh-binding site of α3β2, α3β4 and α7 nAChRs.

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3.2 AIMS

3.2.1 Characterisation of α-conotoxin RegIIA isolated from Conus regius.

To determine the pharmacological profile of RegIIA using two-electrode

voltage-clamp technique in Xenopus oocytes expressing recombinant

nAChR subtypes.

3.2.2 Alanine scan mutagenesis.

To understand the molecular mechanism and critical residues that

determine RegIIA’s specific nAChR subtype selectivity.

3.2.3 Significance

In conjunction with modelling simulation being done by our collaborators,

this study could provide a detailed understanding of RegIIA’s molecular

pharmacology at the α3β4 and α3β2 nAChR subtypes.

This study could also provide valuable information to aid the future design

and development of α3β4-selective drugs to treat lung cancer and nicotine

addiction.

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3.3 RESULTS

3.3.1 Selective α-conotoxin RegIIA inhibition of recombinant nAChR subtypes

RegIIA selectivity was examined by inhibiting ACh-evoked currents mediated by various

nAChRs subtypes expressed in Xenopus oocytes. ACh was applied at 5 min intervals and the

corresponding membrane currents were assessed. The peptide was bath incubated for 5 mins

before co-application of ACh and the peptide. Synthetic RegIIA (1 µM) completely inhibited

ACh-evoked current amplitude produced by α3β4, α3β2 and α7 nAChRs [Figure 3.1].

However, RegIIA did not inhibit muscle (αβγδ) or α4β2 nAChRs (n = 4–5). RegIIA (1 µM)

inhibited only 20 ± 5% of ACh-evoked current amplitude of α9α10 nAChR. Concentration–

response curves of RegIIA display the order of selectivity and their corresponding IC50 values

for α3β2 (11 nM) > α3β4 (47 nM) > α7 (61 nM) [Figure 3.2 and Table 3.1].

Figure 3.1: Concentration-dependent RegIIA inhibition of ACh-evoked current

amplitude mediated by (A) α3β2, (B) α3β4 and (C) α7 nAChRs expressed in oocytes.

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Figure 3.2: Selectivity of α-conotoxin RegIIA inhibition of nAChR subtypes. RegIIA (1

µM) completely inhibited α3β2, α3β4 and α7 receptors, and inhibited the α9α10 receptor by

~20%. Concentration–response curves for RegIIA inhibition gave IC50 of 16 nM for α3β2

(●), 48 nM for α3β4 (▲) and 51 nM for α7 (□). All data represents mean ± SEM; n = 4–7.

Table 3.1: α-Conotoxin RegIIA inhibition of recombinant nAChR subunits expressed in Xenopus oocytes.

nAChR subtype IC50 (95% CI) nH α3β2 10.7 nM (8.8–12.9) –1.1 ± 0.1 α3β4 47.3 nM (39.5–56.6) –1.5 ± 0.2

α7 61.2 nM (47.8–78.4) –1.2 ± 0.2 α9α10 >1 µM α4β2 >1 µM αβδγ >1 µM

RegIIA (1 µM) had no effect on α4β2 or αβγδ, and inhibited α9α10 by only 20%. All data represents n = 4–7.

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3.3.2 Directed peptide synthesis of α-conotoxin RegIIA analogues and NMR

α-Conotoxins have a conserved Cys-framework. Their three-dimensional structure is

dominated by a helical structure. In native α-conotoxins, I–III and II–IV disulfide connectivity

is dominant and folds the peptide into globular conformation [28, 29]. However, α-conotoxin

disulfide connectivity can change upon oxidation and reduction to form ribbon (I–IV and II–

III disulfide bonds) or beads (I–II and III–IV disulfide bonds). These conformations play

significant roles in potency and specificity of α-conotoxins at nAChRs [24, 30].

Under normal conditions of solid-phase peptide synthesis, the percentage of a peptide in a

single conformation varies, which can be undesirable. This problem was solved by using

amino acids with stable protective groups (Acm) under normal oxidative conditions

(0.1 M NH4HCO3, pH 8.2). The use of Cys-Acm amino acids at positions 1 and 3 and a two-

step oxidation procedure, yielded the alanine mutants in a globular conformation (I–III and

II–IV disulfide bonds), shown by HPLC. This was confirmed by 2D NMR (COSY and

NOESY), which showed the negative 1H shift values between the amino acids 3 and 7

positions and indicated the presence of α-helix secondary structure [Figure 3.3].

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Figure 3.3: RegIIA and alanine analogues secondary αH shifts.

3.3.3 Inhibition of nAChR subtypes by α-conotoxin RegIIA analogues:

To understand the RegIIA structure–activity relationship at nAChRs, RegIIA analogues were

tested on α3β2, α3β4 and α7 nAChR subtypes at a concentration of 300 nM [Figure 3.4].

[H14A]RegIIA showed complete loss in activity at all of the above-mentioned nAChR

subtypes. At α7 nAChR subtype, no noticeable change was seen for [P13A]RegIIA inhibition,

whereas inhibition by all other analogues was significantly reduced or completely lost

[Figure 3.4(B)]. In contrast, no change in inhibition of the α3β4 nAChR subtype by RegIIA

analogues was observed except for [N9A]RegIIA which completely lost its activity at α3β4.

Furthermore, [N11A]RegIIA and [N12A]RegIIA selectivity for α3β4 nAChR subtypes

improved. Their inhibition of the α3β2 nAChR subtype was significantly reduced (by

approximately 50%), whereas no change was observed at α3β4 nAChR subtype [Figure

3.4(B)]. This was apparent in the concentration–response curves for [N11A]RegIIA and

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[N12A]RegIIA, which showed the inhibition curve for the α3β2 (red line) nAChR subtype

shifted toward the right [Figure 3.5]. The IC50 values for [N11A]RegIIA and [N12A]RegIIA

at the α3β2 nAChR subtype was 115.9 nM (95% Cl 62.7 – 214.1; nH = –0.9 ± 0.22) and 278

nM (95% Cl 153.4 – 503.8; nH = –1.1 ± 0.25) respectively, and at the α3β4 nAChR subtype is

51.6 nM (95% Cl 44.2 – 60.1; nH = –2.0 ± 0.21) and 112 nM (95% Cl 92.1 – 136.2; nH = –1.7

± 0.32), respectively [Table 3.2].

Figure 3.4. RegIIA and alanine analogue (300 nM) inhibition of various nAChR

subtypes expressed in Xenopus oocytes. (A) Bar graph of inhibition of nAChR subtypes by

RegIIA and its analogues. Data represents mean ± SEM, n = 4–6. (B) Two-way ANOVA

scatter plot illustrating the loss of activity of the RegIIA analogues (300 nM) relative to wild-

type RegIIA at various nAChR subtypes. [H14A]RegIIA completely lost its activity at α3β2,

α3β4 and α7 nAChRs. [N9A]RegIIA was more selective for the α3β2 subtype than RegIIA

was. [N11A]RegIIA and [N12A]RegIIA selectivity for the α3β4 nAChR subtype significantly

improved. All analogues, except [P13A]RegIIA, significantly lost activity at the α7 nAChR

subtype. *** p < 0.001, * p < 0.05; n = 4–6.

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Figure 3.5: [N11A]RegIIA and [N12A]RegIIA exhibiting improved selectivity at α3β4

nAChR subtypes (A) Superimposed traces showing ACh-evoked current inhibition of the

α3β2 nAChR subtype by 100 nM (i) RegIIA, (ii) [N11A]RegIIA and (iii) [N11A]RegIIA . (B)

Concentration–response curves for (ii) [N11A]RegIIA and (iii) [N12A]RegIIA inhibition of

the α3β4 nAChR (black line, open symbols) and α3β2 nAChR subtypes (red line, closed

symbols). [N11A]RegIIA and [N12A]RegIIA shifted the curve to the right for the α3β2 (red

line) nAChR subtype, giving an IC50 value of 116 nM and 278 nM, respectively. All data

represents mean ± SEM; n = 4–6.

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Table 3.2: RegIIA and analogues inhibition of nAChR subtypes.

α3β4 α3β2 α7

IC50 (95% Cl) nH IC50 (95% Cl) nH IC50 (95% Cl) nH

RegIIA 47.3 nM (39.5–56.6) –1.5 ± 0.2

10.7 nM (8.8–12.9) –1.1 ± 0.1

61.2 nM (47.8–78.4) –1.2 ± 0.2

[N11A]RegIIA 51.6 nM (44.2–60.1) –2.0 ± 0.2

115.9 nM (62.7–214.1) –0.9 ± 0.2

ND –

[N12A]RegIIA 112 nM (92.1–136.2) –1.7 ± 0.3

278 nM (153.4–503.8) –1.1 ± 0.2

ND –

[N11A,N12A]RegIIA 370 nM (3.09–442.3) –1.7 ± 0.2

9.87 µM (7.9–12.4) –1.5 ± 0.3

21.5 µM (17.1–27.0) –3.1 ± 0.8

IC50 values with 95% Cl. and Hill slope (nH) obtained from concentration–response curves for RegIIA and analogues at α3β2, α3β4 and α7 nAChR subtypes. All data represent mean of n = 4–6 experiments. ND – not determined.

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3.3.4 Double mutant [N11A,N12A]RegIIA selectively inhibits α3β4 nAChR subtype

Double mutant [N11A,N12A]RegIIA was synthesised to better understand the cumulative

effect of the two residues on nAChR activity. When tested, [N11A,N12A]RegIIA inhibited

the α3β4 nAChR subtype with an IC50 of 370 nM (95% Cl 3.09 – 442.3; nH = –1.7 ± 0.2) and

a 7-fold decrease in potency compared with RegIIA [Figure 3.6]. However, at the α3β2 and

α7 nAChR subtypes, potency decreased by approximately 1,000-fold (IC50 = 9.9 µM) and

360-fold (IC50 = 21.5 µM), respectively [Table 3.2]. This indicates an approximate 27-fold

change in selectivity at the α3β2 nAChR subtype, and an approximate 58-fold change at the

α7 nAChR subtype.

Figure 3.6: [N11A,N12A]RegIIA inhibition of α3β2 and α3β4 nAChR compared with

that of wild-type RegIIA. Concentration–response curve of [N11A,N12A]RegIIA gave an

IC50 of 370 nM at α3β4 (▲) and 9.9 µM at α3β2 (■), with an approximate 27-fold change

in selectivity. All data represents mean ± SEM; n = 4–6.

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3.4 DISCUSSION

3.4.1 Conus regius: characterisation of novel α-conotoxin RegIIA

Since 1994, when the first α-conotoxin ImI was discovered in the worm-hunting Conus

imperialis and found to target neuronal nAChRs, numerous α-conotoxins have been

identified and functionally characterised [14, 31]. Most native α-conotoxins have a ω-shaped,

three-dimensional globular conformation, which results from the two disulfide bonds (I–III

and II–IV) in the peptide’s characteristic cysteine framework CCXnCXmC (classified as

cysteine framework I) [29]. The number of amino acids indicated by n and m designate the

subclass of the peptide. RegIIA belongs to the α4/7 subclass and exhibits the classical ω-

shaped globular structure, with balanced shape, charges and polarity.

Today, various peptides have been identified from the venom of C. regius, a Western Atlantic

worm-hunting cone snail species that belongs to various superfamilies, of which 8 belong to

the A-superfamily with the cysteine framework I [32]. The conotoxin composition of C.

regius venom gained special importance due to the identification of α-conotoxin RgIA, which

selectively targets the α9α10 nAChR subtype and high voltage-activated (HVA) calcium

channel currents via GABAB receptors [33]. α-Conotoxin RegIIA, also isolated from C.

regius, potently inhibits the α3β4 nAChR subtype [34]. HVA calcium channels modulated by

GABAB receptors are involved in pain pathways and the α3β4 nAChR subtype is implicated

in lung cancer pathophysiology. This makes the discovery of RgIA and RegIIA very

significant and puts them at the forefront of potential new therapeutics.

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3.4.2 Alanine mutagenesis reveals the pharmacological role of the –NNP– motif in

RegIIA

RegIIA exhibits high homology to various known peptides [Table 3.8], with two main

features: an –SHPA– conserved sequence and –NNP– motif. While the –SHPA– conserved

sequence is observed in the peptides of different subclasses that target various nAChR

subtypes, the –NNP– motif has been specifically observed in peptides that inhibit α3β2 and

α7 nAChR subtypes, such as OmIA [35], EpI [36], PnIA [37], TxIA [38] and ArIB [39]. This

observation corroborates with the most important finding of this study – the alanine mutation

of the –NNP– motif significantly affected the peptide’s inhibitory activity at α3β2 and α7

nAChR subtypes. Additionally, the [N11A,N12A]RegIIA analogue is the most potent of the

α3β4 nAChR subtype-selective peptides.

Alanine mutations at other positions also provided vital information about the structure–

function relationship between α-conotoxins and neuronal nAChRs. Asparagine at the ninth

position is critical for RegIIA inhibition of the α7 and α3β4 nAChRs, because alanine

mutation of this residue completely abolished RegIIA inhibition of both nAChR subtypes.

3.4.3 Molecular modelling and molecular dynamics reveal structural topology of α3β2

and α3β4 nAChRs and residues that interact with RegIIA

To elucidate the molecular mechanism of RegIIA’s inhibition of the α3β4 nAChR subtype,

an alanine scan mutagenesis protocol was used. This technique is well established, and in

conjunction with atomistic molecular dynamics (MD) simulations, it has previously enabled

spectacular progress in the field of molecular pharmacology [26, 40]. X-ray and NMR studies

of AChBP–α-conotoxin complexes, and the support of synthetic analogues, promoted the

94

understanding of ligand–receptor interactions [41-44]. The extracellular N-terminal domain

(ECD) of β2 and β4 nAChR subunits, to which α-conotoxins bind, exhibited a high sequence

homology (70%). Recent MD simulation studies also reveal a well-preserved structural

topology of α3β2 and α3β4 nAChRs. However, the ACh-binding pocket interface between

the α and β subunits was larger in α3β2 than α3β4 nAChR subtypes [45]. This difference

could signify a distinct shift in [N11A] and [N12A]RegIIA selectivity for α3β2 and α3β4,

even though both of these residues primarily interact with the α3(+) interface.

To elucidate the structure–function relationship of the alanine scan mutagenesis results, our

collaborators carried out atomistic MD simulations of [N11A,N12A]RegIIA. The results

indicated that the double mutant induced a significant loss in contact Y92, S149, Y189, Y196

residues of the α3 subunit, and W57, and F119 residues of the β2 subunit induced by the

double mutant. A comprehensive receptor mutagenesis study of various α-conotoxins (MII,

GID and PnIA) that inhibit the α3β2 nAChR indicated that the β2 subunit pharmacophore,

comprised of T59, E61, V111, F119 and L121 residues, has a significant role in ligand

binding [46].

A more recent molecular docking study of α-conotoxin GIC and the ECD of human α3β2 and

α3β4 nAChR subtypes revealed that all three subunits have residues that interact with the

conotoxin (α3 subunit: Y92, Y150, Y189 and Y196; β2 subunit: W57, V111, F119 and L121;

β4 subunit: W57, I111, L119 (Q119 in rat β4) and L121). It is also interesting to note that the

α3Y196 and β2F119 residues of the α3β2-GIC model were more closely located than the

α3Y196 and β4L119 residues of the α3β4-GIC model [45]. These results are consistent with

the MD simulations displaying the loss of pairwise contacts between wt RegIIA and

[N11A,N12A]RegIIA at α3β2 compared to α3β4 nAChR subtypes. This study provides the

95

first experimental evidence into the molecular pharmacological difference between an α-

conotoxin and these two nAChR subtypes at structural and functional levels.

3.5 SUMMARY AND CONCLUSION

The CHRNA3, CHRNB4 and CHRNA5 gene clusters encoding the α3, β4 and α5 nAChR

subunits gained significant importance in recent years. This may be due to the recent genetic

and physiological studies implicating that the α3β4 nAChR has a direct functional role in the

pathophysiology of lung cancer and nicotine addiction [9, 12]. While the α3β4 nAChR was

initially identified in ganglia, recent studies show it is also distributed throughout the CNS

and other tissues, such as the interpeduncular nucleus and medial habenula [47, 48].

Identifying and successfully synthesising [N11A,N12A]RegIIA, a α3β4 nAChR subtype-

selective antagonist, could help to decipher the physiological role of this receptor. Our study

also extends the understanding of RegIIA interactions with various nAChR subtypes and

elucidated the key residues on the toxin and receptor binding sites. This information is

invaluable in the design and development of α3β4-selective drugs to treat lung cancer and

nicotine addiction.

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Table 3.3: Sequence alignment of α-conotoxins targeting various nAChR subtypes.

α-Conotoxin Sequence nAChR selectivity IC50 for α3β4 nAChR inhibition Reference

RegIIA GCCSHPACNVNNPHIC* α3β2>α3β4> α7 50 nM [34]

OmIA GCCSHPACNVNNPHICG* α3β2>α7>α6β2 - [49]

GIC GCCSHPACAGNNQHIC* α3β2≈α6β2>α7 - [50]

PeIA GCCSHPACSVNHPELC* α9α10>α3β2>α6β2*>α3β4>α7 480 nM [51]

Mr1.1 GCCSHPACSVNNPDIC* α3β2>α3β4>α7 1400 nM [52]

Ls1a SGCCSNPACRVNNPNIC* α3β2>α7 - [53]

AuIB GCCSYPPCFATNPD-C* α3β4 2500 nM [54]

BuIA GCCSTPPCAVLY---C* β2*>β4* 28 nM [55]

PIA RDPCCSNPVCTVHNPQIC* α6β2*>α6β4≈α3β2>α3β4 520 nM [56]

ArIB DECCSNPACRVNNPHVCRRR* α7≈α6β2*>α3β2 - [57]

Amino acids homologous to RegIIA are labelled with grey background. Peptides inhibiting the α3β4 nAChR subtype (orange) and their

corresponding IC50 values are shown. The conserved cysteine framework is highlighted in yellow.

97

3.6 References

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104

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105

CHAPTER 4

Characterisation of LsIA:

First α-conotoxin isolated from Conus limpusi

Conus limpusi

106

4.1 INTRODUCTION

α-Conotoxins are a class of bioactive peptides isolated from the venom of cone snails

belonging to the genus Conus. They inhibit various nAChR subtypes with a high degree of

specificity and potency. This unique pharmacological profile has led to the development of α-

conotoxins as novel molecular probes for the physiological study of nAChR subtypes and

drugs to treat various pathological conditions involving cholinergic mechanisms of action [1].

The pharmacological profile of an α-conotoxin is determined by the peptide’s structure and

sequence. α-Conotoxins of the α4/7 and α4/3 subclass, defined by their characteristic cysteine

framework, inhibit neuronal nAChR subtypes almost exclusively [2].

nAChRs are pentameric transmembrane ion channels. They are constituted by various nAChR

subunits, which classifies them into homomeric receptors, such as α7 or α9, or heteromeric

receptors, such as α4β2 or α3β2 [3]. nAChRs primarily mediate fast synaptic transmissions in

the CNS and PNS. They also modulate cholinergic transmission in non-neuronal cells,

mediating subtype-specific physiological functions [4].

α3β2 and α7 nAChR subtypes play vital roles in various functions, such as neuronal plasticity,

angiogenesis and gene regulation [5]. They also are involved in various pathophysiological

conditions, such as schizophrenia, Alzheimer’s disease and myasthenia gravis [6].

Here I describe the pharmacological properties of the novel α-conotoxin LsIA, the first

peptide isolated from Conus limpusi, a species of worm-hunting cone snail commonly found

on the south east coast of Queensland, Australia. LsIA is an α4/7-conotoxin with the

characteristic I–III and II–IV disulfide connectivity. LsIA exhibited selective and potent α7

and α3β2 nAChR subtype antagonism. In this report, I also examined the structure–function

relationship of the presence of a unique N-terminal serine at position 2 and C-terminal

107

carboxylation. Furthermore, I also investigated the pharmacological implications, involving

incorporation of the α5 subunit towards the inhibition of LsIA at α3β2 nAChR subtypes.

4.2 AIMS

4.2.1 Characterisation of LsIA.

To functionally characterise LsIA – the first α-conotoxin isolated from

Conus limpusi.

To identify the role the N-terminal sequence and C-terminal carboxylation

play towards the pharmacological profile of LsIA.

4.2.2 Significance

To identify and understand the pharmacological profile of novel α-

conotoxins at nAChR.

To understand the structure–functional relationship of unique α-conotoxin

features.

To understand the functional implication of α5 subunit in conotoxin

pharmacology.

108

4.3 RESULTS

4.3.1 LsIA inhibition of recombinant nAChR subtypes

I determined LsIA potency and selectivity at neuronal nAChRs by examining its effect on

ACh-evoked currents mediated by different nAChR subunit combinations expressed in

Xenopus oocytes. ACh was applied every 5 min and ACh-evoked membrane currents were

assessed. LsIA (1 µM) completely inhibited ACh-evoked current amplitude mediated by α7,

α3β2 and α3α5β2 nAChR subtypes. However, LsIA (3 µM) inhibited α3β4 nAChR-mediated

currents by only 40 ± 5% (n = 6) and had no effect on α9α10 and α4-containing nAChR

subtypes (n = 4–8) [Figure 4.1].

500 nA

500 ms

α7ACh

α3β2ACh

300 nA

2 s

100 nA

1 s

AChα3α5β2

30 nM

10 nM

Control

30 nM

10 nM

Control

3 nM

100 nM

30 nM

Control

10 nM

A

-10 -9 -8 -7 -6 -5

0.0

0.2

0.4

0.6

0.8

1.0

1.2α3β2

α7

α3β4α4β2α4β4

α9α10

α3α5β2

[LsIA] (M)

Rela

tive

Cur

rent

Am

plitu

de

B

Figure 4.1: α-Conotoxin LsIA selectivity for various nAChR subunit combinations

expressed in Xenopus oocytes. (A) Superimposed traces of ACh-evoked currents in the

absence (control) and presence of various LsIA concentrations at α7, α3β2 and α3α5β2

nAChR subtypes. (B) Concentration–response curves for LsIA inhibition of different nAChR

subtypes gave IC50 values of 10.3 nM (95% CI, 8.8–12.1 nM; nH = –1.3 ± 0.1) at α3β2; 31.2

nM (95% CI, 26.1–37.3 nM; nH = –1.1 ± 0.1) at α3α5β2; and 10.1 nM (95% CI, 8.7–11.6 nM;

nH = –2.1 ± 0.4) at α7 subtypes. LsIA (1 µM) completely inhibited ACh-evoked currents

mediated by α3β2, α3α5β2 and α7 nAChRs. Data represents mean ± SEM, n = 3–6.

109

LsIA reversibly inhibited α3α5β2 about three times less potently (IC50 = 31.2 nM) than it did

the α3β2 subtype (IC50 = 10.3 nM). α5 subunit expression was confirmed by desensitisation

experiments described in the Material and methods section. As shown in a previous study [7],

the time course response of the α3α5β2 nAChR subtype showed notably faster desensitisation

of ACh-evoked currents compared with those of the α3β2 nAChR subtype [Figure 4.2 Inset].

Concentration–response curves for LsIA inhibition of ACh-evoked currents at different

nAChR subtypes and their corresponding IC50 value exhibit the following selectivity

sequence: α7 (10.1 nM) ≅ α3β2 (10.3 nM) > α3α5β2 (31.2 nM) [Figure 4.1].

The on- and off-rates (kon and koff) for nAChR inhibition by the peptide were obtained from

the time course of responses in the presence and upon washout of the peptide [Figure 4.2 and

4.3 and Table 4.1]. LsIA (10 nM) recovered more slowly from block at the α7 nAChR

subtype than at the α3β2 and α3α5β2 nAChR subtypes, giving a ki of 1.47 x 10-9 M [Figure

4.3 and Table 4.1]. This ki value is 6.8-fold lower than IC50. A similar trend was observed at

the α3α5β2 subtype, which had a ki value 3.3-fold lower than IC50 [Table 4.1].

110

Time (min)

Rela

tive

Inhi

bitio

n

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0.0

0.2

0.4

0.6

0.8

1.0LsIA Washout

A

200 nA

30 s

ACh

α3β2

Time (min)

Rel

ativ

e In

hibi

tion

0 1 2 3 4 5 6 7 8 9 10

0.0

0.2

0.4

0.6

0.8

1.0LsIA washout

B

200 nA

30 s

ACh

α3α5β2

Figure 4.2: Kinetics of LsIA inhibition of peak ACh-evoked current amplitude as a

function of time. Onset (filled bar) of LsIA (30 nM) block and recovery (open bar) upon

washout at α3β2 (A) and α3α5β2 (B) nAChR subtypes. Inset: Representative ACh (50 µM)-

evoked currents in oocytes expressing (A) α3β2 and (B) α3α5β2 nAChR subtypes.

111

Time (min)

Rela

tive I

nhib

ition

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 180.0

0.2

0.4

0.6

0.8

1.0LsIA Washout

B α7

Figure 4.3: Kinetics of α-conotoxin LsIA block and recovery at the α7 nAChR subtype.

(A) Representative ACh-evoked currents in the absence and presence of LsIA in oocytes

expressing α7 nAChR. LsIA (10 nM) was bath applied for 5 min before washout. Responses

to a 1-s pulse of ACh (200 µM) and toxin for on-rate, and ACh alone for off-rate, kinetics was

measured at various time intervals. C is the ACh control response before the toxin was

applied. (B) LsIA inhibition of peak ACh-evoked current amplitude as a function of time.

Onset (filled bar) of block by LsIA (10 nM) and recovery (open bar) upon washout at the α7

nAChR subtype.

112

Table 4.1: α-Conotoxin LsIA’s kinetic constants for blocking nAChR subtypesa.

kon (min–1 M–1) koff (min–1) ki (M) IC50 (M)

α3β2 6.93 (4.99–8.87) x 107 1.052 (0.910–1.194) 15.2 x 10–9 10.3 x 10–9

α3α5β2 1.26 (0.28–2.24) x 108 1.205 (0.766–1.644) 9.6 x 10–9 31.2 x 10–9

α7 1.35 (1.17–1.53) x 108 0.199 (0.182–0.215) 1.47 x 10–9 10.1 x 10–9

a Numbers in parentheses are 95% CI. Mean of data from n = 6–8 experiments.

113

4.3.2 Influence of C-terminal carboxylation and N-terminal truncation of LsIA

Conotoxins typically have a high degree of post-translational modification [8, 9], with

C-terminal amidation shown to influence structure and enhance target specificity [9].

Native α-conotoxin LsIA also undergoes C-terminal amidation. Our collaborators

synthesised a C-terminally carboxylated analogue of LsIA (LsIA#) to examine its

activity at different nAChR subtypes. Interestingly, LsIA# was ~ three-times less

potent at the α7 subtype and ~ three-fold more potent at the α3β2 subtype [Figure

4.4].

α7 nAChR

-9 -8 -7 -6

0.0

0.2

0.4

0.6

0.8

1.0

1.2 LsIA#LsIA[∆1]LsIA[∆1-2]LsIA

[Peptide] (M)

Rela

tive

Cur

rent

Am

plitu

de

α3β2 nAChR

-10 -9 -8 -7 -6 -5

0.0

0.2

0.4

0.6

0.8

1.0

1.2

LsIALsIA#

[∆1]LsIA[∆1-2]LsIA

[Peptide] (M)

Rela

tive

Cur

rent

Am

plitu

deA B

Figure 4.4: Influence of N-terminus truncation and C-terminus carboxylation of

LsIA on ACh-evoked current inhibition at the α7 (A) and α3β2 (B) nAChR

subtypes. Concentration–response curves for LsIA#, [∆1]LsIA and [∆1–2]LsIA

inhibition gave IC50 values of 30.7 nM (95% CI, 21.7–43.4 nM; nH = –1.5 ± 0.4), 23

nM (95% CI, 19.3–27.5 nM; nH = –2.0 ± 0.3) and 44.1 nM (95% CI, 37.1–52.4 nM;

nH = –2.5 ± 0.4) at α7, respectively; and 3.3 nM (95% CI, 2.2–5.1 nM; nH

= –1.0 ±

0.2), 56.2 nM (95% CI, 40.4–78.2 nM; nH = –1.0 ± 0.2) and 92.4 nM (95% CI, 69.3–

123.3 nM; nH = –0.9 ± 0.1) at α3β2, respectively. The broken line represents the

carboxylated LsIA (LsIA#) concentration–response curve. Data represents means ±

SEM, n = 4–7.

114

α-Conotoxin LsIA contains a unique N-terminal serine residue. Truncation of the four

residue N-terminal tail of α-GID has been shown to influence activity at neuronal

nAChRs [10]. Our collaborators synthesised two truncated analogues, [∆1]LsIA

(lacking the serine at position 1) and [∆1-2]LsIA (lacking the serine at position 1 and

glycine at position 2). [∆1]LsIA- and [∆1-2]LsIA-amidated peptides were two-fold

and four-fold less potent at the α7 subtype than LsIA, respectively [Figure 4.4 and

Table 4.2]. They were also five-fold and nine-fold less potent at the α3β2 subtype

than LsIA, respectively [Figure 4.4 and Table 4.2]. These results indicate that the N-

terminal sequence plays a major role in LsIA potency.

Table 4.2: Half-maximal inhibitory concentrations (IC50) and Hill slope (nH)

values from concentration–response curves for LsIA and its analogues at the

α3β2 and α7 nAChR subtypesa.

α3β2 α7 Peptide IC50 (nM) nH IC50 (nM) nH

LsIA 10.3 (8.8–12.1) –1.3 ± 0.1

10.1 (8.7–11.6) –2.1 ± 0.4

[∆1] LsIA 56.2 (40.4–78.2) –1.0 ± 0.2

23 (19.3–27.5) –2.0 ± 0.4

[∆1–2] LsIA 92.4 (69.3–123.3) –0.9 ± 0.1

44.1 (37.1–52.4) –2.5 ± 0.3

LsIA# 3.3 (2.2–5.1) –1.0 ± 0.2 30.7 (21.7–43.4) –1.5 ± 0.4 a Numbers in parentheses are 95% CI. Mean of data from n = 5–8 experiments.

115

4.4 DISCUSSION

α-Conotoxins evolved as selective probes that target nicotinic receptors, parallel with the

phylogenetic evolution of marine cone snails belonging to the genus Conus. This corresponds

with the structural similarities between various conotoxins, including conserved cysteine

framework and residues. The cysteine framework of α-conotoxins (CC-C-C) divides the

peptide sequence into two loops, from which they are classified into various subclasses. A

high-sequence homology or conserved residues are observed across various α-conotoxins

[Table 4.3].

Table 4.3: Sequence alignment of LsIA and α-conotoxins.

α-Conotoxin Sequence nAChR selectivity Reference

Ls1a SGCCSNPACRVNNPNIC* α3β2≈α7>α3α5β2 This study GID IRDγCCSNPACRVNNOHVC# α7≈α3β2>α4β2 [10] ArIA IRDECCSNPACRVNNOHVCRRR# α7>α3β2 [11] ArIB DECCSNPACRVNNPHVCRRR# α7≈α6β2*>α3β2 [11] MII GCCSNPVCHLEHSNLC* α3β2≈α6β2*>α6β4 [12] RegIIA GCCSHPACNVNNPHIC* α3β2>α3β4> α7 [13] OmIA GCCSHPACNVNNPHICG* α3β2>α7>α6β2* [14] GIC GCCSHPACAGNNQHIC* α3β2≈α6β2*>α7 [15] Mr1.1 GCCSHPACSVNNPDIC* α3β2>α3β4>α7 [16] PnIA GCCSLPPCAANNPDYC* α3β2>α7 [17] AuIB GCCSYPPCFATNPD-C* α3β4 [18] BuIA GCCSTPPCAVLY---C* β2*>β4* [19] RgIA GCCSDPRCRYR----CR# α9α10>>α7 [20]

* denotes an amidated C-terminus; #, free carboxyl C-terminus; γ, γ-carboxyglutamate. Cysteine residues are highlighted yellow. Residues homologous to LsIA are highlighted grey.

Previous studies that aimed to understand the molecular interactions involved in α-conotoxin

subtype-selective antagonism of nAChRs used X-ray studies of co-crystal AChBP and ImI

structures, [A10L,D14K]PnIA and [A10L]TxIA. They revealed significant interaction

between loop 1 (residues between Cys II and III) and the principal face of the receptor [21,

22]. The loop 2 residues of the peptide were shown to majorly interact with the

complementary side of the receptor. These studies identified the general role of loop1 in

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peptide affinity, while loop2 contributes to the subtype selectivity of the α-conotoxin. Also,

structurally, loop1 plays a vital role in the secondary structure of the peptide (presence of α-

helix). NMR studies revealed significant disruption of the peptide’s classic ω-globular

structure upon mutagenesis [23]. This was also indicated through the presence of conserved

residues within loop1 of various subclasses of α-conotoxins [Table 4.3]. AChBP co-crystal

studies also showed significant hydrogen bonding and van der Waals interactions between the

Ser-, Pro- and Gly-conserved residues of loop1 in almost all α-conotoxins and the receptor

binding pocket [21].

α-Conotoxin LsIA is a new peptide isolated from C. limpusi that potently inhibits nAChRs. It

is an α4/7 conotoxin and exhibits equipotent inhibition at α3β2 and α7 subtypes. Pairwise

sequence alignment shows strong homology between LsIA and previously characterised α-

conotoxins that target the α3β2 and α7 nAChR subtypes.

LsIA exhibits two sequence motifs, namely –SXPA– and –NNP–. These motifs are also found

among OmIA, RegIIA, Mr1.1 and ArIB conotoxins, which have a similar pharmacological

profile to LsIA [Table 4.3]. The structural and functional significance of the –SXPA– motif

within loop1 of conotoxins was discussed earlier.

Mutation of Ser and Pro residues to Ala in α-conotoxin GID led to significant activity loss at

α7 and α7/α3β2 nAChR subtypes, respectively [24]. A similar interaction between LsIA and

α7 and α3β2 nAChR subtypes is expected.

Along with conserved motifs, LsIA exhibits a unique N-terminal Ser residue. In this study, I

examined the functional implication of the N-terminal sequence, including the most common

post-transcriptional modification found in the α-conotoxins: C-terminal amidation. The

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influence of the N-terminal sequence was previously examined in α-conotoxin GID. Initial

studies with [∆1-4]GID showed significant activity loss at the α4β2 nAChR subtype, but no

change in IC50 was observed at α7 and α3β2 nAChR subtypes [10]. Interestingly, a noticeable

loss in activity at α7 and α3β2 subtypes was observed when the first three residues were

deleted ([∆1-3]GID). However, [∆1] and [∆1-2]GID truncation only affected α3β2 inhibition

[24].

A similar trend was observed in LsIA, where truncation of first and second residues lead to

considerable loss in activity at the α7 and α3β2 nAChR subtypes [Figure 4.4 and Table 4.2].

This activity loss was more prominent at the α3β2 subtype, with ~5-fold reduction, than at the

α7 subtype, which only showed ~2-fold less activity, with [∆1]LsIA. This effect was further

amplified for [∆1-2]LsIA, which triggered ~9-fold and ~4-fold activity loss at α3β2 and α7

nAChR subtypes, respectively. Surprisingly, the most striking effect on peptide function was

observed when the C-terminus was carboxylated (LsIA#). This modification led to a ~3-fold

increase in potency at the α3β2 nAChR subtype, but caused an opposite effect at the α7

nAChR subtype, with a ~3-fold activity loss.

In α-conotoxin GID, C-terminus carboxylation only influenced peptide activity at the α3β2

nAChR subtype [24]. One explanation for this is the formation of intra-molecular salt bridges

or hydrogen bonds when the free C-terminus carboxyl group is incorporated into LsIA.

In this study, I report a difference in pharmacological activity of LsIA at α5-containing

nAChRs, with LsIA being 3-fold less potent at the α3α5β2 subtype than at the α3β2 subtype.

Unlike the α3 subunit, the α5 subunit is an auxiliary subunit that does not form functional

receptors when expressed with β2 or β4 [25]. However, its role as the fifth subunit in nAChR

pharmacology and physiology has recently gained prominence, because it influences

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conductance [26], agonist sensitivity [27] and ion permeability [7]. Previous studies of α-

conotoxin Vc1.1 at α5-containing nAChRs showed no significant IC50 change [28].

Interestingly, kinetic analysis of LsIA inhibition revealed faster onset of block (kon) at the

α3α5β2 subtype than at the α3β2 subtype, which contributes to the 3-fold lower ki value at

α3α5β2. No significant difference in koff was observed between the α3α5β2 and α3β2 nAChR

subtypes. LsIA off-rate kinetics was slower at the α7 subtype than at the α3β2 and α3α5β2

subtypes. Also, a significant ~ 7-fold difference was observed in the ki and IC50 values at the

α7 subtype. This difference could be due to a higher Hill slope (nH) of –2.1 at the α7 subtype

than at the other subtypes, indicating positive peptide binding cooperativity.

4.5 SUMMARY AND CONCLUSION

LsIA is the first peptide isolated from the venom of C. limpusi. Structure–activity data

collected in this study indicates N-terminal and C-terminal sequences have unique and

specific roles in the pharmacology of this peptide. The N-terminal sequence (Ser1 and Gly2

residues) contributes to nAChR binding affinity, while C-terminus modification imparts

subtype selectivity. While a number of α-conotoxins that potently inhibit α3β2 and α7

nAChR subtypes exist [Table 4.3], I report differences between α-conotoxin LsIA’s

pharmacology and kinetics of inhibition in the presence and absence of the auxiliary α5

subunit. In conclusion, this study provides vital information to improve our understanding of

nAChR inhibition and aid development of novel analogues with improved subtype selectivity.

119

4.6 References

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conopeptides targeted to nicotinic receptors: Concerted discovery and biomedical

applications, Channels 2, 143-152.

2. Daly, N. L., and Craik, D. J. (2009) Structural studies of conotoxins, IUBMB life 61,

144-150.

3. Albuquerque, E. X., Pereira, E. F., Alkondon, M., and Rogers, S. W. (2009)

Mammalian nicotinic acetylcholine receptors: from structure to function,

Physiological reviews 89, 73-120.

4. Albuquerque, E. X., Pereira, E. F., Castro, N. G., Alkondon, M., Reinhardt, S.,

Schroder, H., and Maelicke, A. (1995) Nicotinic receptor function in the mammalian

central nervous system, Annals of the New York Academy of Sciences 757, 48-72.

5. Dajas-Bailador, F., and Wonnacott, S. (2004) Nicotinic acetylcholine receptors and

the regulation of neuronal signalling, Trends in pharmacological sciences 25, 317-

324.

6. D'Hoedt, D., and Bertrand, D. (2009) Nicotinic acetylcholine receptors: an overview

on drug discovery, Expert opinion on therapeutic targets 13, 395-411.

7. Gerzanich, V., Wang, F., Kuryatov, A., and Lindstrom, J. (1998) alpha 5 Subunit

alters desensitization, pharmacology, Ca++ permeability and Ca++ modulation of

human neuronal alpha 3 nicotinic receptors, The Journal of pharmacology and

experimental therapeutics 286, 311-320.

8. Craig, A. G., Bandyopadhyay, P., and Olivera, B. M. (1999) Post-translationally

modified neuropeptides from Conus venoms, European journal of biochemistry /

FEBS 264, 271-275.

120

9. Kang, T. S., Vivekanandan, S., Jois, S. D., and Kini, R. M. (2005) Effect of C-

terminal amidation on folding and disulfide-pairing of alpha-conotoxin ImI,

Angewandte Chemie 44, 6333-6337.

10. Nicke, A., Loughnan, M. L., Millard, E. L., Alewood, P. F., Adams, D. J., Daly, N. L.,

Craik, D. J., and Lewis, R. J. (2003) Isolation, structure, and activity of GID, a novel

alpha 4/7-conotoxin with an extended N-terminal sequence, The Journal of biological

chemistry 278, 3137-3144.

11. Whiteaker, P., Christensen, S., Yoshikami, D., Dowell, C., Watkins, M., Gulyas, J.,

Rivier, J., Olivera, B. M., and McIntosh, J. M. (2007) Discovery, synthesis, and

structure activity of a highly selective alpha7 nicotinic acetylcholine receptor

antagonist, Biochemistry 46, 6628-6638.

12. Everhart, D., Cartier, G. E., Malhotra, A., Gomes, A. V., McIntosh, J. M., and Luetje,

C. W. (2004) Determinants of potency on alpha-conotoxin MII, a peptide antagonist

of neuronal nicotinic receptors, Biochemistry 43, 2732-2737.

13. Franco, A., Kompella, S. N., Akondi, K. B., Melaun, C., Daly, N. L., Luetje, C. W.,

Alewood, P. F., Craik, D. J., Adams, D. J., and Mari, F. (2012) RegIIA: an alpha4/7-

conotoxin from the venom of Conus regius that potently blocks alpha3beta4 nAChRs,

Biochemical pharmacology 83, 419-426.

14. Talley, T. T., Olivera, B. M., Han, K. H., Christensen, S. B., Dowell, C., Tsigelny, I.,

Ho, K. Y., Taylor, P., and McIntosh, J. M. (2006) Alpha-conotoxin OmIA is a potent

ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7

nicotinic acetylcholine receptors, The Journal of biological chemistry 281, 24678-

24686.

15. McIntosh, J. M., Dowell, C., Watkins, M., Garrett, J. E., Yoshikami, D., and Olivera,

B. M. (2002) Alpha-conotoxin GIC from Conus geographus, a novel peptide

121

antagonist of nicotinic acetylcholine receptors, The Journal of biological chemistry

277, 33610-33615.

16. Peng, C., Chen, W., Sanders, T., Chew, G., Liu, J., Hawrot, E., and Chi, C. (2010)

Chemical synthesis and characterization of two alpha4/7-conotoxins, Acta biochimica

et biophysica Sinica 42, 745-753.

17. Luo, S., Nguyen, T. A., Cartier, G. E., Olivera, B. M., Yoshikami, D., and McIntosh,

J. M. (1999) Single-residue alteration in alpha-conotoxin PnIA switches its nAChR

subtype selectivity, Biochemistry 38, 14542-14548.

18. Luo, S., Kulak, J. M., Cartier, G. E., Jacobsen, R. B., Yoshikami, D., Olivera, B. M.,

and McIntosh, J. M. (1998) alpha-conotoxin AuIB selectively blocks alpha3 beta4

nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release, The

Journal of neuroscience : the official journal of the Society for Neuroscience 18,

8571-8579.

19. Azam, L., Dowell, C., Watkins, M., Stitzel, J. A., Olivera, B. M., and McIntosh, J. M.

(2005) Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes

among neuronal nicotinic acetylcholine receptors, The Journal of biological chemistry

280, 80-87.

20. Ellison, M., Haberlandt, C., Gomez-Casati, M. E., Watkins, M., Elgoyhen, A. B.,

McIntosh, J. M., and Olivera, B. M. (2006) Alpha-RgIA: a novel conotoxin that

specifically and potently blocks the alpha9alpha10 nAChR, Biochemistry 45, 1511-

1517.

21. Ulens, C., Hogg, R. C., Celie, P. H., Bertrand, D., Tsetlin, V., Smit, A. B., and Sixma,

T. K. (2006) Structural determinants of selective alpha-conotoxin binding to a

nicotinic acetylcholine receptor homolog AChBP, Proceedings of the National

Academy of Sciences of the United States of America 103, 3615-3620.

122

22. Dutertre, S., Ulens, C., Buttner, R., Fish, A., van Elk, R., Kendel, Y., Hopping, G.,

Alewood, P. F., Schroeder, C., Nicke, A., Smit, A. B., Sixma, T. K., and Lewis, R. J.

(2007) AChBP-targeted alpha-conotoxin correlates distinct binding orientations with

nAChR subtype selectivity, The EMBO journal 26, 3858-3867.

23. Millard, E. L., Daly, N. L., and Craik, D. J. (2004) Structure-activity relationships of

alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors, European

journal of biochemistry / FEBS 271, 2320-2326.

24. Millard, E. L., Nevin, S. T., Loughnan, M. L., Nicke, A., Clark, R. J., Alewood, P. F.,

Lewis, R. J., Adams, D. J., Craik, D. J., and Daly, N. L. (2009) Inhibition of neuronal

nicotinic acetylcholine receptor subtypes by alpha-Conotoxin GID and analogues, The

Journal of biological chemistry 284, 4944-4951.

25. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S., and Patrick, J.

(1987) Functional expression of two neuronal nicotinic acetylcholine receptors from

cDNA clones identifies a gene family, Proceedings of the National Academy of

Sciences of the United States of America 84, 7763-7767.

26. Lukas, R. J., Changeux, J. P., Le Novere, N., Albuquerque, E. X., Balfour, D. J., Berg,

D. K., Bertrand, D., Chiappinelli, V. A., Clarke, P. B., Collins, A. C., Dani, J. A.,

Grady, S. R., Kellar, K. J., Lindstrom, J. M., Marks, M. J., Quik, M., Taylor, P. W.,

and Wonnacott, S. (1999) International Union of Pharmacology. XX. Current status of

the nomenclature for nicotinic acetylcholine receptors and their subunits,

Pharmacological reviews 51, 397-401.

27. Wang, F., Gerzanich, V., Wells, G. B., Anand, R., Peng, X., Keyser, K., and

Lindstrom, J. (1996) Assembly of human neuronal nicotinic receptor alpha5 subunits

with alpha3, beta2, and beta4 subunits, The Journal of biological chemistry 271,

17656-17665.

123

28. Clark, R. J., Fischer, H., Nevin, S. T., Adams, D. J., and Craik, D. J. (2006) The

synthesis, structural characterization, and receptor specificity of the alpha-conotoxin

Vc1.1, The Journal of biological chemistry 281, 23254-23263.

124

CHAPTER 5

Novel αD-conotoxin GeXXA from Conus generalis

reveals unique nAChR binding mechanism

Conus generalis

125

5.1 INTRODUCTION

As mentioned earlier (Chapter 1, Section 2), various bioactive peptides have been identified

from the venom of cone snails belonging to the genus Conus, labelled conotoxins. The

disulphide-rich peptides targeting nAChRs are grouped into various superfamilies, based on

their structural and functional properties: the A- (α- and αA-conotoxins), M- (ψ-conotoxins),

S- (αS-conotoxins) and C-conotoxins (αC-conotoxins) [1].

Conotoxins from the D-superfamily were only recently discovered and functionally

characterised [2, 3]. First isolated from the venom C. vexillum, three novel αD-conotoxins,

αD-VxXIIA, αD-VxXIIB and αD-VxXIIC, were functionally characterised as potent

inhibitors of α7- and β2-containing neuronal nAChRs [2]. Kauferstein et al. (2009) identified

and analysed two new αD-conopeptides from the vermivorous snails Conus mustilinus (αD–

M) and Conus capitaneus (αD–Cp). These peptides have a pharmacological profile similar to

that of αD-VxXIIA with a 72% sequence homology [Table 5.1] [4].

In the present study, we identified novel αD-conotoxin GeXXA from the venom of Conus

generalis. Functional characterisation revealed αD-GeXXA non-selectively inhibits nAChRs,

an action not previously identified in αD-conotoxins, which usually exhibit characteristic

subtype selectivity. We also report the first synthesis of a functionally active monomeric αD-

GeXXA isoform.

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Table 5.1: Sequence alignment of αD-conotoxin GeXXA with other, previously discovered αD-conotoxins.

αD-Conotoxin Sequence nAChR selectivity Reference GeXXA DVH-RPCQSVRPGRVWGKCCLTRLCSTMCCARADCTCVYHTWRGHGCSCV- α7>α3*>αβδγ This study Ms20.3 DVR--ECQVNTPGSSWGKCCMTRMCGTMCCARSGCTCVYHWRRGHGCSCPG α7>β2* [4, 5] Cp20.3 EVQ--ECQVDTPGSSWGKCCMTRMCGTMCCSRSVCTCVYHWRRGHGCSCPG α7>β2* [4, 5] VxXXA D--VQDCQVSTPGSKWGRCCLNRVCGPMCCPASHCYCVYHRGRGHGCSC-- α7>α3β2 [2] VxXXB DDE-SECIINTRDSPWGRCCRTRMCGSMCCPRNGCTCVYHWRRGHGCSCPG α7>β2*>αβδγ [2] VxXXC DLR--QCTRNAPGSTWGRCCLNPMCGNFCCPRSGCTCAYNWRRGIYCSC-- α7>β2* [2]

127

5.2 AIMS

5.2.1 Characterisation of αD-conotoxin GeXXA isolated from Conus generalis

To determine the pharmacological potency and selectivity of dimeric and

monomeric GeXXA at recombinant nAChR subtypes expressed in

Xenopus oocytes using the two-electrode voltage clamp technique.

5.2.2 Receptor hybrid studies

To identify the site where dimeric and monomeric αD-conotoxin GeXXAs

bind with nAChRs, and the molecular mechanism behind their inhibition

of nAChRs.

5.2.3 Significance

This study, in conjunction with modelling simulations our collaborators

are doing, could shed light on the molecular mechanism behind the αD-

conotoxins interaction with nAChR subtypes.

αD-conotoxins represent a novel class of nAChR-inhibiting peptides as

potential neurophysiological tools and drug therapeutics. This study could

also provide valuable information that could aid novel peptide design and

the development of future drugs.

128

5.3 RESULTS

5.3.1 Concentration-dependent inhibition of α3-containing nAChRs by αD-conotoxin

GeXXA

To examine the pharmacological activity of αD-conotoxin GeXXA at nAChRs, it was tested on

ACh-evoked currents mediated by different nAChR subtypes expressed in Xenopus oocytes.

GeXXA (1 µM) had no or little effect on α4β2 and α4β4 subtypes, but inhibited α3β4 and

muscle (α1β1εγ) by 70–80%, and inhibited α9α10, α7 and α3β2 subtypes completely [Figure

5.1(A)].

Concentration–response curves showed GeXXA was more selective for α3-containing than

α4-containing nAChR subtypes [Figure 5.1(B)]. It was most potent at the α7 nAChR subtype,

with an IC50 of 210 nM (95% CI, 174–253) and Hill slope (nH) of –1.6 ± 0.2. The IC50 (95%

CI) and Hill slope values from concentration–response curves for the inhibition of various

nAChR subtypes are summarised in Table 5.2.

Table 5.2: Pharmacological profile of dimeric αD-conotoxin GeXXA inhibition of

various nAChRs.

nAChR subtype IC50 (95% CI) nH

α7 210 nM (174–253) –1.6 ± 0.2 α3β2 498 nM (407–609) >> –1.0 α3β4 614 nM (491–768) –2.1 ± 0.4 α4β2 > 3 µM – α4β4 > 3 µM –0.9 ± 0.2 αβδγ 743 nM (606–911) –1.6 ± 0.2

All data points indicate mean ± SEM; n = 4–7.

129

Figure 5.1: Dimeric αD-conotoxin GeXXA inhibition of various nAChR subtypes. (A)

Superimposed current traces obtained in the absence (control) and presence of 300 nM of

dimeric αD-conotoxin GeXXA inhibition at the (i) α7 nAChR subtype and (ii) α3β2 nAChR

subtype. (B) Concentration–response curves obtained for dimeric αD-conotoxin GeXXA

inhibition of nAChR subtypes. Dimeric GeXXA (3 µM) non-selectively inhibited all nAChR

subtypes, except α4β2. The highest potency of inhibition was observed at the α7 nAChR

subtype. The IC50 (95% CI) and Hill slope (nH) values from concentration–response curves

are summarised in Table 5.2. All data points represent the mean ± SEM; n = 4–7.

130

5.3.2 Monomeric αD-conotoxin GeXXA selectively inhibits the α9α10 nAChR subtype

We describe for the first time monomeric αD-conotoxin GeXXA inhibition of nAChRs. When

tested on various nAChR subtypes, 1 µM of monomeric GeXXA completely inhibited the

α9α10 nAChR subtype, but had little or no activity at other nAChR subtypes. This

antagonism of α9α10 was also reversible [Figure 5.2(A)], unlike dimeric αD-conotoxin

GeXXA which irreversibly inhibited α9α10 subtype [data not shown]. The concentration–

response curve obtained for the monomeric GeXXA at α9α10 nAChR gave an IC50 of 198

nM (95% CI, 164–238; nH = –1.7 ± 0.3) [Figure 5.2(B)].

Figure 5.2: α9α10 hybrid nAChR inhibition by monomeric αD-conotoxin GeXXA. (A)

Superimposed ACh-evoked currents obtained in the absence (control) and presence of 1 µM

of monomeric αD-contoxin GeXXA at human and rat α9α10 nAChR subtype. (B) Monomeric

αD-conotoxin GeXXA was 10-fold less potent at the human α9α10 nAChR subtype than at

the rat α9α10 nAChR subtype, whereas no change was observed at the hybrid hα9rα10

receptor. All data points indicate mean ± SEM; n = 4–7.

131

5.3.3 α9α10 hybrid nAChR studies reveal the site of monomeric αD-conotoxin GeXXA

binding

The difference between dimeric and monomeric αD-conotoxin GeXXA inhibition of the

α9α10 nAChR subtype was further investigated on hybrid receptors using human and rat α9

and α10 nAChR subunits. When initially tested on human α9α10, monomeric α-D GeXXA

was 10-fold less potent compared with rat α9α10. However, similar potency was observed on

the hybrid hα9rα10 receptor to that of rat α9α10 subtype [Figure 5.2(B)].

Concentration–response curves for monomeric αD-conotoxin GeXXA inhibition of rat, hybrid

and human α9α10 nAChR subtypes gave IC50 values of 198 nM (95% CI, 164–238; nH = –1.7

± 0.3), 224 nM (95% CI, 194–258; nH = –1.4 ± 0.1) and 2.02 µM (95% CI, 1.82–2.25; nH = –

1.7 ± 0.1), respectively. All data points represent the mean ± SEM; n = 4–7.

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5.4 DISCUSSION AND CONCLUSION

Conotoxins are invaluable pharmacological tools, each with unique structural and functional

features. αA-Conotoxins consist of up to 30 amino acid residues and generally characterised

by their competitive antagonism of muscle nAChRs, while ψ-Conotoxins are known to be

non-competitive muscle nAChR antagonists [6].

While there have been significant advances in developing our understanding of nAChRs,

much of their physiological role and pharmacology of these unique peptides on nAChRs is

still unknown.

D-superfamily conotoxins consist of 45–50 amino acid residues and have characteristic 10

cysteine residue framework [Table 5.1] [5]. They have various structural and functional

features. First, several post-translational modifications, such as carboxylation and

hydroxylation, are seen in native peptides. Second, native peptides occur as homodimers

connected by disulphide bonds [4]. αD-conotoxin GeXXA is a disulphide-linked homodimer

of two identical peptides and consists of 50 amino acid residues with 10 cysteines [Figure

5.3].

Figure 5.3: The sequence and disulphide linkage of αD-conotoxin GeXXA. For clarity,

only the N-terminal sequence of the second subunit is shown.

133

Previous pharmacologically characterised αD-conotoxins were shown to be non-competitive

muscle and neuronal nAChR antagonists, with selectivity for α7- and β2-containing receptors

[2]. However, even though each GeXXA peptide chain shared high homology with other

known αD-conotoxins [Table 5.1], αD-GeXXA exhibited concentration-dependent inhibition

of various nAChR subtypes, with selectivity for α7 and α3-containing nAChRs. αD-GeXXA

inhibits α7 (IC50 210 nM), α3β2 (IC50 498 nM), α3β4 (IC50 614 nM) and muscle (α1β1δγ)

(IC50 743 nM) nAChR subtypes, and weakly inhibits α4β2 and α4β4 nAChR subtypes (IC50 >

3 µM). However, its inhibition of both rat and human α9α10 nAChR subtypes was

irreversible, indicating that it binds tightly to these receptors.

The crystal structure of native αD-GeXXA [contributed to this study by collaborators]

provided further insight into its unique pharmacological activity and mechanisms of action.

Structures at 1.5 Ǻ resolution revealed an N-terminal domain (NTD, residues 1–20) and a C-

terminal domain (CTD, residues 21–50) in each peptide chain, arranged in an approximately

2-fold symmetric architecture. An interchain disulphide bond between Cys6 of one chain and

Cys18 of another chain within the NTD facilitates αD-GeXXA dimerisation [Figure 5.4].

Figure 5.4: The crystal structure of αD-conotoxin GeXXA. The NTDs of two GeXXA

subunits are shown in green and light green. The CTDs are represented in orange and light

orange. The disulphide bonds are colored yellow. [Provided to this study by collaborators].

134

We report the first synthesis of αD-GeXXA’s C-terminal domain (CTD) and its activity. The

isolated CTD adopts a canonical inhibitory cysteine knot (ICK) disulphide linkage. When

tested on various nAChR subtypes, αD-GeXXA-CTD selectively and reversibly antagonises

the rat α9α10 nAChR subtype, with an IC50 of 198 nM, but is 10-fold less potent at the human

α9α10 nAChR subtype (IC50 2.02 µM). Hybrid receptor studies indicate that the α10 nAChR

subunit contributes to the difference between αD-GeXXA’s potency for human and rat α9α10

nAChR subtype.

These results indicate that the α10 nAChR subunit is a major binding site for αD-conotoxin

GeXXA. Peptide sequence alignment revealed residue differences between the extracellular

domains of mature human and rat α10 subunits [Figure 5.5]. Molecular dynamics simulations

[contributed to this study by collaborators] indicate a two-site binding interaction between

αD-GeXXA and the His7 of the α10 subunit. Recently, similar hybrid-receptor studies of the

α9α10 nAChR subtype found that α-conotoxin Vc1.1 and RgIA preferentially bound to the

α10α9 pocket [3, 7]. These findings are also supported by electrophysiological data that show

Hill slope values > 1 for αD-GeXXA interaction with various nAChRs, which indicates a

positive cooperative binding mode. The Hill equation has been extensively used to aid

understanding of the complex pharmacokinetic and pharmacodynamic models of drug–

receptor interaction [8-10].

This study is the first to report monomeric αD-conotoxin activity, and gives insight into αD-

conotoxin GeXXA’s novel binding mechanism at nAChRs.

135

Figure 5.5: Pairwise sequence alignment of the ACh-binding site of the mature α10 subunit peptide. This alignment reveals the difference

between human and rat residues (background white). The mature α10 subunit peptide lacks the signal sequence (first 25 residues). Peptide sequences

were obtained from the NCBI Protein online database.

136

5.5 References

1. Arias, H. R., and Blanton, M. P. (2000) Alpha-conotoxins, International Journal of

Biochemistry & Cell Biology 32, 1017-1028.

2. Loughnan, M., Nicke, A., Jones, A., Schroeder, C. I., Nevin, S. T., Adams, D. J.,

Alewood, P. F., and Lewis, R. J. (2006) Identification of a novel class of nicotinic

receptor antagonists: dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus

vexillum, Journal of Biological Chemistry 281, 24745-24755.

3. Azam, L., and McIntosh, J. M. (2012) Molecular basis for the differential sensitivity

of rat and human alpha9alpha10 nAChRs to α-conotoxin RgIA, Journal of

Neurochemistry 122, 1137-1144.

4. Kauferstein, S., Kendel, Y., Nicke, A., Coronas, F. I., Possani, L. D., Favreau, P.,

Krizaj, I., Wunder, C., Kauert, G., and Mebs, D. (2009) New conopeptides of the D-

superfamily selectively inhibiting neuronal nicotinic acetylcholine receptors, Toxicon

54, 295-301.

5. Loughnan, M. L., Nicke, A., Lawrence, N., and Lewis, R. J. (2009) Novel α D-

conopeptides and their precursors identified by cDNA cloning define the D-conotoxin

superfamily, Biochemistry 48, 3717-3729.

6. Jacobsen, R., Yoshikami, D., Ellison, M., Martinez, J., Gray, W. R., Cartier, G. E.,

Shon, K. J., Groebe, D. R., Abramson, S. N., Olivera, B. M., and McIntosh, J. M.

(1997) Differential targeting of nicotinic acetylcholine receptors by novel αA-

conotoxins, Journal of Biological Chemistry 272, 22531-22537.

7. Yu, R., Kompella, S. N., Adams, D. J., Craik, D. J., and Kaas, Q. (2013)

Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic

acetylcholine receptor, Journal of Medicinal Chemistry 56, 3557-3567.

137

8. Goutelle, S., Maurin, M., Rougier, F., Barbaut, X., Bourguignon, L., Ducher, M., and

Maire, P. (2008) The Hill equation: a review of its capabilities in pharmacological

modelling, Fundamental & clinical pharmacology 22, 633-648.

9. Prinz, H. (2010) Hill coefficients, dose-response curves and allosteric mechanisms,

Journal of Chemical Biology 3, 37-44.

10. Weiss, J. N. (1997) The Hill equation revisited: uses and misuses, FASEB Journal 11,

835-841.

138

CHAPTER 6

Dicarba modification of α-conotoxins exhibits differential selectivity

for nAChRs and GABAB receptors

Conus victoriae

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6.1 INTRODUCTION

6.1.1 Conotoxins - Conus victoriae and Conus regius

α-Conotoxins in the venom of the marine cone snails, are short disulphide-bonded peptides

ranging from 12–30 amino acids. These peptides specifically target nAChR subtypes and have

potential therapeutic applications. Conus victoriae and Conus regius venoms gained a special

interest, due to identification of two novel peptides, α-conotoxins Vc1.1 and RgIA,

respectively. These peptides have exhibited analgesic properties in rat neuropathic pain

models [1, 2].

α-Conotoxins Vc1.1 and RgIA belong to the cysteine framework I family (CCXnCXmC) and

exist in a globular conformation with I–III and II–IV disulphide connectivity in native form

[3]. Both of these peptides were first identified using a cDNA approach [4, 5]. Vc1.1 is 16

amino acids in length and belongs to the α4/7 subclass of conotoxins. Like most conotoxins,

Vc1.1 exhibited various post-translational modifications, including C-terminus amidation,

hydroxylation of Pro6 and γ-carboxylation of Glu14 residues. This modified peptide, called

vc1a, was identified in the venom of Conus victoriae and is the native form of Vc1.1 [6]. On

the other hand, RgIA is an α4/3 conotoxin, consisting of 12 amino acids, and lacks post-

translational modification.

6.1.2 The molecular mechanism of analgesia

The involvement of nAChRs in pain pathways is now well established [7]. However, the role

of nAChR subtypes in pain pathways and the molecular mechanism of these pathways are yet

to be elucidated. Various small molecule compounds acting as α4β2 and α7 nAChR

antagonists are the primary leads for pain therapeutics [8]. However, the identification of α-

conotoxins as nAChR subtype-selective probes has led to the development of novel drugs to

140

treat neuropathic pain [9]. A major breakthrough came with the discovery of α-conotoxins

Vc1.1 and RgIA, which have acute and cumulative anti-nociceptive effects in chronic

constriction nerve injury (CCI) and partial sciatic nerve ligation (PSNL) rat models of

neuropathic pain [1, 2]. Subcutaneous or intramuscular administration of these peptides

alleviated mechanical hyperalgesia and allodynia. This effect lasted up to 24 h post-

administration without the rats developing a tolerance to the drug. In addition, Vc1.1’s

analgesic effects lasted up to one week after treatment was ceased [10].

The pharmacological profiles of Vc1.1 and RgIA were only established three years after their

analgesic properties were identified. Initial electrophysiological studies using synthetic Vc1.1

confirmed the α3β4 nAChR subtype is its pharmacological target, with an IC50 of 3 µM [11].

However, concurrent studies revealed Vc1.1 and RgIA also potently inhibit the α9α10 nAChR

subtype, with an IC50 of 64 nM and 5 nM, respectively [4, 12]. These studies suggested the

α9α10 nAChR subtype as a novel therapeutic target for neuropathic pain.

Recent studies have also uncovered a new biological target for both of these peptides. Vc1.1

and RgIA inhibit high voltage-activated (HVA) calcium channel currents via GABAB

receptor activation, which suggests these α-conotoxins may mediate a novel pain pathway

[13-15].

6.1.3 The α9α10 nAChR subtype: expression and function

Of the various nAChR subunits, only the α7 and α9 subunits form homopentamers, and the

α10 subunit assembles only with the α9 subunit to form functional receptors [16]. Co-

assembly with the α10 subunit also significantly increases the expression of α9α10 nAChRs

[17]. α9 and α10 subunits are expressed in various tissues, such as skin, dorsal root ganglia

(DRG), pars tuberalis of the pituitary gland, and cochlear hair cells [10]. In the auditory

141

system, which is composed of cochlear hair cells, α9-containing receptors mediate the

synaptic transmission, and therefore modulate auditory stimuli [18]. In skin, α9 and α10

subunits regulate the cell-adhesion properties of keratinocytes and modulate wound-healing

(re-epithelialisation) [19, 20]. While α9 and α10 subunits are implicated in cholinergic

signalling in the above-mentioned tissues, their precise subunit stoichiometry is yet to be

determined. Although α9 can form a functional homomeric receptor, the expression of α10

subunit was found to be necessary for synaptic transmission in cochlear cells [21].

Furthermore, Plazas et al. (2005) showed that α9α10 when recombinantly expressed in

oocytes, stoichiometry of (α9)2(α10)3 was observed [22].

6.1.4 α-Conotoxin drug development: limitations and strategies

The nature of the compound is an important aspect of drug development, because it

determines the drug’s side effects. While the peptidic nature of α-conotoxins is a unique

advantage for drug development, it is also their Achilles’ heel. α-Conotoxins are susceptible

to natural peptidic degradation, which drastically affects their bioavailability and half-life.

The functional impediment of α-conotoxins via peptidic-degradation can result from a

proteolytic attack on the N- and/or the C-terminus of the peptide, or from disruption

(scrambling) of the disulphide linkage within the α-conotoxin.

Various strategies to improve α-conotoxin stability have been implemented, such as

cyclisation and selenocysteine modification. Peptide cyclisation has been shown to counter

the proteolytic degradation problem in Vc1.1, RgIA and other peptides [23, 24]. The α-

conotoxin three-dimensional structure is dominated by a helical structure shaped by a

conserved disulphide framework. The most predominant I–III and II–IV disulphide

connectivity is found in native α-conotoxins and folds the peptide into globular conformation.

However, the disulphide connectivity of α-conotoxins can interchange under oxidative and

142

reduced conditions to form ribbon (I–IV and II–III disulphide bonds) or bead (I–II and III–IV

disulphide bonds) conformations.

Peptide exploitation in research and clinical settings has also been hampered by the lability of

the disulphide bridges that are essential for conotoxin structure and activity [25]. Alteration of

disulphide linkage by methods such as selenocysteine modification broke new ground in drug

development [26]. Replacing the cysteine bridges with non-reducible dicarba links has also

been identified as a novel solution to the lability problem. It was shown to significantly

improve α-conotoxin ImI stability, while its functional activity remained comparable with that

of the native peptide.

Here, I explore the functional implications of this approach on novel analgesic α-conotoxin

Vc1.1 and RgIA, which inhibit HVA calcium channel currents via GABAB receptor

activation and α9α10 and α3β4 nAChR subtypes.

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6.2 AIMS

6.2.1 Characterisation of dicarba modified α-conotoxins Vc1.1 and RgIA.

To functionally characterise regioselective dicarba analogues of α-

conotoxins Vc1.1 and RgIA using two-electrode voltage-clamp technique

in Xenopus oocytes expressing recombinant nAChR subtypes.

6.2.2 Significance

This study, in conjunction with structural studies by our collaborators,

could provide a detailed understanding of the functional implications of

dicarba modification on the Vc1.1 and RgIA activity at nAChRs.

It could also provide valuable information about the development of

dicarba peptides as novel drugs to treat neuropathic pain.

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6.3 RESULTS

6.3.2 Regioselective dicarba Vc1.1 analogues exhibit differential activity at α9α10

nAChR subtypes

The functional effects of dicarba modification on Vc1.1 was tested on recombinant α9α10 and

α3β4 nAChR subtypes heterogeneously expressed in Xenopus oocytes using the two-electrode

voltage-clamp technique. When tested, cis- and trans-[3,16]-dicarba Vc1.1 analogues (3 µM)

inhibited the α9α10 nAChR subtype by 24% and 54%, respectively, but the [2,8]-dicarba

Vc1.1 analogue showed no inhibitory effects [Figure 6.1(A)]. A similar trend was observed

for these peptides at the α3β4 nAChR subtype [Figure 6.1(B)].

Figure 6.1: Percentage inhibition of ACh-evoked currents by dicarba Vc1.1 analogues (3

µM) at α9α10 (A) and α3β4 (B) nAChR subtypes. All data represent the mean ± SEM; n ≥

3.

A concentration–response analysis gave an IC50 of 2.8 µM (95% CI, 2.0–4.1 µM) and a Hill

slope of –1.3 ± 0.3 (n = 4) for trans-[3,16]-dicarba Vc1.1. The cis isomer was ~ five-fold less

active, with an IC50 of 12.5 µM (95% CI, 5.6–27.9 µM) and a Hill slope of –0.8 ± 0.2 (n = 4)

[Figure 6.2(C)].

145

Figure 6.2: Dicarba Vc1.1 analogues concentration-dependent inhibition of ACh-evoked

currents at the α9α10 nAChR subtype. Superimposed ACh (50 µM)-evoked current traces

mediated by (A) α9α10 and (B) α3β4 nAChR subtypes in the absence (control) and presence

of 3 µM of [3,16]-trans-dicarba Vc1.1 and [3,16]-cis-dicarba Vc1.1. (C) Concentration–

response curves for Vc1.1 and dicarba Vc1.1 analogue inhibition of the α9α10 nAChR

subtype. All data represents mean ± SEM; n ≥ 4.

6.3.2 Dicarba modification of RgIA confers similar pharmacological effects to those of

Vc1.1

In this study, each of the peptides was compared with native RgIA on α9α10 and α7 nAChRs

expressed in Xenopus oocytes. Native RgIA reversibly inhibited ACh-evoked currents

mediated by α9α10 and α7 nAChRs in a concentration-dependent manner, with an IC50 of 5.5

nM (nH = −1.3) and 3.3 µM (nH = −0.9), respectively [4].

146

At the α9α10 nAChR subtype, cis- and trans-[3,12]-dicarba RgIA analogues (3 µM each)

inhibited ACh-evoked currents by 35.4 ± 1.5% and 31.5 ± 2.1% (n = 3), respectively [Figure

6.3(A)(i)]. The cis isomer exhibited an IC50 of 1.15 µM (95% CI, 0.84–1.55 µM) and a Hill

slope of −1.9 ± 0.4 (n = 3). The trans isomer was 1.3-fold less active, with an IC50 of 1.47 µM

(95% CI, 1.01–2.15 µM) and a Hill slope of −1.2 ± 0.2 (n = 3) [Figure 6.3(B)].

In contrast, at the α7 nAChR subtype, cis-[3,12]-dicarba RgIA inhibition of ACh-evoked

currents was similar to that of native RgIA, with an IC50 of 3.73 µM (95% CI, 1.42–9.82 µM)

and a Hill slope of −1.6 ± 0.6 [Figure 6.3(B)]. The trans-isomer was inactive at α7 when

tested at a concentration of 3 µM [Figure 6.3(A)(ii)]. Replacing the [2,8]-cystine bridge

significantly abolished nAChR activity, with both the cis- and trans-[2,8]-dicarba RgIA

inactive when tested at 10 µM at the α9α10 nAChR subtype [data not shown].

147

Figure 6.3: [3,12]-Dicarba RgIA analogue inhibition of ACh-evoked currents at rat

α9α10 and human α7 nAChRs expressed in Xenopus oocytes. (A) (i) Superimposed ACh

(50 µM)-evoked currents mediated by α9α10 nAChRs in the absence (control) and presence

of 3 µM each of cis-[3,16]-dicarba RgIA and trans-[3,16]-dicarba RgIA. (ii) Superimposed

ACh (200 µM)-evoked currents at α7 nAChRs in the absence (control) and presence of 3 µM

each of cis-[3,16]-dicarba RgIA and trans-[3,16]-dicarba RgIA. (B) Concentration–response

curves for cis-[3,12]-dicarba RgIA gave an IC50 of 1.15 µM (95% CI, 0.84–1.55 µM) and a

Hill slope of −1.9 ± 0.4 at the α9α10 nAChR. trans-[3,12]-dicarba RgIA gave an IC50 of 1.47

µM (95% CI, 1.01–2.15 µM) and a Hill slope of −1.2 ± 0.2 at the α9α10 nAChR. At the α7

nAChR subtype, cis-[3,12]-dicarba RgIA had an IC50 of 3.73 µM (95% CI, 1.42–9.82 µM)

and a Hill slope of −1.6 ± 0.6, but trans-[3,12]-Dicarba RgIA was inactive. All data represent

mean ± SEM; n ≥ 3.

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6.4 DISCUSSION

6.4.1 Dicarba modification imparts differential pharmacological selectivity to α-

conotoxins Vc1.1 and RgIA

Dicarba modification of the disulphide bridge imparted differential and significant changes to

the pharmacological profile of α-conotoxins Vc1.1 and RgIA. Dicarba modification of the

Cys2–Cys8 disulphide bond in Vc1.1 and RgIA, led to complete loss of activity at nicotinic

receptors. Both peptides also lost appreciable activity at the α9α10 nAChR subtype when the

Cys3–Cys16 disulphide bond in Vc1.1 and Cys3–Cys12 disulphide bond in RgIA were

replaced with dicarba linkage. The [3, 12]-dicarba RgIA analogues are more potent than the

corresponding dicarba Vc1.1 analogues, and their IC50 change was more prominent than the

native peptide. NMR structural analysis [experiments carried out by our collaborators;

data not shown] of these peptides shows a clear perturbation of the three dimensional

structure of [2,8]-trans-dicarba Vc1.1 and RgIA analogues, which contributes to its loss of

activity at nAChR subtypes.

While previous point mutation studies of Vc1.1 and RgIA revealed the key residues

interacting with the α9α10 nAChR subtype, this study and molecular dynamics simulations

[experiments conducted by our collaborators; data not shown] propose significant

interaction between the peptides’ disulphide bonds and ACh-binding pocket. Our

collaborators’ molecular docking studies revealed disulphide stacking interaction between the

Cys2–Cys8 bond and disulphide of the C-loop of the principal subunit. This interaction was

shown to be important for α4/7 conotoxin PnIA[A4L,D14K] bound to the AChBP [27]. This

model is corroborated by the complete loss of activity of the [2,8]-dicarba-modified Vc1.1

and RgIA peptides. It also explains the significant variance in peptide function, whereas NMR

studies [conducted by our collaborators] show no significant perturbation except for trans-

149

[2,8]-dicarba Vc1.1. Furthermore, when tested, these peptide analogues exhibited opposite

effects on their corresponding biological targets. [2,8]-Dicarba Vc1.1 analogues inhibited

HVA calcium channel currents via GABAB receptor activation in rat DRG neurons, but

[3,16]-dicarba Vc1.1 peptides were inactive [results published] [28]. Similar results were

also observed with RgIA dicarba analogues [experiments conducted by our collaborators;

data not shown]. These results suggest that the CysII–CysIV disulphide bond may interact

with GABAB receptors in a similar way to that in which the CysI–CysIII bond interact with

nAChRs.

6.4.2 Molecular determinants of Vc1.1 and RgIA pharmacological selectivity

Unlike other nAChR subtypes, α9α10 has very unique pharmacological properties, which are

yet to be fully understood. Various compounds, such as nicotine, cytisine and epibatine,

which are agonists of α7 and other heteromeric receptors, antagonise the α9α10 nAChR

subtype [10]. Furthermore, inhibition of α9α10 nAChR by non-cholinergic antagonists, such

as strychnine (glycine receptor antagonist), represents a distinctive and mixed

pharmacological property for this receptor subtype [17].

Vc1.1 emerged as novel drug to treat neuropathic pain after various studies showed it’s

cumulative and long-lasting alleviation of hyperalgesia and allodynia. However, drug

development of Vc1.1 was stopped at phase 2A of the human clinical trials, because potencies

at rat and human α9α10 nAChR subtype inhibition differed significantly [29]. Various studies

that aimed to explain the molecular determinants of Vc1.1 and RgIA identification of their

pharmacological target, the α9α10 nAChR subtype, were later conducted [3]. Point mutational

analyses of RgIA revealed that region 5–7 interacts with the α9α10 binding pocket [30].

Similar studies using Vc1.1 showed that as well as region 5–7, a second region, 11–15, is

needed for Vc1.1 activity at the α9α10 subtype [31]. This study also showed significantly

150

increased potency at rat and human α9α10 subtypes when Asn9 was changed to hydrophobic

residues in Vc1.1 [31].

These studies had two significant effects on the development of Vc1.1 and RgIA as drug

leads. Firstly, they elucidated the molecular mechanism of inhibition at α9α10, providing vital

information about the structure–function relationship between these two α-conotoxin classes.

Secondly, peptide analogues developed in these studies with increased activity at α9α10

subtype provided new lead compounds to aid in drug development.

6.5 SUMMARY AND CONCLUSION

α-Conotoxins are subtype-selective nAChR antagonists and established leads for drugs to

treat various pain conditions [23]. Here, I have outlined various studies that contributed to our

understanding of the molecular pharmacology of Vc1.1 and RgIA at α9α10 and GABAB

receptors. These studies have significantly progressed development of these novel α-

conotoxins as pain therapeutics. I have also successfully identified the potential use of dicarba

modification of these peptides to improve their bioavailability and half-life, and maintain their

biological activity. My results provide vital information about the molecular mechanism of

peptide inhibition of the α9α10 nAChR subtype. Together with previous mutational studies,

synthesis and functionally characterisation of new dicarba analogues that aim to improve

peptide potency are being carried out.

151

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alpha-Conotoxin Vc1.1 Analogues with Differential Selectivity for Nicotinic

Acetylcholine and GABAB Receptors, ACS chemical biology 8, 1815-1821.

29. Metabolic. (2007) Metabolic discontinues clincal trial programme for neuropathic pain

drug, ACV1.

30. Ellison, M., Feng, Z. P., Park, A. J., Zhang, X., Olivera, B. M., McIntosh, J. M., and

Norton, R. S. (2008) Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10

nAChR: structure and identification of key receptor-binding residues, Journal of

molecular biology 377, 1216-1227.

31. Halai, R., Clark, R. J., Nevin, S. T., Jensen, J. E., Adams, D. J., and Craik, D. J. (2009)

Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at

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the alpha9alpha10 nicotinic acetylcholine receptor, The Journal of biological

chemistry 284, 20275-20284.

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CHAPTER 7

Conclusion & future directions

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Nicotinic research has significantly grown over the past 30 years. [1-3]. However, there are

still numerous challenges and questions to be answered that are related to these ligand-gated

channels [4]. There has also been a prominent shift in the direction of research into nAChRs.

While previous studies focused on nAChR structure and function, present research aims to

understand the physiological roles played by various nAChR subtypes [5, 6]. Today, nAChR

subtypes are implicated in numerous neuronal and non-neuronal diseases, which further

indicate their vital physiological role [1, 7].

α-Conotoxins, a diverse group of peptides that act as subtype-specific nAChR antagonists, are

the ideal tool to identify the role played by nAChRs in various physio- and pathophysiological

processes. Developments in molecular techniques and peptide chemistry have exponentially

increased the number of α-conotoxins being discovered [8]. However, out of the 700 cone

snail species, each with 100s of α-conotoxins, only 0.1% have been functionally characterised

to date [9, 10]. There are also various challenges in the clinical exploitation of these peptides.

In this thesis, the collaborative studies identify and tackle some of the pressing issues related

to the therapeutic development of conotoxins, such as nAChR subtype-specific ligand potency

and selectivity, and α-conotoxin stability under physiological conditions.

α-Conotoxin AuIB is the only peptide known to selectively target the α3β4 nAChR subtype

with an IC50 of 2.5 µM [11]. In this thesis, I successfully characterised a novel α4/7 conotoxin

RegIIA, isolated from the venom of Conus regius. This peptide, although active at α3β2 and

α7 nAChRs, potently inhibited the α3β4 nAChR subtype, making it a significant discovery.

RegIIA is only the fifth α-conotoxin known to target the α3β4 nAChR subtype. Other

characterised α-conotoxins known to inhibit the α3β4 nAChR subtype are BuIA, AuIB, PIA

and PeIA [12]. Unlike α-conotoxin AuIB, which belongs to a unique α4/6 subclass and

exhibits distinct sequence, RegIIA and other α-conotoxins exhibit high-sequence homology.

158

This made RegIIA an ideal candidate to help us identify and understand the molecular

determinants governing its α3β4 nAChR subtype antagonism. Using alanine scanning

mutagenesis, I not only identified the critical residues that interact with each of the nAChR

subtypes, but also successfully synthesised an analogue that potently and selectively targets

the α3β4 nAChR subtype. This will be invaluable in deciphering the physiological role played

by the α3β4 nAChR subtype, and for the design and development of α3β4-selective drugs to

treat lung cancer and nicotine addiction.

In this thesis, I characterised another α-conotoxin, LsIA, isolated from Conus limpusi. This

peptide was of special interest, since it contained a unique N-terminal serine amino acid in

addition to a glycine, which is found in almost all conotoxins. To date, very few α-conotoxins

have been found to exhibit this N-terminal sequence [12]. Therefore, I examined the

significance of this sequence, including C-terminus carboxylation, on LsIA’s functional

activity. While the truncated analogues caused LsIA’s loss in activity at both of its

pharmacological targets, which was similar to previous studies with α-conotoxin GID [13], C-

terminal carboxylation changed selectivity between the α3β2 and α7 nAChR subtypes. I also

extended this study to identify the role the α5 nAChR subunit plays in α-conotoxin

pharmacology. Interestingly, while α5 subunit incorporation into the α3β2 nAChR subtype

decreased LsIA’s IC50 three-fold, a faster onset of block (kon) was observed at the α3α5β2

nAChR subtype. Together, these results present a novel strategy for the development of novel

analogues with improved subtype selectivity and suggest a pharmacological role played by

the auxiliary α5 subunit in the inhibition of nAChRs by α-conotoxins.

I also functionally characterised a novel conotoxin, GeXXA, which belongs to the D-

superfamily. αD-conotoxins are unique homo-dimeric peptides that non-competitively target

nAChRs. However, very few peptides from this family have been characterised. αD-GeXXA

159

selectively inhibits α7 and α3-containing nAChRs and irreversibly inhibits α9α10 nAChR

subtype. This is the first report of monomeric αD-conotoxin activity, selectively inhibiting the

α9α10 nAChR subtype. These results present a novel binding mechanism for an αD-

conotoxin and provide future insights for the development of new nAChR antagonists.

Finally, in a collaborative study, I examined the functional implications of dicarba

modification of the disulphide bonds in α-conotoxins Vc1.1 and RgIA. Both of these peptides

exhibit analgesic properties and inhibit α9α10 nAChR and N-type calcium channel currents

via GABAB receptor activation [14]. However, the lability of the disulphide bond has

hampered clinical exploitation of these and other α-conotoxins.

Dicarba modification of the disulphide bonds has been shown to improve peptide stability

[15, 16]. I identified a significant reduction in activity for Vc1.1 and RgIA at the α9α10

nAChR subtype. However, interestingly, regioselective I–III and II–IV dicarba analogues

exhibited differential selectively towards their biological targets. These results suggest that

the CysI–CysIII disulphide bond of Vc1.1 and RgIA interacts with α9α10 nAChR subtype

receptors. This may help us establish a novel strategy for developing subtype-selective

nAChR antagonists and new leads for stable drugs to treat various pain conditions.

Overall, the studies outlined in this thesis have provided crucial information to improve our

understanding of the molecular pharmacology and potential therapeutic use of α-conotoxins.

160

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