Discovery and Characterisation of Novel

Analgesic Neurotoxins, hCav2.2 Channel Inhibitors, from Cone Snail and Spider Venoms

Silmara Rodrigues de Sousa

BPharm, MPharm

A Thesis Submitted for the Degree of Doctor of Philosophy at

The University of Queensland, 2014

Institute for Molecular Biosciences

ABSTRACT

Cav2.2 is a voltage-gated calcium channel isoform expressed in nerve terminals of excitable

cells and dendrites, where it initiates neurotransmitter release and controls nociception. Cav2.2 is particularly important in controlling signalling in nociceptive pathways such as the ventral and dorsal horn of the spinal cord, dorsal root ganglion (DRG) neurons, and along the dendrites and at presynaptic terminals, where it contributes critically to neurotransmitter release.

Although different Cav channel types are expressed in nociceptive pathways. Cav2.2 is up- regulated at the spinal cord during chronic pain and nerve injury states, along with the auxiliary

α2δ subunit. The Cav2.2 auxiliary subunits α2δ1 and α2δ2 are the targets of the gabapentinoid drugs (gabapentin and pregabalin), which are currently marketed for use in neuropathic pain

treatment. Morphine, a drug used for decades for the treatment of severe pain inhibits Cav2.2 indirectly at the spinal cord level. Ziconotide (Prialt®) is a direct Cav2.2 blocker that produces

efficient analgesia, despite side effect problems. Therefore, Cav2.2 inhibition is a validated

analgesic strategy, and Cav2.2 inhibitors have therapeutic application in the treatment of neuropathic pain.

Venomous animals, such as snakes, scorpions, cone snails and spiders, are rich sources of

remarkably potent and selective Cav2.2 inhibitors. There are aproximately 100 different venom components per species of cone snails, leading to an estimate of 50,000 different pharmacologically active components, present in venoms of all living cone snails. Spiders are the most successful venomous animals with an estimated 100,000 extant species. The vast majority of spiders employ a lethal venom cocktail to rapidly subdue their prey. Some spider species produce venom containing >1000 unique peptides.

The main aim of this thesis was to discover and pharmacologically characterize new Cav2.2 channel inhibitors from the venoms of cone snails and spiders to help finding alternative drug leads for the treatment of chronic severe pain. Initially we established human cell-based miniature high throughput screening assays. These assays allowed accelerated identification and characterization of novel Cav2.2 channel inhibitor peptides extracted from large venom libraries. ω-Conotoxins MoVIA and MoVIB were discovered from a worm hunter cone snail. MoVIA given intrathecally was a potent analgesic in a rat model of induced neuropathic pain.

The first Chapter of this thesis gives a general introduction, describes the pathophysiology of pain as and the role of Cav2.2 in pain pathways. Chapter 1 also introduces venom toxins as

tools to isolate Cav2.2 channels and as drug leads to threat pain. Parts of Chapter 1 have been published in Toxins, 2013. The pdf version has been attached as Appendix 2 at the end of the thesis.

Parts of Chapter 2 have been published in PLoS ONE, 2013 and a pdf version has been attached as Appendix 2. Chapter 2 describes the establishment of robust functional calcium fluorescent assays and binding assays to identify and characterize Cav channel inhibitors. Using these assays the pharmacology of a range of ω-conotoxins were compared and importantly, new hCav2.2 inhibitors were discovered and pharmacologically characterised. Additionaly the influence of auxiliary subunits in the pharmacology of ω-conotoxins was investigated and is described in Chapter 2.

In Chapter 3 it is described the discovery and characterization of two novel ω-conotoxins named MoVIA and MoVIB. These toxins were isolated from the venom of the cone snail Conus moncuri, a worm hunting cone snail. Worm huntings are ancestral cone snails. MoVIA

and MoVIB were highly selective Cav2.2 channel inhibitors in rat DRG cells, the human SH- SY5Y cells and fish brain. Before this thesis it was thought ω-contoxins were only part of fish hunting cone snail venoms and this component of the venom was needed to produce the motor cabal effect needed for the snails to paralyse the prey (fishes), which would otherwise move too quickly. MoVIA and MoVIB were chemically synthesised and the structure-function relashionship and family tree were studied. Interestingly, in vivo studies proved analgesic potential of toxins MoVIA and MoVIB in rats. Therefore these toxins have the potential to be used as lead tools for treatment of neuropathic pain in humans.

Chapter 4 reports the discovery and functional characterisation of Cd1a, a new Cav2.2 channel inhibitor toxin isolated from the venom of Ceratogyrus darlingi, a tarantula African spider. Toxins from related spiders are potent Kv and Nav channels. Cd1a pharmacology was

investigated at hNav channels using assays established in house and confirmed activity at

hCav2.2, but not at other hCav channel subtypes endogenously expressed in SH-SY5Y cells.

Interestingly, Cd1a inhibited a range of hNav channel subtypes with high potency, suggesting

this toxin is more a Nav than a Cav channel inhibitor.

The results described in this thesis will provide new pharmacological tools for the discovery

and characterization of Cav inhibitors and may lead to the development of better analgesic options for the treatment of severe pain conditions.

Declaration by the author

This thesis is composed of my original work and contains no material previously published or written by another person, except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

Publications during candidature

1. Peer-Reviewed Articles

Sousa SR, Vetter I, Lewis RJ (2013a). Venom Peptides as a Rich Source of Cav2.2 channel blockers. Toxins 5(2): 286-314.

Sousa SR, Vetter I, Ragnarsson L, Lewis RJ (2013b). Expression and Pharmacology of

Endogenous Cav Channels in SH-SY5Y Human Neuroblastoma Cells. PLoS One 8(3): e59293.

2. Conference Abstracts

Sousa SR, Ragnarsson L, Vetter I, Herzig V, King GF, Lewis RJ (2012). Development of High Throughput Calcium Channel Assays to Accelerate the Discovery of Novel Toxins

Targeting Human Cav2.2 Channels. In: 17th World Congress of the International Society on Toxinology & Venom Week 2012 Vol. 60, p 111. Hawaii.

Publications included in this thesis

No publications included as chapters. The peer reviewed articles listed above have been attached as Appendix 2.

Statement of contributions by others to the thesis

Invaluable input in establishing research projects, designing experiments and interpreting results, proof reading the thesis, has been given by my advisory team Professor Richard Lewis, Dr Irina Vetter, and Dr Lotten Ragnarsson. Dr Irina Vetter has contributed with making the

Cav2.2 topology figure (Figure 1-2, Chapter 1). Dr Anderson Wang helped making the figures of the toxins structure (Figure 1.4 and Figure 1-4, Chapter 1) and Mr Olivier Allart helped preparing the pain pathway figure (

Figure 1-1, Chapter 1).

Through collaboration with Prof. Paul Alewoods laboratory, University of Queensland, Dr Andreas Brust synthesized the ω-conotoxins and the spider toxin Cd1a. Dr Sebastien Dutertre assisted with LC/MS data collection (Figure 3.3 and Fig 4.3) and Dr Lotten Ragnarsson assisted with molecular biology experiments (Figure 3.3, Chapter 3). The DRG neurons extraction, culture, and electrophysiological assays (Figure 3.11, Chapter 3) were performed by Dr Jeffrey Mc Arthur and me through collaboration with Prof. David Adams laboratory, at the RMIT University, in Melbourne. Dr Johan Rosengren from the School of Biomedical Sciences, University of Queensland, calculated the NMR structure and assisted with the NMR structure analysis (Figure 3-12 and 3-13, Chapter 3). The in vivo rat model of neuropathic pain was performed by Rebecca Smith and me (Figure 3-11, Chapter 3), in collaboration with Prof. Macdonald Christie’s laboratory, at the Kolling Institute of Medical Research, University of Sydney. Prof. Glenn King kindly provided the spider venoms.

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

Acknowledgements

I would like to acknowledge my supervisor Prof. Richard Lewis for guiding and giving me the opportunity to complete my PhD in this field of research that I am passionate about. Richard’s suggestions in presentations, designing experiments, interpreting data and writing the thesis and manuscripts, were invaluable; and I could not ask for a friendlier environment to do my PhD.

I would like to express my deepest appreciation to Dr Irina Vetter for teaching in the lab, presentations and writing. Irina has had an essential role in my PhD, helping me to coordinate projects, priorities and timelines. Most importantly, I thank Irina for encouraging, supporting and guiding during the years of my PhD. Thanks Irina, we made it and it has been a great journey!

I would also like to acknowledge with much appreciation the crucial role of Dr Lotten Ragnarsson. Dr Lotten’s expertise in molecular biology and training were essential in learning lab techniques used for the first result chapter of this thesis. I could not ask for a better teacher. Thanks Lotten for expert and friendly advice, and for being always there when I needed you!

Very special thanks to Asa Anderson for patiently teaching the binding assays. Thank my Dr Marco Inserra, who explained cell culture and helped with FLIPR data analysis. Thanks to Thanks Dr Josh Wingerd and Irina Vetter for guiding in HPLC use. Thanks Thea for patiently helping with administrative work and thesis formatting. Thanks to my friend Lea Indjein for advice with PCR methods.

I would like to thank Prof Paul Alewood and his group from the University of Queensland, especially Dr Andreas Brust for making peptides MoVIA, MoVIB, [R13Y]-MoVIB analogue, and the spider toxin Cd1a. With this synthetic peptides version, I was able to characterize these toxins. Thank Prof David Adams and Prof. Mac Donald Christie for allowing me the opportunity to work in their lab in collaboration with experts in the fields of electrophysiology, Dr Jefferson Mc Arthur, and animal models of neuropathy, veterinary Dr Rebecca Smith. The results of this collaboration were essential for my thesis and I have had excellent trainings in Melbourne and in Sydney.

Furthermore I would also like to acknowledge with much appreciation the crucial role of Dr Amanda Carozzi, who gave great support and went much further beyond her work duties to make sure PhD students are successful.

Thanks to Prof. Glenn King and Matt Sweet for participating on my PhD milestones committee, and for giving inestimable suggestions for my candidature.

I would like to acknowledge the financial support from The University of Queensland providing a UQRS scholarship. I would also like to acknowledge The Institute for Molecular Biosciences for a travel grant, which allowed me to attend an international conference in Hawaii; and my supervisor Richard Lewis for providing financial support to complete my PhD projects, and to attend conferences and collaborate with other laboratories in Australia, which were excellent experiences.

Last but not least, many thanks go to the most important people in my life: my family and my husband Olivier Allart. I thank my family for supporting my decision to come to Australia to pursue my dream PhD. Olivier my love, this journey has been much smoother and fun thanks to your company, support, loving, and dedication and caring.

Keywords

Pain, Cav2.2 channels, analgesia, pharmacology, conotoxins, ω-conotoxins cone snail toxins, spider venom toxins, SH-SY5Y cells, FLIPR assays, and binding assays.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 030406, Proteins and Peptides, 30%

ANZSRC code: 110999, Neurosciences, 30%

ANZSRC code: 111599, Pharmacology and Pharmaceutical Sciences, 40%

Fields of Research (FoR) Classification

FoR code: 1115 Pharmacology and Pharmaceutical Sciences, 40%

FoR code: 1109 Neurosciences, 30%

FoR code: 0601 Biochemistry and Cell Biology, 30%

Table of Contents

Chapter 1 Literature Review...... 2

Cav2.2 Role in Pain Pathophysiology and Analgesic Toxin Inhibitors ...... 2

1.1 FOREWORD ...... 2

1.2 INTRODUCTION ...... 2 1.2.1 Pain ...... 2 1.2.1.1 Gate Control Theory of Pain ...... 3 1.2.1.2 Classifying Pain ...... 4 1.2.1.3 Acute Pain ...... 4 1.2.1.4 Chronic Pain...... 4 1.2.1.5 Nociceptive and Neuropathic pain ...... 4 1.2.1.6 Physiology of Pain Signaling ...... 5 1.2.1.7 Descending Facilitation ...... 6 1.2.1.8 Peptide versus Small Molecule Neurotransmitters ...... 6 1.2.1.9 Social and Economic Impact of Pain ...... 8 1.2.1.10 Managing Pain ...... 8

1.2.2 Cav Channels...... 11

1.2.2.1 Cav Channels Molecular Basis ...... 12

1.2.2.2 Cav2.2 in Pain Pathophysiology ...... 13

1.2.2.3 Cav2.2 Inhibitors in Animal models of Pain ...... 14

1.2.2.4 Cav2.2 Channels Regulation by Auxiliary Subunits ...... 18

1.2.2.5 Other Cav Regulatory Pathways...... 21

1.2.2.6 The Role of Other Cav Channels in Pain ...... 22

1.2.3 Cav2.2 Channel Inhibitors ...... 23 1.2.3.1 Indirect Inhibitors...... 23 1.2.3.2 Direct Inhibitors ...... 23

1.3 CONCLUSION ...... 38

1.4 THESIS AIMS ...... 39

Chapter 2 Development of High Throughput Cav2.2 Assay & Characterization of ω- Conotoxins Pharmacology ...... 41

2.1 FOREWORD ...... 41

2.2 AIMS OF THIS CHAPTER ...... 41

2.3 INTRODUCTION ...... 41

2.4 MATERIAL AND METHODS ...... 43 2.4.1 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) ...... 43 2.4.2 RNA Isolation, Synthesis of cDNA, PCR Reaction and Controls ...... 44 2.4.3 Agarose Gel Electrophoresis ...... 45 2.4.4 Sequencing PCR Products ...... 45 2.4.5 Cell Culture ...... 45 2.4.6 Whole Cell Preparation for the Radioligand Binding Assay ...... 45 2.4.7 Membrane Preparation for the Radioligand Binding Assays ...... 46 2.4.8 [125I]-GVIA Radioligand Binding Assay ...... 46 2.4.9 Intracellular Ca2+ Measurement using the FLIPR ...... 47 2.4.10 Statistical Analysis ...... 48 2.4.11 Z’ Score as a Measurement of Robustness of Assays ...... 48

2.4.12 SH-SY5Y cells express multiple Cav isoforms and auxiliary subunits ...... 48 2.4.13 125I-GVIA Displacement from SH-SY5Y Cell Membranes ...... 55

2.4.14 Functional Characterization of Native Cav channels ...... 57 2.4.15 Ca2+ Signaling Detected by the FLIPR ...... 58

2.5 DISCUSSION AND CONCLUSIONS ...... 65

Chapter 3 Novel Analgesic ω-Conotoxins Discovered from the venom of Conus moncuri. Functional, Molecular and Structural Studies...... 71

3.1 FOREWORD ...... 71

3.2 INTRODUCTION ...... 71

3.3 MATERIALS AND METHODS ...... 73 3.3.1 Drugs and Chemicals ...... 73 3.3.2 Crude Venom Extraction ...... 73 3.3.3 Cell Culture ...... 74 3.3.4 Rat Dorsal Root Ganglia Neurons (rDRG) ...... 74 3.3.5 Discovery of MoVIA and MoVIB ...... 75 3.3.6 HPLC Isolation of Active Peptides ...... 75 3.3.7 Edman Sequencing Analysis ...... 76 3.3.8 Preparation of C. moncuri RNA ...... 76

3.3.9 Preparation of cDNA and Polymerase Chain Reaction (PCR) ...... 76 3.3.10 Chemical Synthesis of MoVIA and MoVIB ...... 77 3.3.11 NMR Spectroscopy ...... 77 3.3.12 3D Structure Calculations ...... 78 3.3.13 Electrophysiology Recording of HVA Ca2+ Currents from rDRG ...... 78 3.3.14 Preparation of Membrane for Radioligand Binding Assays ...... 79 3.3.15 Radioligand Binding Assays ...... 79 3.3.16 Intrathecal MoVIB in Rats with PNL-Induced Neuropathy ...... 79 3.3.17 Behavioral Tests in Rats PNL treated ...... 80 3.3.18 Side Effect Score ...... 81 3.3.19 Statistical Analysis ...... 81 3.3.20 Calculating peptide net charge ...... 82

3.4 RESULTS ...... 82 3.4.1 Discovery and Assay-Guided Isolation of MoVIA and MoVIB ...... 82 3.4.2 MoVIA and MoVIB Chemical Synthesis and Mass Spectrometry ...... 84 3.4.3 MoVIA and MoVIB Displace 125I-GVIA from Cell Membranes ...... 90

3.4.4 MoVIA and MoVIB Selectively Inhibit Functional hCav2.2 Channels ...... 92 3.4.5 MoVIA and MoVIB Selectively Inhibited HVA N-type in Rat DRG ...... 94 3.4.6 1H NMR Solution Structure of MoVIB ...... 96 3.4.7 Secondary Chemical-Shifts ...... 99 3.4.8 MoVIB produces dose dependent analgesia in the PNL model...... 100

3.5 DISCUSSION AND CONCLUSIONS ...... 102 3.5.1 Other Factors that May Influence ω-Conotoxins Efficacy ...... 109

3.6 CONCLUSION ...... 110

Chapter 4 Discovery and Functional Characterization of a Novel Cav2.2/Nav Channels Inhibitor Toxin, from a Tarantula Spider ...... 112

4.1 FOREWORD ...... 112

4.2 INTRODUCTION ...... 112

4.3 MATERIAL AND METHODS ...... 113 4.3.1 Drugs and Chemicals ...... 113 4.3.2 Cell Culture and FLIPR Ca2+ Imaging Assays ...... 114 4.3.3 Screening Bioactive Spider Venoms ...... 114

4.3.4 HPLC Fractionation ...... 114 4.3.5 N-terminal Sequencing Analysis ...... 115 4.3.6 Chemical Synthesis of Spider Toxin ...... 115 4.3.7 Preparation of Membranes for the Radioligand Binding Assays ...... 115 4.3.8 125I-GVIA Radioligand Binding Assays ...... 115

4.3.9 Membrane Potential Nav Assays ...... 115 4.3.10 Statistical Analysis ...... 116

4.4 RESULTS ...... 116 4.4.1 Screening and Assay-Guided Isolation ...... 116 4.4.2 Mass Spectrometry and Synthesis ...... 119 4.4.3 Cd1a did not displace 125I-GVIA ...... 120

4.4.4 Cd1a Inhibits Functional Cav2.2 Preferentially in SH-SY5Y Cells ...... 121

4.4.5 Cd1a Inhibits Native and Recombinant Nav Channels ...... 122

4.5 DISCUSSION AND CONCLUSION...... 125

Chapter 5 General Discussion ...... 131

Chapter 6 References ...... 140

List of Figures

Figure 1-2. Role of Cav2.2 in Ascending Pain Pathway...... 14

Figure 1-3. Cav2.2 channels Topology ...... 27 Figure 1-4. ω-Conotoxins 3D NMR Structure and Important Residues in Side Chains...... 28 Figure 1-5. NMR Solution Structure of the Spider ω-theraphotoxin-Hh1a (HWTX-X) and the assassin bug toxin Ptu1...... 37

Figure 2-1. RT-PCR of Cavα and Auxiliary Subunits in SH-SY5Y Cells...... 54 Figure 2-2. 125I-GVIA Displacement by ω-Conotoxins from SH-SY5Y Cells...... 56

Figure 2-3. Functional Cav2.2 FLIPR Assay Plate Layout...... 58 Figure 2-4. Ca2+ Signal from FLIPR Assays in SH-SY5Y Cells...... 59

Figure 2-5. Functional Cavs Expressed in SH-SY5Y cells...... 61 Figure 2-6. Characterization of Resistant Ca2+ Responses in SH-SY5Y Cells...... 62

Figure 3-1. FLIPR Cav2.2 HTS Assay. Plate layout...... 83 Figure 3-2. Assay-guided Fractionation. Active Cone Snail Toxins...... 84 Figure 3-3. LC/MS Analysis of Synthetic and Native MoVIA and MoVIB...... 86 Figure 3-4. AA precursor sequences of MoVIA Predicted from cDNA Sequences and Aligned with Sequences of Other Conotoxins Belonging to the O-Superfamily...... 88 Figure 3-5. Cladogram Tree of the O-Superfamily...... 89 Figure 3-6. 125I-GVIA Radioligand Binding Assays...... 91

Figure 3-7. Functional Cav2.2 Assays...... 93

Figure 3-8. High Voltage-Activated (HVA) Cav Currents Rat DRG Neurons...... 95 Figure 3-9. The 20 superimposed NMR structures of MoVIB...... 96 Figure 3-10. Hα and NH Chemical Shifts of MoVIB and [R13Y]-MoVIB...... 99 Figure 3-11. Effects of Intrathecal MoVIB on Pain of PNL-Induced Rats...... 101 Figure 3-12. NMR Solution Structure and Side Chains of MoVIB...... 104 Figure 3-13. Electrostatic Surface Mappings...... 105

Figure 4-1. Cav2.2 Responses Are Inhibited by Ceratogyrus darlingi Venom...... 117 Figure 4-2. Cd1a Isolation by Reverse Phase Liquid Chromatography...... 118 Figure 4-3. Cd1a Co-elutes With Synthetic Form...... 119 Figure 4-4.125I-GVIA Displacement Assays...... 120 2+ Figure 4-5. KCl Activated Ca Responses Mediated by Cav2.2 in SH-SY5Y Cells...... 121

List of Tables

Table 1.1. Summary of Cav channel modulators in clinical use for pain management...... 10

Table 1.2. Physiological function and pharmacology of Cav channel subtypes...... 16 Table1.3. Analgesic Conotoxins: Classification, Amino acid Sequence and Clinical Potential...... 26 Table 1.4. ω-Conotoxins AA sequences and Affinity at Displacing 125I-GVIA or 125I-MVIIA in Binding Assays...... 29

Table 1.5. Cav2.2 Inhibitor Toxins from Spider Venoms...... 35

Table 2.1. Primers Used for Identification of Cav Channels Isoforms Expressed in SH-SY5Y Cells...... 50 Table 2.2. 125I-GVIA Displacement from Rat Brain and SH-SY5Y Cells ...... 57 2+ Table 2.3. Potency (IC50 ± SEM) of Cav Channel Modulators at Functional Ca Imaging Assays...... 64 Table 3.1. ω-Conotoxins MoVIA and MoVIB AA Sequences and Alignment...... 85 125 Table 3.2. ω-Conotoxin Affinity (IC50 ± SEM, [nM]) at Displacing I-GVIA Binding...... 92

Table 3.3. Potency of ω-Conotoxins Inhibiting Functional Cav2.2 Channels...... 95 Table 3.4. Energies and structural statistics for the family of 20 MoVIB structures with highest overall MolProbity score...... 98 Table 4.1. Amino acid sequence of Cd1a, Cm1a and Cm1b...... 122 Table 4.2. Promiscuous Pharmacology of Toxins Cd1a, Cm1a and Cm1b...... 124

List of Abbreviations

Cav Voltage-gated calcium channels

hCav2.2 Human voltage-gated calcium channel isoform 2.2

Nav Voltage-gated sodium channels

Kv Voltage-gated potassium channels

Ca2+ Calcium ion

Na2+ Sodium ion

Ba2+ Barium ion

Ni2+ Nickel ion

Zn2+ Zinc ion

HIV Human immunodeficiency virus

AIDS Acquired immunodeficiency syndrome

NSAIDS Non-steroidal anti-inflammatory drugs

DHP Dihydropyridine

PHA Phenylalkylamine

BTZ Benzothiazepine

DRG Dorsal root ganglia neurons

rDRG Rat dorsal root ganglia neurons

CGRP Calcitonine gene-related peptide

VWF-A Von Willebrand Factor A

AID Alpha Interaction Domain

BID Beta interaction Domain

ER Endoplasmic reticulum

SNARE Soluble N-ethylmaleimide-sensitive factor activating protein receptor

PKC Protein kinase C

NMDA N-methyl-D-aspartate

nAChR Nicotinic acetylcholine receptor

3D Three-dimensional

sp. Species

IC50 Half-maximal inhibitory concentration

ED50 Half-maximal excitatory concentration

pIC50 Positive half-maximal inhibitory concentration

Kd Dissociation constant

ND None determined

NDR Non detectable response

HTS High throughput screening

RNA Ribonucleic acid

mRNA Messenger ribonucleic acid

dNTP Deoxyribonucleotide triphosphate

cDNA Complementary deoxyribonucleic acid

gDNA Genomic deoxyribonucleic acid

PCR Polymerase chain reaction

BLAST Basic Local Alignment Search Tool

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

bp Base pairs

TBE Tris-borate EDTA

BCA Bicinchoninic acid

BSA Bovine serum albumin

PEI Polyethyleneimine

cpm Counts per minute

PSS Physiological salt solution

AA Amino acid

SEM Standard error of the mean

125I Iodine (radioisotope)

FLIPR Fluorescent plate imaging reader

KCl Potassium chloride

CaCl2 Calcium chloride

ICK Inhibitory knot motif

RP-HPLC Reverse-phase high performance liquid chromatography

MALDI-TOF Matrix-assisted laser desorption-time of flight

KHz Kilohertz

pClamp Patch clamp

LC/MS Liquid chromatography/mass spectrometry

NMR Nuclear magnetic resonance

SAR Structure activity relationship

ACN Acetonitrile

TFA Trifluoroacetic acid

Ref Reference

DMEM Dulbecco’s modified eagle medium

PWT Paw withdraw threshold

MPE Maximum possible score

RFU Relative fluorescence unit

ANOVA Analysis of variance

PTM Post-translational modification

TTX-s Tetrodotoxin sensitive

TTX-r Tetrodotoxin resistant

°C Degree Celsius

DTT DL-Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

FBS Foetal bovine serum

FDA Food and Drug Administration

RPMI Roswell park memorial institute medium

CHO Chinese hamster ovary

HEK Human embryonic kidney

CO2 Carbon dioxide

KDa Kilo Daltons

DMSO Dimethyl sulfoxide

CNS Central Nervous System

TTA P23,5-dichloro-N-[1-(2,2-dimethyltetrahydro-pyran-4-ylmethyl)-4- fluoro-piperidin-4-ylmethyl]-benzamide

NSAIDs Non-steroidal anti-inflammatory drugs

List of Amino Acid Abbreviations

Ala A Alanine

Arg R Arginine

Asn N Asparagine

Asp D Aspartate

Cys C Cysteine

Glu E Glutamate

Gln Q Glutamine

Gly G Glycine

His H Histidine

Ile I Isoleucine

Leu L Leucine

Lys K Lysine

Met M Methionine

Phe F Phenylalanine

Pro P Proline

Ser S Serine

Thr T Threonine

Trp W Tryptophan

Tyr Y Tyrosine

Val V Valine

Chapter 1

Chapter 1

1

Chapter 1 Chapter 1 Literature Review

Cav2.2 Role in Pain Pathophysiology and Analgesic Toxins Inhibitors

1.1 FOREWORD

This chapter gives an introduction of what is pain and pain as a pathophysiology. This chapter includes parts of a peer-reviewed publication in Toxins, 2013 [1]. A pdf version of this paper has been attached in the end of the thesis, appendix 2. To fit the format of this thesis, some parts have been modified compared to the published review publication. Most of the work presented in this chapter is my own. However, contribution of others, including the co-authors of the review paper Prof. Richard Lewis and Dr Irina Vetter, consisted of proof reading and editing the manuscript. This has been detailed in the statement of contributions by others, at

the beginning of this thesis. Dr Irina Vetter has helped preparing the Cav2.2 topology figure. Dr Anderson Wang helped preparing the figures of the toxins structures and Mr. Olivier Allart helped preparing the pain pathway figure.

1.2 INTRODUCTION

1.2.1 Pain

Pain is an unpleasant feeling that is an essential component of the body’s defence system. It provides a rapid warning to the nervous system to initiate a motor response to minimize physical harm. Lack of the ability to experience pain, as in the rare condition congenital insensitivity to pain with anhidrosis causes very serious health problems such as self-mutilation and auto-amputation (for review see: [2]).

Up until the twentieth century there was a debate about the nature of pain. One side held that sensory stimuli, which activate ordinary sense organs, such as those for touch, would initiate pain through the same sense organs if the stimuli were strong enough. It was not until the twentieth century that the debate was settled and it was shown conclusively that there were specialized sensory organs that signalled pain. The main characteristic of pain identified was

2

Chapter 1 the affective aspect. Emotion, motivation, and consequent behaviour connect with pain [5]. This implies an interaction between neural structures involved in pain sensation and autonomic control (for review see:[6]).

In 1979 the Association for the Study of Pain (IASP) included in annual recognised in annual report writing that emotional feelings influence pain [7]. Pain was since defined by IASP as “an unpleasant sensory and emotional” experience associated with actual or potential tissue damage, or described in terms of such damage [7, 8].

1.2.1.1 Gate Control Theory of Pain

Melzack and Wall, 1965 originally described the theory of gate control [3] a theory that stimulated interest and debate, because it introduces peripheral and central “gating” concepts [3], the interaction between ascending peripheral afferent fibers with a modulation system in the spinal cord and grey matter, in addition to a descending modulatory system (for review see: [4]). This theory was certainly an improvement on the early theories to explain pain transmission.

Melzack and Wall, 1965 group proposed that small nerve fibers (pain receptors) and large nerve fibers (other receptors) synapse on projection cells (P), which go up the spinothalamic tract to the brain and inhibitory interneurons (I), within the dorsal horn. The interplay among these connections determines when painful stimuli go to the brain. When no input comes in, the inhibitory neuron prevents the projection neuron from sending signals to the brain (gate is closed). Normal somatosensory input occurs when there is mostly or only large-fiber stimulation. Both the inhibitory neurons and the projection neurons are stimulated, but the inhibitory neurons prevent the projection neurons from sending signals to the brain (gate is closed). Nociception (pain perception) occurs when there is mostly small-fiber stimulation or only small-fiber stimulation. This inactivates the inhibitory neuron, and the projection neuron sends signals informing of pain to the brain (gate is open) [3].

Neurons in the brainstem and cortex have efferent pathways to the spinal cord, and the impulses they send can open or close the gate. The effects of some brain processes, such as those in anxiety or excitement, probably have a general impact, opening or closing the gate for all inputs from any areas of the body. Descending pathways from the brain close the gate by inhibiting the projection neurons and diminishing pain perception [3].

3

Chapter 1 1.2.1.2 Classifying Pain

Pain may result from any illness, trauma, surgery or other external stimuli. Pain may also be experienced in the absence of noxious stimuli. Together with temperature and other bodily feelings, pain constitutes the interoception, a physiological condition of the body.

1.2.1.3 Acute Pain

Acute pain is the type of pain of short onset, lasting from hours to days and disappearing once the underlying cause is treated. Acute pain occurs as an important physiological response to protect individuals, as it signals to identify that there is damage. With this information, behaviour changes can be initiated, so that further injury can be avoided [5].

1.2.1.4 Chronic Pain

Chronic pain affects a large percentage of the world’s population and is a major therapeutic challenge [13]. Different from acute pain, chronic pain persists or recurs after the healing phase. Pathophysiological conditions such as diabetes, viral infections, nerve injury and inflammation can cause abnormal neuronal activity and give rise to chronic pain, which is often refractory to currently available treatment options [9, 10]. A range of pathophysiological conditions, such as diabetes, viral infections, nerve injury and inflammation can cause abnormal neuronal activity and give rise to persistent, chronic pain, that are often refractory to currently available treatment options [9] [10]. Additionally, chronic pain may result from abnormalities in rostrum ventral medial (RVM) facilitatory cells in the central nervous system [11].

1.2.1.5 Nociceptive and Neuropathic pain

Depending on the origin, pain can be classified as nociceptive or neuropathic. Nociceptive pain originates from tissue damage, while neuropathic pain originates from nerve damage [12]. Neuropathic pain produces abnormal neuronal activity, which underlies symptoms of peripheral and central sensitization and spontaneous ectopic nociceptors activation [9]. Sensitization in chronic pain is characterized by a reduced threshold for noxious stimuli (allodynia), and increased action potential firing in response to supra-threshold stimuli (hyperalgesia) [13].

4

Chapter 1 The activation of nociceptors or peripheral pain-sensing neurons by noxious peripheral stimuli elicits nociceptive pain and depending on the origin of the innervating neurons this pain can be subdivided in visceral (internal organs) or somatic (cutaneous or deep tissues) pain. Pain emanating from the skin and deeper tissues (e.g. joints and muscle) is referred to as somatic pain, while pain emanating from the internal organs is referred to as visceral pain [7]. Somatic pain is usually well localized, whereas visceral pain is harder to identify.

Normal individuals often do not perceive signals emanating from their intestinal tract; however, enteric and extrinsic visceral afferents become hypersensitive in pain-processing disorders such as functional bowel syndromes [21], or in diseases associated with inflammation including inflammatory bowel disease and pancreatitis [21]. Both inflammatory bowel disease and cancer-related metastases to viscera may produce persistent pain despite resolution of the underlying disease state.

A significant proportion of the visceral spinal afferent fibres are high-threshold or silent nociceptors, which respond selectively to noxious stimuli. Spinal visceral afferent action potentials propagate along the fibres stimulating increases in Ca2+ intracellular, resulting in the release of neurotransmitters, mainly calcitonin gene-related protein (CGRP) and substance P (SP) from varicose nerve terminals, which in turn modulate blood flow and enteric motor reflexes; the end result is a neurogenic inflammatory response [14, 15].

1.2.1.6 Physiology of Pain Signaling

The primary afferent sensory neurons (mainly C-fibers and A-δ fibers) are localized in the peripheral nervous system and are classified as thermal, mechanical, polymodal and silent [16]. The speed at which an individual nerve fibre conducts action potentials is related to the diameter of the fibre [16]. Consequently, the type of pain carried by the pain-sensing neurons, or nociceptors, differs. Aδ fibers have a large diameter, are myelinated and conduct fast pain information, while the C fibres are smaller, non-myelinated and display slow conduction [17]. Activation of the rapidly-conducting Aδ-fibers leads to localized, pricking “first pain”, whereas activation of C-fiber results in burning or dull “second pain” with poor localization [18].

Nociceptors are pain receptors localized in the nerve endings of primary afferent fibres, which respond to noxious cold, noxious heat and high threshold mechanical stimuli as well as a variety of chemical mediators and are implicated in the sensation of a variety of noxious painful stimuli including thermal, mechanical, and inflammatory [7, 16]. Activation of diverse 5

Chapter 1 classes of peripheral nociceptors by a painful stimuli initiates a train of action potentials in the membrane of neuronal cells, which propagate along the axons of the primary afferent nerve fibres to nerve terminals embedded in lamina I and II of the dorsal horn of the spinal cord [1, 19]. In this region, nociceptive primary afferent neurons terminate and form primary synapses with secondary afferent fibres. These fibres relay nociceptive information to the thalamus and brain centres through releasing pro-nociceptive neurotransmitters, such as substance P and calcitonin gene-related peptide (CGRP) [20]. These substances activate postsynaptic receptors on neurons of the spino-thalamic tract. These neurons are second-order neurons that project to the thalamus. [15, 19, 21, 22] Projection to the brain allows pain perception.

1.2.1.7 Descending Facilitation

Classically, the formalin test is considered as a model of acute inflammatory pain that produces two well-identified phases, phase I and phase two of nociceptive behaviour [23]. However, after days of sub cutaneous injection into rodents, formalin induces a ‘third phase,” which reflects secondary mechanical hyperalgesia [24]. The formalin-induced secondary allodynia and hyperalgesia are maintained by activation of descending facilitatory mechanisms [25].

Descending facilitatory neurons maintain behavioural hypersensitivities to mechanical and thermal stimulus during the late stages of nerve injury [26]. Once initiated, maintenance of nerve injury-induced central sensitization in the spinal dorsal horn requires descending pain facilitation mechanisms arising from the RVM (rostral ventromedial medulla) [11, 27]. Since RVM facilitatory neurons are integral to a spino-bulbo-spinal loop that reaches brain area, co- ordinating the sensory and affective components of pain, activity in these areas may also influence painful outcome following nerve injury and the response to treatments [26].

1.2.1.8 Peptide versus Small Molecule Neurotransmitters

There are many different types of neurotransmitter substances in the CNS, over 100 or so, and sometimes a presynaptic element releases more than one type [28]. Neurons have developed ability to regulate synthesis, packaging, release, and degradation of neurotransmitters to achieve the desired levels of transmitter molecules [28]. Small molecule neurotransmitters and neuropeptide transmitters are the two main types of neurotransmitter substances in the CNS. The different steps in synthesis and packaging of neuropeptide

6

Chapter 1 transmitters versus classical small molecule transmitters lead to differential release and replacement mechanisms [30].

The synthesis of small-molecule transmitters such as GABA, Glutamate and Glycine, occurs via specialised enzymes within pre-synaptic nerve terminals. Glutamate is an excitatory neurotransmitter that mediates EPSP (excitatory postsynaptic potential), a “depolarizing” rapid response from neurons and is a “classical” example of a small molecule transmitter [31]. After synthesis small-molecule neurotransmitters are packaged into small clear-core vesicles (often referred to as ‘synaptic vesicles’) and thus can be rapidly replaced after release [30]. The enzymes needed for small molecule transmitter synthesis are synthesized in the neuronal cell body and transported to the nerve terminal cytoplasm by a mechanism called slow axonal transport [32]. The precursor molecules used by these synthetic enzymes are usually taken into the nerve terminal by transporter proteins found in the plasma membrane of the terminal. These enzymes generate a cytoplasmic pool of neurotransmitter that must then be loaded into synaptic vesicles by transport proteins in the vesicular membrane. For some small-molecule transmitters, the final synthetic steps actually occur inside the synaptic vesicles [31].

Peptide-secreting neurons generally synthesize polypeptides, called pre-propeptides (or pre- proproteins), in the cell bodies that are much larger than the final, “mature” peptide. Processing these polypeptides takes place by a sequence of reactions in several intracellular organelles. Pre-propeptides final stages of peptide neurotransmitter processing occur after packaging into vesicles and involve proteolytic cleavage, modification of the ends of the peptide, glycosylation, phosphorylation, and disulphide bond formation. Peptide transmitters are synthesized in the endoplasmic reticulum localized in the cell body, transported to the synapses, and then packaged in large dense-core vesicles. This process requires gene expression. Neuropeptide synthesis is, therefore, much like the synthesis of proteins secreted from non- neuronal cells. A major difference, however, is that the neuronal axon often presents a very long distance between the site of a peptide's synthesis and its ultimate secretion. The peptide- filled vesicles must therefore be transported along the axon to the synaptic terminal by a mechanism responsible for such movement, known as fast axonal transport [33]. Intrathecal administration of Cav modulators will have effects on peptide neurotransmitters, such as substance P, but not on non-peptide, small molecule, transmitters [29].”

7

Chapter 1 1.2.1.9 Social and Economic Impact of Pain

Environmental factors and individual differences make pain a subjective experience that is difficult to assess. Chronic pain has a large socio-economic impact. More than one and a half billion people worldwide suffer from moderate to severe chronic pain [8]. Besides the emotional and financial impact it has on patients and their families, chronic pain also imposes a significant economic burden to the world’s health systems [34] due to reduced productivity and income from both patients suffering from pain and their families.

The majority of people suffering from chronic unrelieved pain are from countries with low- and middle-incomes and an increasing burden of chronic diseases such as cancer and HIV/AIDS. The 2007 report from the Australian MBF Foundation in collaboration with University of Sydney Pain Management Research Institute highlighted the need for chronic pain to be elevated as a health priority and made a number of major recommendations for addressing the impact of chronic pain in the community [30, 35]. An estimated 20% of the adult Australians suffer from chronic pain. The cost of chronic pain in Australia reaches $34.3 billion, or $10,847 per person affected per year [30]; while worldwide the pain cost to the society is estimated to be $55 billion per year and includes a loss of productivity from full-time workers [32]. Furthermore, in the United States (US) a survey indicated that at least 50 million people suffer from chronic pain[36]. The cost of pain for the health services in the US has been ~ 100‒130 billion US$ per year. This cost results from long hospital stays, increased rates of re-hospitalization, increased outpatient visits and decreased capability to work fully, leading to income and insurance coverage losses [7]. In comparison with some other major health problems, pain affects more Americans than diabetes, heart disease and cancer combined [37, 38].

1.2.1.10 Managing Pain

Pain management is complex and represents a significant challenge. It can involve pharmacological, interventional (e.g., anaesthetic, surgery) and psychophysical measures or radiotherapy. However, the pharmacological treatment using analgesic drugs is the mainstay treatment and is also the main focus of this thesis. The first line option for pharmacological treatment is oral administration of analgesics, such as paracetamol, NSAIDs (Non-steroidal anti-inflammatory drugs) and/or opioids. The treatment of neuropathic pain includes, in addition, specific antidepressants or anticonvulsants [39]. Unfortunately, in a range of patients,

8

Chapter 1 oral drug administration fails to achieve adequate and sustained pain relief, as do other systemic (e.g. transdermal or parenteral) routes of administration. This occurs because unfortunately most of the current available analgesics act through targets that are not only essential for treating pain, but also critical in mediating physiological functions, which leads to significant side effects [39]. When the side effects from systemically administered analgesics are intolerable or when pain relief is insufficient, the second line of option includes interventional approaches such as nerve block, surgeries, spinal or intrathecal injection of drugs such as morphine, hydromorphone, fentanyl, clonidine and a range of local anaesthetics given alone or in combination [40]. However, serious adverse events can restrict the use of systemic analgesics and its long-term usage is often associated with additional side effects and the development of tolerance.

Cav2.2 is expressed at the spinal cord level, where its opening is essential for

neurotransmitter release [46, 67]. Cav2.2 channels inhibition mediates analgesia [46, 69]. While

other Cav isoforms expression are also found in nociceptive pathways, Cav2.2 has an important role in controlling signalling in nociceptive pathways such as the ventral and dorsal horn of the spinal cord and DRG (dorsal root ganglion) neurons. Especially, along dendrites and at

presynaptic terminals, Cav2.2 contributes critically to neurotransmitter release, essential for neurotransmission of pain information [68]. The opioid analgesic morphine has been used for many years as the first line option to treat severe pain, and this drug indirectly modulates Cav2.2 channels.

Several lines of evidence support Cav2.2 as analgesic target. Cav2.2 knock-out mice is insensitive to formalin-induced pain, visceral pain. Cav2.2 knock-out mice is resistant to development of neuropathic pain, as shown using the spinal nerve ligation model [20, 41].

Ziconotide is a direct Cav2.2 blocker. Ziconotide is a synthetic version of MVIIA a ω-conotoxin extracted from the venom of marine cone snail, conus magus, which is currently commercialised as Prialt® (Table 1.1). Prialt has been effectively used on the treatment of severe chronic pain associated with cancer, HIV/AIDS and a range of other neuropathies. [29]. Ziconotide acts synergistically with opioids reducing dose-related side effects and improving

tolerance or addiction [13]. In contrast to Cav2.1 knock-out mice, Cav2.2 had normal sensory

and motor functions [20, 41]. Therefore Cav2.2 is an important analgesic target.

Although ziconotide has provided effective relief for severe neuropathic and inflammatory pain, in laboratory animal models and in humans when injected intrathecally (directly into the

9

Chapter 1 spinal cord) [20, 42-45], associated side effects limits ziconotide use. Ataxia and other supra- spinal neurological side-effects such as dizziness, confusion, ataxia, abnormal gait, memory impairment, nystagmus and hallucinations can develop after ziconotide is given intrathecally, if sufficient amounts move rostrally up the neuraxis and reach the brain [46].

Table 1.1. Summary of Cav channel modulators in clinical use for pain management.

Mechanism of Drug FDA Indications Action Opiates analgesics inhibit presynaptic Relief of a range of moderate to severe

Cav2.2 channels in the acute and chronic pain conditions, otherwise Morphine spinal dorsnal horn via intractable with other common analgesic and

Gi/o protein coupled µ- NSAIDS opioide receptos (MOR)

Binding to the α2δ auxiliary subunit of the

Cav2.2 to reduce Cav2 Postherpetic neuralgia, epilepsy and Gabapentin activity, partially pediatric partial seizures interfering with trafficking Neuropathic pain associated with diabetic Binding to the α2δ peripheral neuropathy and postherpetic Pregabalin auxiliary subunit of the neuralgia; adjunctive therapy for partial Cav2.2 onset seizures and fibromyalgia HIV/AIDS, Cancer, management of severe chronic pain in patients who are

Ziconotide Cav2.2 direct blocker intolerant or refractory to other treatment such as systemic analgesics, adjunctive therapies, or/and intrathecal morphine

Cav2.2 is still underexploited as an analgesic target despite proved analgesic property of MVIIA in treating human severe pain. Avoiding side effects is still a problem with this class. Therefore some effort needs to be made for the discovery of new alternative analgesics. Venom

10

Chapter 1 are rich libraries sources of bioactive toxins. Many animal toxins have shown activity on ion channels, including Kv, Nav and Cav channels. This theses will discuss the use of venom libraries as tools to understand Cav2.2 channels function and to discover new analgesic leads. 2+ Cav2.2 channels are part of the Cav (voltage-gated Ca channels) superfamily, which consists of membrane proteins essential for the control of Ca2+ signalling events such as muscle

contraction, gene expression and neurotransmitter and hormone release. Dysfunction of Cav channels is related to a variety of heart, circulatory and neurological diseases, including arrhythmias, hypertension, some forms of epilepsy, migraine and other chronic diseases such as cancer, diabetes, ischemic brain injury and neuropathic pain [13, 47].

1.2.2 Cav Channels

Cavα1 subunit is the Cav pore-forming subunit and determines the main electrophysiological

and pharmacological properties of the channels. Cavα1 is part of the same protein family as the

ion channels Nav and Kv (sodium and potassium voltage-gated). Mammalian genes encoding

10 pore-forming α1 subunit isoforms [α1A̲ α1I and α1s], as well as four auxiliary α2δ, four β auxiliary subunits [β1-4] [48] and eight γ subunits have been identified for Cav channels (for

review see: [49]). Cav auxiliary subunits (α2δ and β, mainly) modify Cavα gating properties and have a significant influence on the channels function [50-53].

Early electrophysiological recordings from neurons, muscle and endocrine cells, revealed voltage-activated calcium currents with distinct characteristics, suggesting the existence of two

major classes of Cav channels based upon the membrane potentials at which they first open [7,

54, 55]. Late studies in other cell types identified a third Cav channel subtype [56], a fourth

[34] and a fifth [37]. Thus it became clear that versatility of function was reflected by Cav channels diversity, expressed on membranes of individual cells [8].

Consistent with distinct electrophysiology properties, ten different Cav channels genes have

been identified in vertebrates. Cav1 subfamily (Cav1.1– Cav1.4) which mediate high voltage

2+ activated responses, includes  subunits 1S, 1C, 1D, and 1F, which mediate L-type Ca currents. L-type Ca2+ channels were initially described in peripheral neurons and cardiac cells, but appear to be present in all excitable as well as many types of non-excitable cells [57]. The

Cav2 subfamily (Cav2.1–Cav2.3) includes  subunits 1A, 1B, and 1E, which also mediate 2+ high voltage activated P/Q-type, N-type, and R-type Ca currents, respectively. The Cav3

11

Chapter 1 subfamily (Cav3.1–Cav3.3) includes  subunits 1G, 1H, and 1L, which mediate low voltage activated T-type Ca2+ currents (Table 1.2) (for reviews see: [49, 58, 59].

HVA channels activate at membrane potentials near ‒30 mV and include Cav1.1–4 (L-type),

Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 (R-type). Low voltage activated channels

include Cav3.1–3 (T-type), which transiently activate at relatively negative potentials (~ –50 to –70 mV). LVA channels lack high-affinity selective blockers, thus knowledge of its physiological role lags behind those of the high-voltage activated isoforms [15, 34, 37, 55-57].

1.2.2.1 Cav Channels Molecular Basis

Cav channels are hetero-oligomeric proteins comprising the core pore-forming α1B subunit, which determines the main biophysical and pharmacological properties of the channel, and

typically two or three auxiliary subunits (α2δ, β and and γ). The 55 kDa Cavβ subunit is

cytoplasmic, while the α2δ has an extracellular disulfide-linked α2δ dimer of 170 kDa, and the γ four transmembrane segments subunit of 33 kDa [51, 60]. The presence or absence of the auxiliary subunits modulate the α1 subunit function and play an important functional role, modifying and regulating the kinetic properties of the channels [61, 62].

While no high-resolution crystal structure of the Cav channels is available to date, structures from related ion channels, in particular the recently determined bacterial Nav channels [65, 66],

promise to provide significant insight into the structure and function of Cav channels.

The primary structure of the Cav superfamily (Table 1.2) has been determined by a combination of protein chemistry, cDNA cloning and sequencing [49, 59, 63, 64]. Their hetero- oligomeric nature was established from biochemical glycosylation and hydrophobicity

analyses. The ~ 190 kDa pore-forming transmembrane α1 subunit (~ 2000 amino acids) is organized in four homologous domains (I–IV) (Figure 1-2) comprising six transmembrane α helices (S1–S6) including the pore-forming P loop between S5 and S6 [59]. The S4 segments form a key part of the voltage sensor module (Figure 1-2), moving outward and rotating under the influence of the electric field and initiating a conformational change that opens the pore. The external entrance to the ion conducting pore of the channel is lined by the P loop, which contains a pair of glutamate residues in each domain, which are required for Ca2+ selectivity. The inner pore is lined by the S6 segments, which form the receptor sites for the pore-blocking

Cav1 antagonist drugs [49].

12

Chapter 1

Figure 1-1. Cav channels Topology, α1B, β, α2δ and γ Subunits.

Topology of Cav channels figure was adaptated from Catteral et al [49, 59]. The four domains

of the α1B subunit are shown in light blue colour, each domain (DI–D4) consisting of six

segments (S1–S6) and a pore between where it is believed to be the binding site of Cav2.2 blockers. The α2δ auxiliary subunit is formed by an extracellular disulfide-linked dimer and a cytoplasmic C-terminus (shown in dark blue). The γ subunit, consisting of four transmembrane segments is showing in green and the cytoplasmic auxiliary β subunit in orange.

1.2.2.2 Cav2.2 in Pain Pathophysiology

The α1B subunit of the Cav2.2 channels is densely expressed at the terminals of peptidergic of primary afferent neurons (PAN), which project to superficial laminae I and II of the dorsal horn of the spinal cord [12]. Consistent with the anatomical localization, toxins that block Cav2.2 channels reduce the release of glutamate and neuropeptides from PAN terminals and decrease noxious stimulus-evoked activity of dorsal horn neurons [20] (for review see: [15, 21, 22]). Sensitivity to toxin block is significantly enhanced after nerve injury [10]. In behavioural models of neuropathic pain, spinal administration of Cav2.2 blockers profoundly attenuates hyperalgesia and allodynia in response to mechanical, chemical or thermal stimulation (for review see: [15, 21, 22]). The mechanism by which Cav2.2 is activated to control nociception is simplified in Figure 1-1.

13

Chapter 1

Figure 1-1. Role of Cav2.2 in Ascending Pain Pathway.

Most neurons in the superficial dorsal horn are interneurons and not projection neurons. Cav2.2 channels are localized at synapses at the projection fibers, which consist of efferent and afferent fibers, uniting the cortex with the lower parts of the brain and with the spinal cord. Therefore intrathecal administration of N-type calcium channel blockers mostly affect the transmission onto interneurons and not directly gating synapses with projection neurons [15]. ω-Conotoxins

GVIA, CVID and MVIIA (Prialt) inhibit N-type currents by selectively antagonizing Cav2.2 intraspinally. The inhibitory effect of the ω-conotoxins at the first synapse in the nociceptive system effectively interrupts pain information by interruption of spinal transmission [13].

1.2.2.3 Cav2.2 Inhibitors in Animal models of Pain

Neuropathic pain states are often produced in animals by peripheral nerve lesion with spinal nerve ligation or nerve-crush procedures, or by peripheral tissue damage with injection of inflammation-producing chemicals, such as formalin or Freud’s adjuvant [33, 71]. Many of the classical pain models were developed to reflect human diseases, allow interpretation of the type

14

Chapter 1 of pain involved and for identification of drugs that may treat these types of pain. In neuropathic pain models such as the spinal nerve ligation (SNL) and sciatic nerve chronic constriction injury (CCI), animals develop peripheral sensitization that results in neuronal hypersensitivity to mechanical and thermal stimuli [18]. Evidence from early studies suggests that Cav2.2 is not directly implicated in most acute thermal pain sensation (e.g., Cav2.2 knockout mice still had pain on hot plate and tail flick assays in rats), but instead plays a more significant role in inflammatory and chronic neuropathic conditions [10, 70].

Animals lacking Cav2.2 gene do not display pain-related behaviours following surgical- or

chemical-induced pain [72-74]. These studies suggest that Cav2.2 channels are more directly involved in chronic nociception [71]. Surgical- and chemical-induced tactile and thermal hyperalgesia are suppressed in Cav2.2 knockout (−/−) mice, although variable acute nociceptive

responses depended on the mutant lines [72-74]. The mutant Cav2.2−/− mice exhibit significantly attenuated response in Phase 2 of the formalin model, but normal pain behaviours in Phase 1 [73]. The response to visceral inflammatory pain caused by acetic acid was also reduced in Cav2.2 knockout mice, suggesting that Cav2.2 plays a major role in pain perception by acting at the spinal level, but not at the supra-spinal level [73].

In addition, the α2δ1 auxiliary subunit of the Cav2.2 channels has been reported to be up- regulated after pain states in dorsal root ganglia (DRG) neurons and spinal dorsal horn after nerve injury [75-78], suggesting an involvement of this isoform with pathophysiological mechanisms of pain [78]. Although as discussed later in this chapter, other mechanisms may underlie the involvement of the α2δ subunit with pain; studies using transgenic mice have found

that the pro-algesic effects of α2δ subunits are mediated at least partially by enhancing Cav2.2 activity in sensory neurons [78]. In this study the author suggested it occurred possibly through enhanced formation of the functional Cav complex in lipid raft micro domains, as well as through hyper-excitability in dorsal horn neurons in response to peripheral stimulation [78].

15

Chapter 1

Table 1.2. Physiological function and pharmacology of Cav channel subtypes. Cav Current Antagonist Localization Physiological Function Subtype Type Class/Name Excitation-contraction coupling, gene Cav1.1 L Skeletal muscle, transverse tubules DHP, PHA, BTZ regulation Cardiac myocytes, smooth muscle Excitation-contraction coupling, hormone Cav1.2 L myocytes, endocrine cells, neuronal cell DHP, PHA, BTZ secretion, gene regulation bodies, proximal dendrites Endocrine cells, neuronal cell bodies Hormone secretion, gene regulation, tonic Cav1.3 L and dendrites, cardiac atrial myocytes and DHP, PHA, BTZ transmitter release pacemarker cells, cochlear hair cells Retinal rod and bipolar cells, spinal Cav1.4 L DHP, PHA, BTZ Tonic neurotransmitter release cord, adrenal gland, mast cells Nerve terminals and dendrites, Neurotransmitter release, dendritic Ca2+ Cav2.1 P/Q ω-agatoxin IVA neuroendocrine cells transient currents Nerve terminals and dendrites, ω-conotoxin CVID, Neurotransmitter release, Ca2+-dependent Cav2.2 N neuroendocrine cells MVIIA,GVIA action potentials Synaptic plasticity, insulin release. Mediate Central nervous system, vascular ω-theraphotoxin- neurotransmitter Cav2.3 R system and endocrine systems. Neuronal Hg1a release through action potential generation cell bodies and dendrites (SNX-482) in dendrites and Ca2+ influx in dendritic spines

16

Chapter 1

Neuronal cell bodies and dendrites, Pimozide,

Cav3.1 T cerebellum and thalamus, cardiac and mibefradil,TTA-P2, Pacemaking, repetitive firing smooth muscles Ni2+, Zn2+

CNS: neuronal cell bodies Kurtoxin, pimozide,

Cav3.2 T and dendrites, heart, liver, kidney, mibefradil, Z123212, Pacemaking, repetitive firing lung, skeletal muscle, pancreas TTA-P2, Ni2+, Zn2+

CNS: neuronal cell bodies and Pimozide, TTA-P2, Cav3.3 T Pacemaking, repetitive firing dendrites Zn2+ Ni2+, mibefradil DHP: Dihydropyridine, PHA: Phenylalkylamine, BTZ: Benzothiazepine, Ni2+: Nickel, Zn2+: Zinc. Table adapted from: Caterall et al., 2005 [79] and Lewis et al., 2012 [80].

17

Chapter 1

1.2.2.4 Cav2.2 Channels Regulation by Auxiliary Subunits

While the pore-forming α1 subunit determines the main electrophysiological and

pharmacological properties of the Cav channels, the auxiliary α2δ and β-subunits have a significant influence on Cav channel function, intracellular trafficking and posttranslational

modifications [81-83]. To date four α2δ auxiliary subunits (α2δ1–4) consisting of extracellular

disulfide-linked α2δ dimers of 170 kDa; four β auxiliary subunits (β1–4) [84] forming a 55 kDa

cytoplasmic complex with the α1 subunit; and eight 33 kDa γ subunits (γ1–8) comprising four transmembrane segments have been identified [60] (for review see [49, 59].

Pain signals originating from peripheral C and Aδ afferent fibers evoke Cav2.2-mediated synaptic vesicle release of neurotransmitters such as glutamate, substance P, and CGRP which activate spinal neurons, altering sensory excitability and leading to pain sensations. Direct block of Cav2.2 channels by ω-conotoxins from cone snail venoms stops the link between the origin of pain and the transmission of pain sensation to the brain, because it decreases excessive calcium signalling during hyperactive excitation. Figure adapted from Zamponi et al. [68].

Clinically important, it has been reported that the affinity of the Cav2.2 inhibitors, the ω-

conotoxins, is often profoundly affected by the presence of auxiliary subunits, in particular 2 and β subunits [82, 85, 86]. However, the degree to which co-expression of auxiliary subunits affects ω-conotoxin potency can vary significantly; with MVIIA, GVIA, CVID and CVIF being particularly susceptible to affinity reductions in the presence of 21 subunits, while the potency of CVIE is affected to a lesser degree by co-expression with auxiliary subunits [82, 85]. Such pharmacological effects can have profound implications for the therapeutic potential of ω-conotoxins, as the 21 subunit’s expression is increased in dorsal root ganglion and spinal cord in several animal models of neuropathic pain [77, 82]. However, voltage-dependent reversibility varies significantly between ω-conotoxins, with CVIE and CVIF showing voltage-

dependent reversal particularly in the presence of 21 and β subunits, while reversibility of block by GVIA and MVIIA are only weakly influenced by the 21 and β2a or β3 subunit co- expression [82, 86]. Residue in the Cav channel identified to contribute to this reversibility of the ω-conotoxins block include in particular Gly1326, as well as intracellular domain II-III linker regions (Figure 1-2) [87, 88].

18

Chapter 1

1.2.2.4.1 α2δ Subunit

The α2δ proteins are auxiliary subunits of Cav2.2 that enhance Cav2.2 trafficking and insertion in the plasma membrane [89], but also influence the biophysical and pharmacological

properties of the channel (for review see: [81]). A single gene product translates the α2δ subunit, which is post-translationally cleaved into the α2 and δ parts that remain associated via

disulphide bridges. The α2 protein (~ 950 amino acids) is entirely extracellular, while the δ part

has a small extracellular part that is attached to α2 and a transmembrane domain with a very

short cytoplasmic tail [90]. The α2δ protein was originally isolated from skeletal muscle as a non-essential subunit of the L-type calcium channel complex [89]. Later it was found to be

expressed in many tissues, with the α2δ isoforms 1 and 2 being highly expressed by many CNS neurons [91]. Importantly, the isoform 1 is overexpressed after peripheral sensory nerve injury,

and thus involved in neuropathic pain conditions [75, 77]. α2δ1 and α2δ2 are the targets for the gabapentinoid drugs (gabapentin and pregabalin), which are drugs currently used in the treatment of neuropathic pain [38, 75, 92].

The α2δ subunits increase the Cav2.2 inactivation rate to different extents [93]. Specifically,

co-expression of α2δ subunits has been reported to cause hyperpolarization of the steady-state inactivation as well as an increase in the voltage-dependence [90, 93]. Importantly, co-

expression of α2δ subunit decreases the potency of ω-conotoxins [82, 86], which has implications for the therapeutic potential of these peptides. Both the physiological functions of

α2δ subunits and the mechanisms, by which the binding of gabapentinoid drugs, such as

gabapentin and pregabalin to α2δ subunits translates into therapeutic action, are not fully

understood. Intriguingly, despite binding to α2δ subunits, gabapentin and pregabalin produce

little acute inhibition of calcium channel currents. Inhibition of Cav2.2 currents after chronic

treatment with these drugs is generally attributed to down-regulation of Cav2.2 trafficking (for review see [90, 93, 94].

Although most of the role of α2δ1 in the regulation of pain has been related to regulation of

Cav2.2 function and trafficking, an alternative analgesic mechanism was proposed recently [95]. The presence of a large extracellular region containing a protein-protein interaction fold

in the α2δ1 subunit, the Von Willebrand Factor A (VWF-A) domain, suggests that the α2δ1 subunit could serve as a receptor for extracellular ligands itself [90, 95]. Indeed, it has been reported that the VWF-A domain of the α2δ1 subunit binds to proteins of the thrombospondin

family [95]. Thus, α2δ1 is proposed to be the neuronal thrombospondin receptor, which is

involved in CNS synaptogenesis (synapse formation) and synaptic maturation [95]. The α2δ1 19

Chapter 1 inhibitor gabapentin was also found to disrupt the interaction of the α2δ1 subunit with proteins of the thrombospondin family, thus leading to inhibition of synapse formation [95]. This occurred both in vitro and in vivo when neonatal mice were treated with gabapentin [93, 95], and it has been proposed that the inhibition of synapse formation represents an additional mechanism by which α2δ1 inhibitors produce analgesia [95].

1.2.2.4.2 β Subunit

Four different genes encode the β subunits (β1–β4) and numerous splice variants are known [89]. The β subunit is the only subunit of the channel that is entirely cytosolic. It has been

proposed that these subunits associate with the α1 subunit predominantly through a highly conserved high affinity interaction that is mediated by the Alpha Interaction Domain (AID) in

the α1 subunit [96] and a corresponding Beta Interaction Domain (BID) in the β subunit [97].

The β subunit aids the trafficking of α1 to the plasma membrane. This was initially thought to

occur due to its ability to mask an endoplasmic reticulum (ER) retention signal in the α1 subunit domain I-II linker [97, 98]. However, several recent studies have reported data which is inconsistent with this hypothesis (for review see [99]). Transplanting the I-II linker of the

Cav2.2 α1 subunit into Cav3.1, which does not require the β subunit for its function, caused current up-regulation rather than down-regulation [100]. Similarly, domain I-II linkers from

several different Cavα1 subunits do not cause ER retention of CD8 or CD4 reporter protein [101, 102]. In addition, several studies have implicated regions other than the I-II linker in

Cavα1 trafficking [102-104], and another study suggested that the mechanism of β subunit-

mediated Cav trafficking involves proteosomal degradation [105].

β subunit co-expression has a large effect on the level of expression, voltage dependence

and kinetics of gating of cardiac and neuronal Cav channels. In general, the level of Cav expression is increased, the voltage dependence of activation and inactivation is shifted to more negative membrane potentials, and the rate of inactivation is increased. However, these effects are β subunit specific [49], with the β2 subunit slowing channel inactivation in combination

with α1B-b/α2δ1, while the β3 induces faster inactivation in combination with α1B-b/α2δ1 [86]. Recovery from ω-conotoxin block is also influenced to varying degrees by the different β subunit isoforms co-expressed with α1B-b/α2δ subunits [85, 86].

20

Chapter 1 1.2.2.4.3 γ Subunit

The γ subunit was originally known to only be associated with the skeletal muscle voltage- gated channel complex. However, recently, expression of γ isoforms 2 and 3 were established in the brain [53, 106]. In addition, γ subunits have been found as part of a neuronal membrane

complex with Cav1.2 [107]; and genetic studies revealed the existence of a γ subunit isoform in the brain whose lack of expression is responsible for the epileptic and ataxic phenotype of the stargazer mouse [53]. The γ5 and γ7 subunits represent a distinct subdivision of the γ subunit family of proteins identified by structural and sequence homology to stargazing.

The γ subunits share a conserved four transmembrane domain topology, with predicted intracellular amino and carboxy termini, and a consensus site for cAMP/cGMP

phosphorylation [89]. Although the effects of γ subunits on the pharmacology of Cav channels

have not been as well studied as the β and α2δ subunits, a γ isoform-dependent negative effect on Cav3.1 low voltage-activated current density has been described [108]. In addition, patch-

clamp recordings showed that transient transfection of γ1 drastically inhibited macroscopic

currents through recombinant N-type calcium channels (Cav2.2/α2δ–1/β3) expressed in HEK-

293 cells [109]. The effects of most of the γ subunit isoforms at Cav2.2 have not been

determined. However, co-expression of the γ7 subunit almost abolished the functional

expression of Cav2.2 in either Xenopus oocytes or COS-7 cells [110, 111]. The γ4 subunit

affected only the Cav2.1 channel [53, 112]. The γ5 subunit may be a regulatory subunit of

Cav3.1 channels (for review see: [113].

1.2.2.5 Other Cav Regulatory Pathways

In addition to regulation by auxiliary subunits, Cav superfamily members produce currents that are regulated by direct interaction with G proteins and SNARE proteins, and in less extent by phosphorylation and protein kinases [114-116]. Whereas G-protein Gβγ subunits inhibit N- type channels by binding to the α1B domain I–II linker and the C-terminus regions [117]; SNARE proteins such as syntaxin 1A, SNAP-25, and cysteine string protein tightly interact

with the large cytoplasmic loop connecting domains II and III of both Cav2.2 and Cav2.1 channels [118-120], and it is believed that their interaction with this region of the channel is a key step in neurotransmission [121, 122]. Moreover, the association of these proteins with the channel modulates their inhibition by G-proteins [120, 123, 124] and can decrease channel availability [125-127].

21

Chapter 1

The binding of Gβγ subunits to Cav2.2 is inhibited by phosphorylation of the channel by protein kinase C (PKC) and by increase in electrical spiking activity [114, 128]. Both the domain I–II and II–III linker regions are targets for protein kinase C-dependent

phosphorylation [129, 130]. Hence, cytosolic structures of the Cav2.2 channel α1 subunit are important regulatory elements of channel activity, and any sequence variation in those regions might be expected to alter channel function and regulation [131].

2+ 2+ Ca entry through a single Cav channel can trigger vesicular release [132], and Ca - triggered synaptic vesicle exocytose depends on the assembly of the SNARE complex, in which the vesicle-associated v-SNARE protein synaptobrevin (VAMP) interacts with two plasma-membrane-associated t-SNARE proteins, SNAP-25 and syntaxin-1 [133-136]. Maturation into a release-ready SNARE complex requires synaptotagmin, an integral Ca2+- binding protein of the synaptic vesicle membrane that provides Ca2+-dependent regulation of the fusion machinery. Ca2+ influx into the presynaptic terminal binds to the Ca2+ sensor, synaptotagmin, and the SNARE complex changes conformation from a trans to a cis state, resulting in the fusion of opposing membranes and the release of neurotransmitter [134]. Different types of calcium channels are found in nociceptive pathways and have been implicated in pain.

1.2.2.6 The Role of Other Cav Channels in Pain

Some pre-clinical studies have suggested that Cav channels other than Cav2.2 also contribute

for pain pathophysiology. Cav3 channels are found in cell bodies of DRG primary afferent neurons and in free nerve endings; they participate in the initiation of the action potential in these locations by lowering the required threshold for activation (for review see ref [137]). The involvement of Cav1 channels in pain pathways have also been described and recently, clinical trials showed that L-type blockers, such as topiramine, are effective in the treatment of neuropathic pain (for review see ref [137]). However, either the studies are inconclusive, controversial, there is a lack of selective blockers, or the presence of severe side effects have

prevented these Cav channel blockers to be approved for therapeutic use (reviewed by ref [137]).

Cav2.1 is widely distributed in synapses of the central and peripheral nervous system, suggesting involvement of these channels in the perception and maintenance of some types of

pain [14, 67]. Regardless of its localization, Cav2.1 channels are certainly not considered a

good analgesic option. When a knock-out mice model was generated to study the role of Cav2.1 22

Chapter 1 channels in neurotransmission, nociception and motor function, the elimination of P/Q-type calcium channels currents altered synaptic transmission completely, resulting in severe ataxia.

The animals died at the age of four weeks [21, 138] and due to the short lifespan of Cav2.1 knockout mice, the role of this calcium channel has not been tested systematically in pain models. Furthermore mutations on Cav2.1 channels gene CACNA1A cause recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in mice. And in humans, these mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy.

1.2.3 Cav2.2 Channel Inhibitors

1.2.3.1 Indirect Inhibitors

Morphine modulates Cav2.2 channels. Binding of morphine to μ-opioid receptors leads to

inhibition of Cav2.2 through Gβγ-mediated signalling, which reduces the ability of DRG sensory neurons to propagate pain signals centrally [68, 139]. A large G-protein-dependent inhibition of Ca2+ currents results in inhibition of neurotransmitter release [49], and reduces the ability of the DRG sensory neurons to propagate pain signals. The combined administration of ziconotide with opioids produces synergistic analgesia when administered intrathecally [44, 137]. This combination therapy may help to reduce opioid tolerance and the potential for opioid-induced hyperalgesia, all of which limit the long-term usefulness of µ-opioid receptor agonists in pain medicine [57, 137].

Spinal noradrenaline injection can reduce Cav2.2-mediated transmission by presynaptic and

postsynaptic inhibition and by α1 adrenoceptor-mediated activation of inhibitory interneurons

[68]. α2-Adrenergic agonists (such as Clonidine) and non-selective small molecule transporter inhibitors (such as duloxetine and the conotoxin Xen2174), have shown effectiveness as

analgesics due to their mechanism of indirect Cav2.2 blockade.

1.2.3.2 Direct Inhibitors

A wide diversity of venomous animals, such as cone snails and spiders, have evolved a large

range of peptide toxins that target ion channels, including Cav channels, expressed in the neuronal and neuromuscular systems of prey and predators as part of efficient prey

23

Chapter 1 immobilization and deterrent strategies [80]. Accordingly, many of the most selective ion channel modulators known originate from venoms (reviewed by [80]).

1.2.3.2.1 Cav2.2 Direct Inhibitors from Cone Snails Venoms

The peptide toxins from cone snails (conopeptides or conotoxins) are small peptide neurotoxins (8‒35 amino acid residues, Table 1.4) that have evolved from a relatively small number of structural frameworks that are particularly well suited to address crucial issues such as potency and stability [1]. It is estimated that the venom of a single Conus species contains a unique array of more than 100 peptides, with less than 1% characterized pharmacologically [140], providing a myriad library of natural compounds. Based on their amino acid sequence, these small peptides can be classified as disulfide-rich and non-disulfide peptides [141].

The ω-conotoxin family consists of a range of disulfide rich peptides from cone snails that

are reference compounds used in the identification of Cav2.2, because this family preferentially

inhibit Cav2.2 channels (see Table1.3; reviewed by ref [8, 9]). GVIA from Conus geographus,

for example, has been used for many years as a probe to discriminate Cav2.2 from other closely

related Cav channel subtypes (for review see ref [14] [10–13]). In addition, MVIIA from Conus

magus, have provided the first Cav2.2 inhibitor currently on the market. Peptide toxins from the ω-family (ω-conotoxins) are part of the O-superfamily, which also includes several other

families with diverse pharmacological activities, such as inhibition of Nav channel currents

(µO-conotoxins), inhibition of Nav channel fast inactivation (δ-conotoxins) and inhibition of

Kv channels (κ-conotoxin). Interestingly, other families of conotoxins also modulate different

pain targets, such as Kv and Nav channels, nicotinic acetylcholine receptors, noradrenaline transporter, NMDA receptors, vasopressin receptors and neurotensin receptors (Table1.3) [39, 140, 141]. Indirectly, these peptides can also regulate G-protein coupled receptors, which play important roles and are frequent up-regulated in pain states, including cannabinoids, serotonin and opioids [140].

1.2.3.2.1.1 ω-Conotoxins Binding Site

While it is now appreciated that ω-conotoxins represent some of the most selective known

inhibitors of neuronal Cav2.2 and many of the key residues involved in binding have been

identified, the precise peptide binding determinants and binding sites on the Cav channels are 24

Chapter 1 still not clear. It is generally accepted that ω-conotoxins act as pore blockers [88, 142], although the binding site has been mapped primarily to the external vestibule of the channel in the domain III pore-forming S5-S6 region (see [80] for docking model). In addition, inhibition of

Cav2.2 by ω-conotoxins can be notably more complex than would be expected from simple pore blockers sharing a homologous binding site. While some of the best-characterized ω-

conotoxins, such as GVIA and MVIIA, bind nearly irreversibly to Cav2.2, reversibility can be induced by voltage protocols which take advantage of the preferential binding of ω-conotoxin

to the inactivated rather than resting state of Cav2.2 [143].

The large putative extracellular loop between IIIS5 and IIIH5 has been shown to be critically

important for the block of Cav2.2 by the extensively studied peptide inhibitor of Cav2.2, ω- conotoxin GVIA (Figure 1-2). In particular, residues Gln1327, Glu1334, Glu1337, Gln1339 [142] and Gly1326 and Glu1332 of this region were identified as being important for blockage by GVIA [144]. The latter group proposed that Gly1326 may form a barrier that controls access of peptide toxins to the outer vestibule of the channel pore and stabilizes the toxin-channel

interaction [144]. In addition, the complex between toxins MVIIA or GVIA and the Cav2.2 channel has been proposed to involve a central aromatic residue: tyrosine in the peptide, which seems to be critical for high affinity interactions, plus lateral basic residues that form salt bridges with Glu1332 and perhaps Glu1334 and Glu1337 on the Cav2.2 channels [144].

25

Chapter 1

Table1.3. Analgesic Conotoxins: Classification, Amino acid Sequence and Clinical Potential. Conotoxin Name/ Classification Target & Action Clinical Potential

µ-SIIIA Nav inhibitor Pain (preclinical lead)

µO -MrVIB Nav1.8 inhibitor Pain (preclinical lead)

δ-EVIA Nav enhancer Pain (preclinical lead)

ω- MVIIA/CVID Cav2.2 inhibitors Pain (phase IV) Pain (phase II)

χ-Xen2174 NET inhibitor Pain (phase II)

α -Vc1.1 nAChR inhibitor Pain (i.v., phase II) Conantokin – con-G NMDA antagonist Pain and epilepsy (preclinical) Contulakin Cont-G Neurotensin R agonist Pain (phase II) Table modified from Lewis, 2009 [145]. The table indicates the clinical potential and clinical phase of each pharmacologically targeting group of conotoxins.

26

Chapter 1

1.2.3.2.1.2 ω-Conotoxins Selectivity versus Side Effects

Factors that define ω-conotoxins selectivity at Cav isoforms have been the major target of neuroscientists and ω-conotoxins such as GVIA, CVID and MVIIA (Cav2.2 blocker), or

MVIIC (Cav2.1 blocker) have played a key role in the differentiation of these Cav subtypes.

While Cav2.2 is important in mediating synaptic transmission at nociceptive synapses, the other

Cav channel subtypes, most notably Cav2.1 and Cav2.3, also mediate significant neurotransmitter release at neuronal synapses [18, 146]. Thus, non-selective block of these Cav isoforms can contribute to severe side effects arising from inhibition of neurotransmitter release in non-nociceptive neurons. Accordingly, a significant challenge in targeting Cav2.2 for therapeutic drug discovery is likely to be the achieving selectivity over other Cav subtypes, especially Cav2.1, which is highly homologous to Cav2.2 (~ 64% identical).

Figure 1-2. Cav2.2 channels Topology

Represented is the pore-forming α1 subunit of the Cav2.2 channels. This large protein consists of four homologous transmembrane domains (I–IV), each domain contains six segments (S1–S6) and a membrane-associated P loop between S5 and S6 (represented in orange/grey), where the binding site of ω-conotoxins is localized. Circles, triangles and rectangles represent the localization of specific residues described to be important for binding of ω-conotoxin GVIA to the

Cav2.2 [88].

27

Chapter 1

Figure 1-3. ω-Conotoxins 3D NMR Structure and Important Residues in Side Chains. Represented in two different orientations are ω-conotoxins side chains; GVIA (PDB 1TTL, green A–B), MVIIA (PDB 1 TTK, blue C-D) and MVIIC (PDB 1CNN, pink D–E). Disulfide bridges are shown in yellow and important amino acid residues; including Y13 (tyrosine13) and K2 (lysine2), and several positively charged residues exposed in the side chain are labelled.

28

Chapter 1

Table 1.4. ω-Conotoxins AA sequences and Affinity at Displacing 125I-GVIA or 125I-MVIIA in Binding Assays. ω-conotoxin 125I-Ctx binding assays to rat brain

name ω-conotoxin Sequence IC50/Kd (nM) Reference

CnVIIA CKGKGAOCTRLMYDCCHGSCSSSKGRC* 0.4 (2.2 > 2.1) [82]

CVIA CKSTGASCRRTSYDCCTGSCRS--GRC 0.6 (2.2 > 1.2) [8]

CVIB CKGKGASCRKTMYDCCRGSCRS--GRC 7.7 (2.2~2.1 > 2.3) [8]

CVIC CKGKGQSCSKLMYDCCTGSC-SRRGKC 7.6 (2.1~2.2) [8]

CVID CKSKGAKCSKLMYDCCSGSCSGTVGRC 0.07 (2.2 > 2.1) [8]

CVIE CKGKGASCRRTSYDCCTGSCRS--GRC 0.025 (2.2 > 2.1 > 1.2~1.3~2.3 [16]

CVIF CKGKGASCRRTSYDCCTGSCRL--GRC 0.098 (2.2 > 2.1 >1.2~1.3~2.3) [16]

FVIA CKGTGKSCSRIAYNCCTGSCRS--GKC ND (2.2 > 2.1 > 3.2) [83]

GVIA CKSOGSSCSOTSYNCCRS-CNOYTKRCY* 0.04 (2.2 >2.1) [84–88]

GVIB CKSOGSSCSOTSYNCCR-SCNOYTKRCYG* ND [88,89]

GVIIA CKSOGTOCSRGMRDCCTS-CLLYSNKCRRY* 3.7 (ND) [88–90]

29

Chapter 1

GVIIB CKSOGTOCSRGMRDCCTS-CLSYSNKCRRY* ND [88]

MVIIA CKGKGAKCSRLMYDCCTGSCRS--GKC 0.055 (2.2 > 2.1) [13,81,87]

RVIA CKPPGSPCRVSSYNCCSS-CKSYNKKCG 0.25 (2.2) [10]

TVIA CLSXGSSCSXTSYNCCRS-CNXYSRKCR ND (2.2 > 2.1) [91,92]

Source: Conoserver database: www.conoserver.org.125I-Ctx = 125I-GVIA or 125I-MVIIA displacement assays to define ω- conotoxins binding to Cav2.2 expressed in different brain preparations including rat, chicken and mouse brain. ND = not determined; in brackets the order of Cav type selectivity for each ω-conotoxin is described.* O = hydoxyproline (PTM: post-translational modification). Conserved cysteine residues are indicated in bold.

30

Chapter 1

1.2.3.2.2 Cav2.2 Inhibitor Toxins from Spiders

While the venoms from spiders, such as Phoneutria nigriventer are dominated by Nav inhibitors [2], in general Cav inhibitors dominate the pharmacology of spider venoms (reviewed

by ref [3]). Unfortunately, the activity of many spider toxins at Cav2.2 has not been

characterized extensively, and many of the spider toxins preferentially target Cav channels other than Cav2.2, such as Cav2.1, Cav2.3 or invertebrate Cav [4–7].Cav2.2 inhibitor toxins from spiders are structurally diverse and share a common structural motif with the ω-conotoxins with 3 disulfide bonds forming an inhibitory cysteine knot (ICK) and as many as 7 disulfide bonds (e.g.: ω-ctenitoxin-Pn3a [148, 149]. To date the only few peptides Cav2.2 channels inhibitors identified from spiders were found in the venoms of the spiders Haplopelma huwenum, Agelenopsis aperta, Phoneutria nigriventer, Phoneutria reidyi and Segestria

florentina [148-162]. The majority of these peptides display activity at Cav1, Cav2.1 and Cav2.3 isoforms. For example, ω-agatoxin-Aa3a is equipotent at Cav1 and Cav2.2, and ω-ctenitoxin-

Pn2a from Phoneutria nigriventer blocks voltage-gated calcium channels Cav2.1, Cav2.3, Cav1 and Cav2.2 in decreasing order of potency [150]. Also, ω-ctenitoxin-Pn4a (PnTx3-6, Phα1β,

neurotoxin 3-6), a toxin from the spider Phoneutria nigriventer inhibits neuronal Cav with a

rank order of potency of Cav1.2>2.2>2.1>2.3 [149, 159, 163, 164]. In earlier studies, Phα1β elicited prolonged analgesia after intrathecal administration in an animal model of incisional pain [165, 166]. ω-ctenitoxin-Pn4a had no effect on mean arterial blood pressure, heart rate or

gross neuronal performance, suggesting that Cav inhibitors from spider venoms may also find therapeutic application with reduced side effects for the treatment of pain [165, 166].

Another non-selective toxin from Phoneutria nigriventer, ω-ctenitoxin-Pn2a (rank order of

potency: Cav2.1>2.3>1>2.2) [157], showed prevalent antinociceptive effects in neuropathic pain models and did not cause adverse motor effects at efficacious doses [167]. However, as

Cav2.1 and Cav3 channels have been proposed as analgesic targets in their own right [168, 169], the contribution of non-Cav2.2 channels to these observed in vivo effects remains to be determined. Given the similar overall structure shared by cone snail and spider venom peptides (Figure 1.4 and Figure 1-4), this divergent selectivity profile is surprising. However, small

differences in primary and tertiary structure may result in profound effects on Cav channel selectivity, as illustrated by conotoxins. Conotoxins MVIIA, MVIIC, GVIA and CVID share a high degree of sequence similarity and are structurally closely related. However, MVIIA,

GVIA and CVID are highly selective Cav2.2 blockers, while MVIIC is a selective Cav2.1 31

Chapter 1

blocker [85]. Table 1.5 lists a number of toxins from spiders with activity at Cav2.2 channels, its amino acid sequences, potency and selectivity profile.

1.2.3.2.2.1 ω-Agatoxin-Aa2a

The venom of Agelenopsis aperta provided the first source of Cav inhibitors, making the

agatoxins some of the best-studied spider Cav channel antagonists. Based on their structural homology and pharmacological properties, agatoxins have been classified into four distinct

groups (agatoxins I –IV). While type I and III agatoxins are selective for Cav1 and Cav2.1, respectively, type II and III agatoxins display activity at Cav2.2. However, while type III

agatoxins such as ω-agatoxin-Aa3a are active at all high-threshold Cav channel isoforms,

including Cav2.1, Cav2.2, Cav2.3 and Cav1, type II agatoxins target Cav2.2 over other Cav isoforms [153, 154]. ω-agatoxin-Aa2a, an 11 kDa mature toxin comprised of 92 residues, displaced ω-conotoxin GVIA binding and synergistically blocked neurotransmitter release with the unrelated L-type toxin ω-AGTX-Aa1a [153]. While more detailed selectivity studies have not been carried out, this suggests that the toxin targets primarily Cav2.2 channels [154].

The structural requirements for high affinity inhibition of Cav2.1 by type IV agatoxins such as ω-agatoxin-Aa4a have been relatively well defined, and are proposed to involve a positively charged area, formed by several basic amino acid residues near the hydrophobic C-terminus [170], as well as a crucial tryptophan residue in position 14. In contrast, nothing is known about the structure-activity of ω-agatoxin-Aa2a. Thus, future studies are necessary to improve our understanding of the molecular interaction between ω-agatoxin-Aa2a and Cav2.2 channels.

1.2.3.2.2.2 ω-Theraphotoxin-Hh1a

ω-Theraphotoxin-Hh1a (Huwentoxin 10 or HWTX-X) was isolated from the venom of the Chinese bird spider and shares several properties with the ω-conotoxins [148]. In contrast to ω-conotoxins, the C-terminus of HWTX-X is not amidated (Table 1.5, Figure 1-4) [148].This relatively small 28 residue peptide is stabilized by three disulfide bonds, and shares a functional motif, defined by a critical aromatic residue and several basic residues, with the ω-conotoxin GVIA (Figure 1.4 and Figure 1.5). Intriguingly, huwentoxin 10 was unable to inhibit twitch responses of electrically stimulated rat vas deferens, suggesting selectivity for a different

Cav2.2 splice variant isoform [148] . While the analgesic potential of ω-theraphotoxin-Hh1a

32

Chapter 1 has not been assessed to date, intraperitoneal injection of this toxin in mice produced no toxic effects [148], suggesting that this peptide could be a promising therapeutic lead for the treatment of pain.

1.2.3.2.2.3 ω-Segestritoxin-Sf1a

ω-Segestritoxin-Sf1a (SNX-325) has been described as a selective Cav2.2 inhibitor, based

on its ability to inhibit Cav2.2 responses in oocytes as well as KCl-mediated neurotransmitter release in hippocampal slices [162]. Interestingly, although ω-segestritoxin-Sf1a shares little structural homology with the ω-conotoxins and is a large peptide stabilized by 4 disulfide bonds, it shares a common binding site with the ω-conotoxins and was able to displace

radiolabelled MVIIA from a rat brain synaptosome preparation [162]. Cav channel inhibitors from other venomous animals

Cav modulators are also found in the venom of other venomous species, including snakes, scorpions, and centipedes. However, these peptides, including calcicludine from the green mamba [171] and kurtoxin from the venom of the scorpion Parabuthus transvaalicus [172],

show no selectivity for Cav2.2, or in the case of glycerotoxin [173]from the marine blood worm

Glycera convulata, are Cav2.2 enhancers and are thus included here only for completeness.

1.2.3.2.2.4 Venom peptides from scorpion

Kurtoxin, a 63-residue peptide isolated from the venom of the scorpion Parabuthus transvaalicus is related to the α-scorpion toxins, a family of toxins that slow inactivation of

Nav channels. While kurtoxin has shown selectivity for heterologous expressed Cav3 or T-type

calcium channels[174], activity at Cav2.2 has been described in rat sympathetic and thalamic

neurons [172]. In contrast to ω-conotoxins with activity at Cav2.2 channels, scorpion toxins

with activity at Cav channels, including kurtoxin, act as gating modifiers. Thus, selectivity differences observed in overexpression systems compared to neurons may be due to the influence of auxiliary subunits, although this has not been assessed to date.

33

Chapter 1

1.2.3.2.2.5 Venom Peptides from Snakes

Calcicludine (60 residues, 3 disulfide bonds) and calciseptine (60 residues, 4 disulfide bonds) were isolated from the venom of the black mamba, Dendroaspis polyepsis, and the green mamba, Dendroaspis angusticeps, respectively. While calciseptine inhibits L-type

calcium channels and has no activity at Cav2.2 [175], calcicludine is less selective and potently

inhibits all high-voltage-activated calcium channels, including Cav2.2 [176].

1.2.3.2.2.6 Venom Peptides from Centipedes

Recently, two peptides with activity at neuronal Cav channels were isolated from the venom of the centipede Scolopendra subspinipes mutilans. ω-SLPTX-Ssm1a, an 83 residues peptide

containing 7 cysteines was found to act as an activator of Cav channels [177], while a smaller

peptide, ω-SLPTX-Ssm2a (54 residues) was shown to inhibit Cav channels expressed in DRG

neurons [177]. However, the Cav subtype selectivity of these peptides is currently unknown but

it may include Cav2.2, which is expressed in peripheral sensory neurons.

1.2.3.2.2.7 Venom Peptides from Assassin Bugs

The predatory assassin bugs (Hemiptera: Reduviidae) contain a complex mixture of small and large peptides in its toxic saliva which is used to immobilize and pre-digest their prey, and for defence against predators [178]. Three novel peptide toxins, named Ado1, Ptu1 and Iob1, isolated from three species of assassin bugs (P. turpis, A. dohrni, and L. obscurus), were biologically active in electrophysiological assays using BHK-N101 cells stably expressing rabbit Cav2.2, β1A subunit, and α2δ subunit [178]. Ptu1 binds reversibly to Cav2.2 with lower affinity than ω-conotoxin MVIIA. Ptu1 lacks most of the residues shown to be important for ω-conotoxin binding to the N-type calcium channel, including equivalents of Tyr13 or Lys2 (Figure 4−5). This peptide belongs to the ICK family, which consists of a four-loop Cys scaffold forming a compact disulfide-bonded core [179]. Thus, as for the other venom peptides

compared here, the structure of Ptu1 aligned with related Cav2.2 blockers MVIIA and GVIA, indicating that a common functional motif can be supported by a common three-dimensional structure despite the lack of sequence homology (Table 1.5).

34

Chapter 1

Table 1.5. Cav2.2 Inhibitor Toxins from Spider Venoms. Functional Toxin name/ Refere (IC50)/ Binding Amino acid sequence Synonym nces (Kd) at Cav2.2 μ/ω-theraphotoxin- 100nM/ ACKGVFGACTPGKNECCPNRVCSDKHKWCKWKL [110,111] Hh1a/Huwentoxin-1 (ND) μ/ω-theraphotoxin- (ND) ACKGVFGACTPGKNECCPNRVCSDKHKWCKWKL [112] Hh1b/Huwentoxin1a3 μ/ω-theraphotoxin (ND) ACKGVFDACTPGKNECCSNRVCSDKHKWCKWKL [112,113] Hh1c/Huwentoxin1a10 μ/ω-theraphotoxin- (ND) ACKGVFDACTPGKNECCPNRVCSDEHKWCKWKL [112] Hh1d/Huwentoxin-1a6 ω-agatoxin-Aa2a/ Binding GCIEIGGDCDGYQEKSYCQCCRNNGFCS [114,115] ω-agatoxin IIA 10 nM/(Y) ω-agatoxin-Aa3a 1.4nM/ SCIDIGGDCDGEKDDCQCCRRNGYCSCYSLFGYLKSGCKCV [116,133] /ω-agatoxin IIIA 170 pM (N) VGTSAEFQGICRRKARQCYNSDPDKCESHNKPKRR ω-agatoxin-Aa3b/ 140nM/ SCIDFGGDCDGEKDDCQCCRSNGYCSCYNLFGYLKSGCKCE [116,117] ω-agatoxin IIIB 2.4 nM (N) VGTSAEFRRICRRKAKQCYNSDPDKCVSVYKPKRR ω-agatoxin-Aa3d/ 35 nM (N) SCIKIGEDCDGDKDDCQCCRTNGYCSXYXLFGYLKSG [116] ω-agatoxin IIID ω-agatoxin-Aa3f/ SCIDIGGDCDGEKDDCQCCRRNGYCSCYSLFGYLKSGCKCV 1.4 nM (N) [116] ω-agatoxin IIIA (58T) VGTSAEFQGICRRKARTCYNSDPDKCESHNKPKRR ω-agatoxin-Aa3g/ SCIDFGGDCDGEKDDCQCCRSNGYCSCYNLFGYLRSGCKCE 2.4 nM (N) [116] ω-agatoxin IIIB (35R) VGTSAEFRRICRRKAKQCYNSDPDKCVSVYKPKRR ω-agatoxin-Aa3h/ SCIDFGGDCDGEKDDCQCCRSNGYCSCYSLFGYLKSGCKCE 2.4 nM (N) [116] ω-agatoxin IIIB (29S) VGTSAEFRRICRRKAKQCYNSDPDKCVSVYKPKRR ω-ctenitoxin-Pn2a/ [7,118,11 > 320nM (N) GCANAYKSCNGPHTCCWGYNGYKKACICSGXNWK Neurotoxin Tx3–3 9] ω-ctenitoxin-Pn3a/ SCINVGDFCDGKKDDCQCCRDNAFCSCSVIFGYKTNCRCEV 50 pM (N) [119] Neurotoxin Tx3–4 GTTATSYGICMAKHKCGRQTTCTKPCLSKRCKKNH 35

Chapter 1

ω-ctenitoxin-Pn4a/ ACIPRGEICTDDCECCGCDNQCYCPPGSSLGIFKCSCAHANK Neurotoxin Tx3–6 122 nM (N) [119,120] YFCNRKKEKCKKA PnTx3–6/Phα1β ω-ctenitoxin- ACAGLYKKCGKGVNTCCENRPCKCDLAMGNCICKKKFVEF >1000 nM (N) [121,122] Pr1a/Neurotoxin PRTx3–7 FGG

ω-segestritoxin- GSCIESGKSCTHSRSMKNGLCCPKSRCNCRQIQHRHDYLGK ~ 10 nM (Y) [123] Sf1a/SNX-325 RKYSCRCS

ω-theraphotoxin- 40 nM (Y) KCLPPGKPCYGATQKIPCCGVCSHNKCT [113] Hh1a/Huwentoxin-10

Source: Arachnoserver spider venom database: http://www.arachnoserver.org [4,5]. Cav2.2 channels selectivity. (Y) = yes, (N) = no, ND = Not determined; Binding: 125I-Ctx performed in different brain preparations, including rat, chicken and mouse brain.

36

Chapter 1

Figure 1-4. NMR Solution Structure of the Spider ω-theraphotoxin-Hh1a (HWTX-X) and the assassin bug toxin Ptu1. The structure of huwentoxin 10 (HWTX-X) (blue A–B) and Ptu1 (pink C–D) showing two different orientations. The position of the four loops is indicated, and disulfide bridges are shown in yellow. Important amino acid residues described to have similar function to tyrosine 13 and lysine 2 in the ω-conotoxins are represented, including Y10 and K7 in Ptu1, as well as phenylalanine 13 (F13) and K17 (lysine 17) and several positively charged residues exposed in the side chain are labelled.

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Chapter 1

1.3 CONCLUSION

Chronic or persistent pain is a major therapeutic challenge, because currently available therapeutics are either ineffective in some patients, have reduced efficacy over time due to

development of tolerance, or induce intolerable side effects [7, 34]. Cav2.2 controls nociception and neurotransmission, as it is expressed in nociceptive fibres, where Cav2.2 is up-regulated

after nerve injury. Cav2.2 inhibition is a validated analgesic strategy in animal and humans.

Ziconotide, the only Cav2.2 inhibitor currently on the market, has been discovered from the snail Conus magus venom and it is active in patients with severe neuropathic chronic pain. The use of Ziconotide has been limited by its dose- related toxicity and the requirement of intrathecal administration [43-45, 180]. Ziconotide can be used as an alternative to patients that have developed tolerance to opioids, or do not respond to other treatment types. An important advantage of ziconotide over other common drugs in the market is that it does not cause dependence or tolerance. Additionally, the association of ziconotide and morphine seems to be additive, as this association increases efficacy and reduces side effects of both drugs.

Spider and cone snail venoms are unexploited rich sources of potent Cav2.2 inhibitor toxins. Despite its analgesic properties the potential of these toxins is still unexplored, as is their potential for use in the treatment of neuropathies. The investigation of toxins naturally occurring in animal venoms may provide tools for understanding mechanisms involving pain pathophysiology, certainly will at least guide drug design and might lead to the development of new drug leads.

38

Aims

1.4 THESIS AIMS

1. The main aims of this thesis were: 2. To establish new functional assays for the discovery and pharmacological characterization

of novel Cav2.2 antagonists.

3. To isolate novel Cav2.2 antagonists from venom libraries. Specifically identify novel

Cav2.2 inhibitor toxins from cone snail and spider venoms. 4. To characterize the pharmacology of these toxins, as well as investigate the role of the splice variants and auxiliary subunits on their pharmacology.

5. To better understand the mechanisms regulating Cav2.2 channels and its auxiliary subunits on the pathophysiology of pain.

6. To characterize the pharmacology of Cav2.2 endogenously expressed in human neuronal cells using these toxins 7. To study the pharmacology, structure and function of the novel analgesic toxins and compare with studies of similar toxins; to increase our understanding of the mechanisms involving potency, reversibility and selectivity of the ω-like toxins. And finally, to try to discover novel analgesic toxins and provide information for the rational design of analogues.

39

Chapter 2

Chapter 2

40 Chapter 2 Chapter 2 Development of High Throughput

Cav2.2 Assay & Characterization of ω-Conotoxins Pharmacology

2.1 FOREWORD

This chapter is based on a peer reviewed journal article, which has been published in Plos one, 2013 [3]. A pdf version of the article has been attached as Appendix 2. In comparison with the peer reviewed article this chapter has some expanded methods, results, discussion and additional unpublished data. Work contributed by others consisted mostly of proof reading and editing, as indicated in the statement of contribution by others to the thesis, at the preliminary pages of this thesis. Specifically, Dr Lotten Ragnarsson assisted with PCR designing, manuscript proof reading and editing. Dr Irina Vetter assisted with pharmacological assays’ design, guidance and manuscript proof reading and editing. Prof Richard Lewis assisted with guidance in experiment designing, manuscript proof reading and editing.

2.2 AIMS OF THIS CHAPTER

 To establish a human functional high-throughput screening assay suitable for the

identification and isolation of novel Cav2.2 inhibitor toxins from venoms.  To characterize the pharmacology of and involvement of auxiliary subunits in the pharmacology of the ω-conotoxins.

2.3 INTRODUCTION

Despite the physiological significance and proved therapeutic relevance of Cav2.2, it still remains under-exploited as a drug target. Animal venoms represent complex natural compound libraries which have evolved over millions of years, and contain some of the most subtype-

selective Cav2.2 channel modulators known [140]. Assay-guided fractionation, the process which bioactive compounds from venoms are isolated based on sequential rounds of fractionation and activity testing, has been used for decades as a successful strategy in the

41 Chapter 2 discovery of novel therapeutic leads (reviewed by ref [181]). This process requires sensitive, accurate and robust assays to detect activity at biological targets.

A range of assay types have been used for the discovery and characterisation of new Cav2.2 inhibitors, including electrophysiology, radioligand binding and fluorescence-based Ca2+ assays [182-186]. Although electrophysiology has been the most popular method used, and this method provides important information, such as the kinetics, voltage and state-dependency, this technique is generally labour-intensive and low-throughput. Despite advances in high throughput electrophysiology for primary drug screening, these new technologies are difficult to use and costly to implement [185, 187]. Therefore, while electrophysiology remains the ideal technology for ion channels characterization in a second phase, it remains relatively poorly suited to high-throughput screening at the early phases of discovery. Therefore, incorporation of functional cell-based high-throughput screening (HTS) assays can significantly accelerate the identification of novel candidates from large compound libraries [183, 185], such as the animal venoms.

Radioligand binding assays are low cost and amenable to HTS, therefore it can be considered for use in a first phase of discovery. However, no functional information is provided by binding assays, and thus compounds that bind but do not modulate function can be detected, which may increase the rate of false positives [188]. Nevertheless, the membrane potential, as well as the association of the pore-forming  subunit of Cav2.2 with auxiliary subunits is disrupted in this assays, which may influence binding affinity determination [189]. Additionally, voltage and state-dependent mechanisms cannot be measured using radioligand

binding assays. Given the narrow safety window of some Cav2.2 pore blocking peptides and small molecules, state-dependent activity may provide improved therapeutic margins (reviewed by: [190]).

The co-expression of the auxiliary α2δ subunit with the α subunit of the Cav2.2 channels has been extensively described to influence the pharmacology of ω-conotoxins, reducing the affinity for recombinant and native channels [3, 82, 86]; additionally, the auxiliary β subunit was found to be involved in determining reversibility of the ω-conotoxins blockade [86].

Considering that α2δ, β and γ auxiliary subunits and their splice variants have varied expression levels in different tissues and disease states (for review see: [83]), for example the α2δ subunit is overexpressed in nerve injury and pain states [82], auxiliary subunits must be co-expressed with α subunits for the study and identification of novel ω-conotoxins. Although auxiliary subunits can be co-transfected with the main subunit in heterologous expression systems, and

42 Chapter 2 this system provides better control of the specific receptors and ion channel types expressed, native systems are more complex models which, when characterized, can provide valuable information of the pharmacology of compounds interfering with the pathophysiology of the channels. Particularly in native systems, auxiliary subunits are present in a more complex context, similar to the native organism.

Functional HTS fluorescent-cell based assays can address some of the problems above mentioned for other types of assays. For example, native and heterologous expression systems can be used, and the Cav2.2 α subunit can be co-transfected with the auxiliary α2δ, β and γ subunits, to produce channels with biophysical properties more similar to the native channel

[182]. In one study the potassium channel Kir2.3 was co-expressed with Cav2.2 α and the auxiliary subunits, to control membrane potential [183]. This mechanism allowed additional identification of novel state-dependent inhibitors [183]. Nevertheless, cell lines expressing

Cav2.2 and auxiliary subunits endogenously can be used in HTS to provide information on the mechanisms of novel toxins inhibition, in a native context. Thus, functional HTS assays are

expected to accelerate the identification and pharmacological characterization of novel Cav2.2 modulators.

SH-SY5Y human neuroblastoma cells are derived from human sympathetic neuronal tissue and maintains essential properties of nerve cells in culture, providing a useful model for the characterisation of molecules affecting function of neuronal targets, including endogenously expressed human Cav channels [191-193]. Particularly, SH-SY5Y cells have been widely used

to study Cav2.2 modulators [191, 194] and additionally, the SH-SY5Y cell line is amenable to

fluorescent HTS assays [195]. However, still little is known about Cavα subtypes, splice isoforms and auxiliary subunits expressed natively in these cells, limiting the interpretation of pharmacological data. Thus, this model system needs to be characterized before it can be used in HTS assays.

2.4 MATERIAL AND METHODS

2.4.1 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Cav channel subtype and auxiliary subunits mRNA expression profiles were investigated in SH-SY5Y cells using standard RT-PCR and specific primers. The primers were designed using The Basic Local Alignment Search Tool (BLAST) [196, 197], or otherwise specified as

43 Chapter 2 previously described in the literature. The primers used to identify Cav subtypes and auxiliary subunits were designed so that all splice variants of specific isoforms would be amplified. On

the other hand, primers to amplify Cav2.2 splice variant isoforms were designed to be specific to each isoform. PCR conditions to detect splice variants were set as previously described [63, 87], with gradient PCR performed for all sets of primers, allowing the identification of optimal annealing temperatures. Primer sequences, Gene Bank reference numbers, predicted PCR product sizes, and optimum annealing temperatures are shown in Table 2.1. Different sets of primers were used to identify the full length and isoforms Δ1 and Δ2 (see Table 2.1) [87].

These primers were designed based on the region of the domain II-III linker of Cav2.2 channels,

as previously described [87]. Primers used to identify the full-length α1B1 and short α1B2 isoforms were designed based on the C-terminus region [63]. Data is representative of at least three independent experiments.

2.4.2 RNA Isolation, Synthesis of cDNA, PCR Reaction and Controls

SH-SY5Y cells (1 x 106) were harvested and total RNA isolated using Trizol® Reagent (Invitrogen, Carlsbad, CA). The isolated RNA was subsequently treated with RNase-free DNase to remove any genomic DNA contamination. RNA concentration was determined by absorbance measurements at 260 nm. DNA purity/integrity was accessed by analyzing the ratio 260/280 nm with a Nanodrop® (Thermo Scientific). Synthesis of first strand cDNA was performed using 1 µg of the extracted RNA and the Omniscript Reverse Transcription Kit (Qiagen), according to the manufacturer’s instructions. cDNA amplifications were performed using Taq Polymerase (New England Biolabs, US). The reaction mix (total 25 µL) included (µL): 1 cDNA (100 ng), 0.125 of the enzyme, 0.5 reverse and 0.5 forward primers (10 µM), 0.5 dNTPs (10 mM), 2.5 Thermopol reaction buffer (10x) and nuclease free water. RT-PCR was carried through as an initial denaturation step at 95°C for 3 min followed by 35 cycles of the steps: 95°C for 30s, optimal annealing temperature, as previously determined (Table 2.1) for 60s, 68°C extension for 60s, plus an extra 5 min elongation step at 68°C. PCR products were analyzed by 1% agarose gel and predicted sizes estimated by comparison with DNA molecular weight makers (50 and 100 bp ladder, New England Biolabs). Target-specific primers for the housekeeping gene GAPDH were designed as previously described [198]. PCR master mix using random primers without cDNA was used as negative gDNA control in all PCRs. Specificity of primers was demonstrated in a range of control experiments (data not shown), including detection of Cav2.2 plasmid but no other Cav subtypes by Cav2.2 primers;

44 Chapter 2 and absence of detectable levels of Cav2.2 in HEK cells. β1 and α2δ1 primers were positive for

β1 and α2δ1 plasmids, while the same primers were negative for β2–4 and α2δ2–3 (data not shown),

indicating primers were selective for β1 and α2δ1 auxiliary subunits. In addition, identity of PCR products was further confirmed by sequencing analysis (data not shown). Figures 1A–D is representative of the average of 3–10 individual experiments.

2.4.3 Agarose Gel Electrophoresis

PCR reactions were run by electrophoresis on a 2% agarose gel (Sigma-Aldrich) prepared with 0.5% TBE (Tris-Borate-EDTA) buffer, containing in addition 10% SYBR® Safe DNA gel stain for band fragment visualisation. An aliquot (10 µL) of each PCR reaction was mixed with 2 µL 6x loading dye (New England Biolabs), applied to the gel and run at 100 V for approximately 40 min. Bands were visualized on the Gel Doc System (BioRad).

2.4.4 Sequencing PCR Products

PCR amplicons were first separated on agarose gels and bands of expected sizes identified. PCR products were purified using the Wizard SV Gel and PCR clean-up system (Promega), and a sample of each purified PCR product was sent for sequencing at the Australian Genome

Research Facility. cDNA sequences of human Cav subtypes and auxiliary subunits were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Entrez/) and BLASTn [199] was used for comparison and confirmation of the identity of human Cav subtypes and auxiliary subunits.

2.4.5 Cell Culture

The human neuroblastoma SH-SY5Y cells (Victor Diaz, Goettingen, Germany) were

cultured and routinely maintained at 37°C and 5% CO2 in RPMI 1640 antibiotic-free medium (Invitrogen, Victoria) supplemented with 10% heat-inactivated FBS and 2 mM GlutaMAX™ (Invitrogen, CA, US). Trypsin/EDTA was used to detach the cells from the T-75 or T175 flasks and cells were split in a ratio of 1:5–1:10 every 34 days or when ~ 80% confluent.

2.4.6 Whole Cell Preparation for the Radioligand Binding Assay

Whole cells were prepared as described for SH-SY5Y cell membranes with the following modifications: After cells were harvested and centrifuged, the supernatant was discarded and the pellet re-suspended in binding buffer to plate 600.000 cells per well in 50 µL, in triplicates

45 Chapter 2 on 96 well-plates. Specific ω-conotoxins binding was determined using the same concentration of protein as that used for SH-SY5Y cell membranes (20 µg/50 µL), corresponds to 600.000 cells per well.

2.4.7 Membrane Preparation for the Radioligand Binding Assays

Radioligand binding assays were performed using rat brain or SH-SY5Y cell membranes prepared as described by Wagner, et al., 1988 [200] with slight modification. For rat brain membranes, male Wistar rats weighing 175‒250 g were sacrificed by cervical dislocation and the whole brain was rapidly removed and dissected on ice. At 4°C, tissue was re-suspended in 50 mM HEPES, pH 7.4 (50 mg wet weight tissue/ml buffer), homogenized using a Brinkmann Polytron homogenizer and centrifuged for 15 min at 40,000 x g. The pellet was re-suspended in 50 mM HEPES and 10 mM EDTA at pH 7.4, incubated on ice for 30 min and centrifuged at 40,000 x g for 10 min. The pellet was then re-suspended in 50 mM HEPES (pH 7.4), containing 10% glycerol. Aliquots were made and kept at –80°C prior to use. SH-SY5Y cell membranes were harvested using trypsin/EDTA, washed once with DPBS, and centrifuged for 4 min at 500 x g. After centrifugation, the supernatant was discarded and the pellet re- suspended in 10 ml binding assay buffer at pH 7.2 containing (mM): 20 HEPES, 75 NaCl, 0.2 EDTA, 0.2 EGTA and complete protease inhibitor (Roche Diagnostics, AU) and sonicated. The homogenates were then centrifuged for 30 min at 40,000 x g and 4°C. The supernatant was discarded and the pellet dissolved in aliquots of binding assay buffer containing 10% glycerol stored at –80°C prior to use. Bicinchoninic acid (BCA) assay reagent (Pierce Rockford, IL, USA) was used for protein quantification.

2.4.8 [125I]-GVIA Radioligand Binding Assay

Tyr22-[125I]-GVIA, was prepared using IODOGEN, as previously described by Ahmad [201], purified using reverse phase HPLC and stored at 4°C for use within 3 weeks. On the day of the assay, membranes were thawed on ice and reconstituted to 10 µg/50 µL (rat) or 10–20 µg/50 µL (SH-SY5Y) in binding assay buffer containing 2% complete protease inhibitor and 0.1% bovine serum albumin (BSA). Stock [125I]-GVIA was diluted to 20000 cpm/50 µL or 30 pM. For displacement studies, [125I]-GVIA was incubated with rat brain or SH-SY5Y membranes or whole cells and varying concentrations of the competing ligand in triplicates in 96 well plate formats. The plates were incubated with shaking for 1 h at room temperature and vacuum filtered through a glass fibre filter pre-soaked in 0.6% polyethyleneimine (PEI), to

46 Chapter 2 reduce non-specific binding and washed with buffer containing (mM) 20 HEPES and 125 NaCl at pH 7.2 using a vacuum system (Tomtec harvester). The filters were then dried at 37°C before being placed in sample bags and soaked in liquid scintillant. Radioactivity was counted using a Microbeta Jet (Wallac, Finland). The non-specific binding was determined in the presence of 50 µL of unlabelled peptides.

2.4.9 Intracellular Ca2+ Measurement using the FLIPR

SH-SY5Y cells were seeded onto 96-well or 384-well flat, clear bottom, black-walled imaging plates (Corning, Lowell, MA, US) at a density of 160,000 (96-well plate) or 40,000 (384-well plate) cells/well, respectively, resulting in 90–95% confluent monolayer after 48 h. On the day of the Ca2+ imaging assays, cells were loaded for 30 min in the dark at 37°C with 5 µM Fluo-4 acetomethoxyester (Fluo-4-AM), in physiological salt solution (PSS composition:

NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM, MgCl2 1.4 mM, NaH2PO4 1.2 mM, NaHCO3

5 mM, CaCl2 1.8 mM, HEPES 10 mM, pH 7.4) containing in addition 0.3 % BSA and 10 µM nifedipine. After the incubation period, the cells were washed once with 100 µL assay buffer (no Fluo-4-AM) and replaced with 100 µL of the same buffer containing in addition 10 µM nifedipine. Plates were transferred to the FLIPRTETRA (Molecular Devices, Sunnyvale, CA) fluorescent plate image reader; camera gain and intensity were adjusted for each plate to yield between 800–1000 arbitrary fluorescence units (AFU) baseline fluorescence, and Ca2+ responses were measured using a cooled CCD camera with excitation at 470–495 nM, and emission at 515–575 nM. Ten baseline fluorescence readings were taken prior to the addition of antagonists, and then fluorescent readings every 2 s for 300 s before 90 mM KCl/5 mM

CaCl2 stimulant buffer was added and fluorescence readings again recorded each second for

further 300 s. To ensure full inhibition of Cav1 responses, the cells were pre-incubated for 40–

60 min with 10 µM nifedipine. To ensure full inhibition of Cav2.2 responses, the cells were pre-incubated for 10 min with 1–3 µM CVID. This time was found to be important for

nifedipine and CVID to reach literature IC50 values, however, to reach potencies similar to that described in the literature, mibefradil and pimozide were pre-incubated for 60 min, before the addition of stimulant buffer and 5 extra minutes of fluorescence readings were taken.

47 Chapter 2

2.4.10 Statistical Analysis

Concentration-response curves were determined following nonlinear regression analysis using a 4-parameter Hill equation, with variable Hill slope fit to the functional assays data, and one site fit to the radioligand binding assays; and normalized using GraphPad Prism (Version 5.00, San Diego, California). In displacement studies the concentrations of displacer producing

50% displacement of specific binding (IC50) is obtained by computer-assisted curve fitting (GraphPad Prism). Specific binding was calculated as the difference between total and non- specific binding. In the functional assays, negative and positive controls (PSS buffer and KCl

90 mM + 5 mM CaCl2, respectively) were used to normalize data. All data is presented as mean ± SEM of 6–10 independent experiments performed in triplicate, unless otherwise stated. Statistical significance was determined using analysis of variance (ANOVA) or student’s t- test, with statistical significance defined as p < 0.05, unless otherwise stated.The F/F values were calculated for all experiments using Microsoft Excel to normalise the raw fluorescence data generated by the FLIPRTetra and the following formula: The baseline (B) value for each individual well is subtracted from the relative fluorescence unit (RFU) generated by the FLIPR. This value is then divided by the baseline: F/F = (RFU – B)/B.

2.4.11 Z’ Score as a Measurement of Robustness of Assays

The Z’ factor was determined as previously described [202]. For each 96 well plate, 48

replicates of a Cav2.2 negative control (CVID + 10 uM Nifedipine) and 48 replicates of a Cav2.2 positive control (KCl 90 mM+ CaCl2 5 mM + 10 μM nifedipine) were assessed. Mean and standard deviation for positive and negative controls were determined using GraphPad Prism (Version 4.00, San Diego, California) and the Z’ factor for each plate determined according to

the following equation: Z’ = 1 – ((3SDpositive + 3SDnegative)/(meanpositive – meannegative)) (see Figure 3–3) .

2.4.12 SH-SY5Y cells express multiple Cav isoforms and auxiliary subunits

The expression of mRNA transcripts for Cav subtypes and auxiliary α2δ, β and γ subunits isoforms assessed in SH-SY5Y cells by performing RT-PCR using specific primers (Figure

2-1 A–D). Bands with the predicted sizes were detected for Cav1.3, Cav2.2, and Cav3.1, while

Cav1.1, 1.2, 1.4, 2.1, 2.3, 3.2 and 3.3 were not detected (Figure 2-1 A). In addition, bands of

expected sizes for β1, β3, β4, α2δ1–3, γ1, 4, 5 and γ7 auxiliary subunits (Figure 2-1 C–D) were

48 Chapter 2 also identified. Since splice variants can be generated by alternative RNA processing, which can influence function and pharmacology [87], we also investigated the expression of some human splice variants [63, 87]. PCR bands with the predicted sizes for Cav1.3 isoforms 1 and

2; full length Cav2.2, α1B1 (Gene bank accession number M94172.1), shorter α1B variant, α1B2

(Gene bank accession number M94173.1) [63]; and Δ1 (but not Δ2) [87, 203] were detected for the first time in the SH-SY5Y cells (Figure 2-1 A).The best annealing temperature for each gene analysed was determined using gradient PCR protocols in a range of rounds of control

experiments, prior to testing each Cav gene-specific primer in its best temperature and condition for the PCR using specific primers. Target-specific primers for the housekeeping gene GAPDH were designed as previously described [198] and GAPDH was detected in all PCRs, indicating amplifications were cDNA specific. PCR master mix using random primers without cDNA was used as negative gDNA control in all PCRs. Specificity of primers was demonstrated in a range

of control experiments (data not shown), including detection of Cav2.2 plasmid but no other

Cav subtypes by Cav2.2 primers; and absence of Cav2.2 in HEK cells. β1 and α2δ1 primers were

positive for β1 and α2δ1 plasmids, while the same primers were negative for β2–4 and α2δ2–4 (data not shown), indicating primers were selective for β1 and α2δ1 auxiliary subunits. The identity of each of these PCR products, including 1, 4, 5 and 7, was confirmed by sequencing.

49 Chapter 2

Table 2.1. Primers Used for Identification of Cav Channels Isoforms Expressed in SH-SY5Y Cells. Subtype/ Tempe Current Accession/Ref Forward/ Reverse Size Auxiliary rapture type Number Primers (bp) Subunit (οC)

Cav1.1 L NM_000069.2 CGCATCGTCAATGCCACCTGGTTTA 623 ND

AGCACATTGTCGAAGTGGAAGTCGC 58 (Chiou, Cav1.2 L CTGCAGGTGATGATGAGGTC 502 2006) GCGGTGTTGTTGGCGTTGTT

Cav1.3 L EU 363339.1 ACCCCCACCTGTAGGATCTCTCTCC 541 68

TCCTGACACTAGTCGAAGTGGTCGC

Cav1.3 L NM_001128840.1 GCTGCTGTGGAAGTCTCTGTCAAGC 343 68

TCAGTGATTCCACCACACACCACGA

Cav1.4 L NM_005183.2 AGGGACCCCTAAGCGAAGAAACCAG 899 ND

ACCCCATGGCATCTTGCATCCAGTA

Cav2.1 P/Q FJ040507.1 AGGACGAGGACAGTGATGAA 365 ND GCAGAGGAAGATGAA GGA AA

Cav2.2 N NM_000718.2 GGAACTGACTTCGACCTGCGAACAC 754 60 (α1B1) M94172.1 CCTCCTCTGCGTGGATCAGGTCATT

Cav2.2 NM_001243812.1 Altered GCTCAGCCGTGGGCTTT 854 62 (α1B2) (Williams et al., 1992a)

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N AGCTTGCCTGAGTGAGCATT Altered Cav2.2 α1BΔ Bp 22 + 34 AGGAGATGGAAGAAGCAGCCAATCA 900 58 N CCTTTCTGGTGTTTCATCTGGTGCA Bp 23+ 33 (Kaneko et al., CCAGAGGATGCAGACAATCAGCGGA 900 60 2002) GCATCTTCTACCTGTCGAGGTACGC Altered CAGCCAATCAGAAGCTTGCTCTGCAAA/AGCTTTCGT Cav2.2 α1BΔ Bp 21 + 31 700 65 N TTGCGGTGGTCCCGCGGT Bp 24 + 33 (Kaneko et CAAGGATGAAGAGGAGATGGAAGAA 1300 ND al., 2002) GCGTACCTCGACAGGTAGAAGATGC

Cav3.1 T BC110995.1 GCTGCTGGAGACACAGAGTACAGGT 397 60

CTCGTGGTATTCGATGCCCATGCTG

Cav3.2 T NM_021098.2 CCTGATCCCTACGAGAAGATCCCGC 433 60

CACGGCTGAAGTACTTGCTGTCCAC

Cav3.3 T AF393329.1 AGATGCCCTTCATCTGCTCCCTGTC 526 60

AAGATCTCCTCGTAGCAGTCGCCAG

β1 NM_000723.3 ATGCACGAGTACCCAGGGGAG 331 60

CAGCGCAGTAGCGGGCCTTATT

β2 NM_000724.3 TCGCTTGCCAAACGCTCGGT 909 ND

ATGACGGCTGCGCTGCTTGT

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β3 NM_000725.2 GCAGCAGCTCGAAAGGGCCA 594 65

ATGCTGGAGCGGGCAGAGGA

β4 NM_001005747.2 TGAAGACTCGGAGGCTGGTTCAGC 731 ND

TGGACCGGGTGTTCGAACGT

α2δ1 NM_000722.2 TGCTCATCGGCCCCTCGTCG 252 60

CCAGGCGCACCAGGGCTTTAG

α2δ2 NM_001174051.1 AGCCTAGGCAGGCGCACACT 878 60

TCTGCACTAGCTCACACTGCTCCGG

α2δ3 NM_018398.2 GGACGAGAGGCTGCGTTTGCA 132 65

GGGCCGGCTAAGCACGTGAA

α2δ4 NM_172364.4 TGGCCTGGGCCTTTGTGCAG 328 (?) ND

GCCTCCTCGGCAGCTTCCAC

γ1 NM_000727.3 TGCTGGCCATGACAGCCGTG 367 60

AACATGGACGCGGGTCGCAG

γ2 NM_006078.3 TCTCTGGGCCTTAATTTTCCCC 439 ND

TTTTCACAGACCCCCAAAGACA

γ3 NM_006539.3 CTCCCCTTCCCCTTTCCTTAAC 840 ND

AGCTGGGATTTCCTTTCTGGAG

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γ4 NM_014405.3 TTTGCACGAAGGTTGTGCTG 909 62–64

TTGCTCTCCTGGCGTTGATT

γ5 NM_145811.2 GATCAAGATGTCCCTGCACTCA 257 64

CAGAGACAAAGGCCAGTATCGT

γ6 NM_145814.1 TGCTCAGTAAAGGTGCAGAGTT 334 ND

CTCGGTGGTTGCTTAGAGAAGT

γ7 NM_031896.4 ACTGGCTGTACATGGAAGAAGG 910 65

TGAAATAAGGGAGTCTGTGGGC

γ8 NM_031895.5 TGCTGAAGCATAGTCATGGTGT 987 ND

CCTCTGCCTTCTCAGTGAACTT 54 (Chiou, GAPDH (Chiou, 2006) GAAGGTGAAGGTCGGAGTC 225 2006), 60 GAAGATGGTGATGGG ATTTC [this study] ND= isoform not detected. Unknown annealing temperature.

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Figure 2-1. RT-PCR of Cavα and Auxiliary Subunits in SH-SY5Y Cells.

Expression of Cavα subtypes, auxiliary β, α2δ and γ subunits, as well as Cav2.2 splice variant isoforms were determined in SH-SY5Y cells using standard RT-PCR and specific primers for

each isoform. A. SH-SY5Y cells endogenously expresses, Cav1.3 isoform 1, Cav1.3 isoform 2,

Cav2.2 and Cav3.1, but not Cav1.1 and Cav1.2, Cav1.4, Cav2.3, Cav3.2 and Cav3.3. Expected band sizes were (bp): Cav1.3 isoform 1, 541; Cav1.3 isoform 2, 343; Cav2.2, 754; and Cav3.1,

397, as indicated with arrows. B. SH-SY5Y cells endogenously express different Cav2.2, α1B splice variant isoforms. Bands with predicted sizes were (bp): α1B1, 728; α1B2, 854; Δ1, 900 bp.

No band was detected for splice Δ2. C–D. SH-SY5Y cells express the auxiliary β1, β3, and β4

but not β2; in addition to α2δ1–3, but not α2δ4; and γ1, γ4–5 and γ7 but not γ2–3 and γ8 subunits.

Expected band sizes were (bp, base pairs): β1, 331; β3, 594; β4, 731; α2δ1, 252; α2δ2, 878; α2δ3,

132 and γ1, 367; γ4, 909; γ5, 257; and γ7, 910.

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2.4.13 125I-GVIA Displacement from SH-SY5Y Cell Membranes

GVIA is the most widely used neurotoxin in neuroscience, with ~ 2000 research papers in the literature reporting the use of this ω-conotoxin as a pharmacological tool [141], to

discriminate N-type currents in electrophysiological assays, or other type of Cav2.2 channel- mediated functional response, from other Cav channel subtypes. The radiolabelled version of the peptide, 125I-GVIA, has been used in binding assays for many years to identify molecules

that bind to Cav2.2 channels, mostly using rat brain membranes [82, 85, 86]. We performed binding assays using the human SH-SY5Y cells and 125I-GVIA to investigate if this cell line 125 contained I-GVIA binding sites, which should be displaced by a range of Cav2.2 inhibitor -conotoxins, including CVIA, CVIB, CVID, CVIE, CVIF, GVIA MVIIA and MVIIC. Each of these conotoxins fully displaced 125I-GVIA binding from crude rat brain membranes with

high affinities: pIC50 ± SEM (M) values: CVIA 8.81 ± 0.05, CVIB 9.02 ± 0.81, CVID 10.53 ± 0.15, CVIE 10.49 ± 0.21, CVIF 10.27 ± 0.28, GVIA 10.43 ± 0.16, MVIIA (10.19 ± 0.04) and MVIIC 8.73 ± 0.072 (Figure 2-2, Table 2.2), consistent with earlier studies [85, 86]. Affinities of these ω-conotoxins were next compared between human and rat Cav2.2 channels. Intriguingly, while the affinity of most of the ω-conotoxins to displace 125I-GVIA binding from SH-SY5Y membranes was similar to that shown in rat brain, both CVID and MVIIA had

significant higher affinity for the human cell membranes (pIC50 of 11.51 ± 0.12 and 11.29 ± 0.23, respectively) than for rat brain membranes (Figure 2-2 A, B and D, Table 2.2). In addition, the affinities of both ω-conotoxins CVID and MVIIA dramatically decreased when determined on the intact SH-SY5Y cells instead of membranes, with GVIA affinity shifted ~ 10-fold, and

CVID and MVIIA affinity shifted ~ 100-fold (see Figure 2-2, Table 2.2).

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A Rat brain membranes B Human SH-SY5Y membranes 100 100

80 80 CVIA CVIA CVIB 60 CVIB 60 CVID CVID 40 CVIE 40 CVIE CVIF CVIF GVIA 20 GVIA I BINDING GVIA (CPM) 20 MVIIA MVIIA 125 MVIIC MVIIC 0 0 -16 -14 -12 -10 -8 -6 -16 -14 -12 -10 -8 -6 LOG LIGAND (M) LOG LIGAND (M)

C D Human SH-SY5Y whole cell 10 Rat brain membrane 100 Human SH-SY5Y membrane 80 1 (nM) 50 60 0.1 40 CVID 0.01 20 GVIA MVIIA Conotoxin IC I-GVIA BINDING (% CPM) (% BINDING I-GVIA 0 0.001 125 -14 -12 -10 -8 -6 IA IB IE IF IC V V V LOG [-CONOTOXIN] (M) C C CVID CV C GVIA MVIIA MVI

Figure 2-2. 125I-GVIA Displacement by ω-Conotoxins from SH-SY5Y Cells. 125 Displacementof I-GVIA binding to Cav2.2 expressed in rat brain and SH-SY5Y intact/whole cell and membranes. A. Displacement of 125I-GVIA from rat brain membranes. B. Displacement of 125I-GVIA from human SH-SY5Y cell membranes. C. Displacement of 125I- GVIA from human SH-SY5Y whole cell. D. The bar graph represents the affinities of the ω-

Conotoxins CVIA, CVIB, CVID, CVIE, CVIF, GVIA, MVIIA and MVIIC (IC50 ± SEM) to displace 125I-GVIA binding from rat brain membranes and human SH-SY5Y cell membranes. Data are mean ± SEM of triplicate data from a representative experiment best fitted to a single- site competition model using GraphPad Prism.

56 Chapter 2 Table 2.2. 125I-GVIA Displacement from Rat Brain and SH-SY5Y Cells ω- SH-SY5Y cells Conotoxin Rat brain membrane membrane SH-SY5Y whole cell IC50 (nM) IC50 (nM) IC50 (nM) CVIA 0.30 ± 0.094 0.40 ± 0.0748 ND CVIB 7.0 ± 0.120 6.76 ±2.22 ND CVID 0.034 ± 0.013 0.0034 ± 0.009 3.2 ± 0.2 CVIE 0.031 ± 0.004 0.029 ± 0.008 ND CVIF 0.078 ± 0.012 0.098 ± 0.007 ND GVIA 0.043 ± 0.013 0.033 ± 0.012 0.27 ± 0.084 MVIIA 0.064 ± 0.007 0.0065 ± 0.001 10 ± 0.085 MVIIC 1.3 ± 0.11 0.935 ± 0.085 ND

ND: Not determined. Concentration values are represented in nM (IC50  SEM).

2.4.14 Functional Characterization of Native Cav channels

To investigate if the Cav channels endogenously expressed in SH-SY5Y cells were functional, and to further study the pharmacology of these channels, we assessed KCl-evoked 2+ Tetra Cav responses using a fluorescent high-throughput Ca imaging assay on the FLIPR

(Fluorescent plate image reader, Molecular Devices, Sunnyvale, CA) (Figure 2-5 A–D). CaCl2 (5 mM) was added to the KCl stimulation solution in all experiments to maximize the Ca2+

influx signal. Co-addition of 90 mM KCl and 5 mM CaCl2 evoked a large transient response indicating increase in intracellular Ca2+ (Figure 2-5 A–B). Concentration-response curves for

KCl-mediated stimulation showed activation of Cav responses with an EC50 of 17.3 mM (pEC50 1.73 ± 0.06 M, Hill slope of 2.5) (Figure 2-5 A, Table 2.3), similar to previously described values [182, 183, 185].

57 Chapter 2

Al l 123456789101112

A

B

C

D

E

F

G

H

Figure 2-3. Functional Cav2.2 FLIPR Assay Plate Layout. FLIPR (Fluorescent Plate Imaging Reader) assays were performed as described in methods section and in ref [3] on SH-SY5Y cells. The columns 1–6 include wells of the FLIPR 96 well-

plate containing KCl 90 mM + CaCl2 5 mM as the activation buffer solution. KCl activates 2+ Ca -mediated Cav2.2 responses in the human SH-SY5Y cells (in the presence of nifedipine 10 µM). The columns 7–12 include wells of the FLIPR plate containing CVID (1µM, in the presence of nifedipine 10 µM), and shows inhibition of Ca2+ responses elicited by KCl. The assays were highly robust, as indicated by a Z-score (a measurement of robustness of assays) of ~ 0.72 [202].

2.4.15 Ca2+ Signaling Detected by the FLIPR

Electrically excitable cells utilize three major Ca2+ signalling pathways: Ca2+ entry through 2+ Cav channels, release of intracellular Ca stores bearing inositol 1,4,5-trisphosphate receptors (InsP3Rs) that are sensitive to the second messenger inositol 1,4,5-trisphosphate (InsP3), and release of Ca2+ from Ca2+ stores bearing ryanodine receptors (RyRs) that are sensitive to caffeine [204, 205]. Pre-treatment of the preparation with caffeine and ryanodine will cause depletion of the caffeine-sensitive Ca2+ store [204]. Thapsigargin is an inhibitor of the cardiac isoform sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pumps, which has been extensively used to study the intracellular Ca2+ pool participating in the generation of the

58 Chapter 2 agonist-induced Ca2+ signals in various cell types. We have used thapsigargin to identify the 2+ percentage of responses occurring due to extracellular Ca flowing through Cav channels in SH-SY5Y cells.

Figure 2-4. Ca2+ Signal from FLIPR Assays in SH-SY5Y Cells. FLIPR (Fluorescent Plate Imaging Reader) assays were performed as described in the methods section and in ref [187], using SH-SY5Y cells to identify how much of the Ca2+ signal from the FLIPR plates was due to Ca2+ induced Ca2+ release as opposed to direct Ca2+ extracellular flowing through Cav channels. Pre-treatment with thapsigargin produced a total of ~25% inhibition in SH-SY5Y cells indicating that the majority of the Ca2+ responses from the FLIPR were due to direct influx through Cav channels.

Our results indicated that the majority of the Ca2+ responses from the FLIPR were due to direct influx through Cav channels, as pre-treatment with thapsigargin produced only ~25% inhibition of the responses in SH-SY5Y cells in the presence of nifedipine (n=2 experiments x 12 replicates/group).

59 Chapter 2

To assess the contribution of each of the Cav channel expressed in SH-SY5Y cells to the KCl-evoked Ca2+ responses, we determined concentration-response curves for KCl/Ca2+ stimulation in the presence of subtype-specific inhibitors. The Cav1 (L-type) inhibitor

nifedipine was used at a concentration (10 μM) that does not affect responses of other Cav subtypes (N, R, P/Q or T-type) [206], to isolate non-L-type responses. The KCl concentration- response curve was shifted to the right in the presence of nifedipine (EC50 of 20.4 mM, pEC50

1.69 ± 0.12 M, Hill slope of 2.9) (Figure 2-5 A, Table 2.3). Conversely, the Cav2.2 (N-Type) inhibitor ω-conotoxin CVID was used at a concentration that does not affect responses of other

Cavs (up to 3 µM) [56, 85], to isolate non-N-type responses. Compared to responses in the presence of nifedipine, the KCl concentration-response curve was shifted to the left in the

presence of CVID (EC50 of 18.6 mM, pEC50 1.86 ± 0.10, Hill slope of 3.5) (Figure 2-5 A, Table 2.3). These differences can be accounted for by the electrophysiological properties of each Cav channel subtype identified, since L-type requires a larger depolarization than N-type to be activated [56], and the control KCl responses is a result of activation of both channel

types. These results confirm that SH-SY5Y cells express functional Cav subtypes, including

Cav1 and Cav2.2, which can be pharmacologically isolated using selective inhibitors. The observed pharmacology is consistent with the subtypes identified in our PCR experiments and with previous reported electrophysiological data [192, 193].

Since 90 mM KCl/5 mM CaCl2 elicits maximal Cav1 and Cav2.2 responses (Figure 2-5, A),

we used this combination to further characterize the Cav channel subtypes expressed in SH-

SY5Y cells. Concentration-response curves for nifedipine at Cav1 channels were generated in the presence of saturating concentration of CVID (1–3 µM). Under these conditions, nifedipine 2+ inhibited KCl evoked Ca responses with an IC50 of 0.28 µM (pIC50 6.5 ± 0.052 M) (Figure 2-5 C, Table 2.3), consistent with reports for nifedipine block of L-type responses in neuronal cells [207]. To characterize Cav2.2 pharmacology, inhibition by -conotoxins was determined in the presence of a near saturating concentration of nifedipine (10 µM). Under these

conditions, the potency of CVIA was IC50 (µM) 0.018 (pIC50 7.99 ± 0.16 M), CVIB 0.22 (pIC50

7.02 ± 0.12 M), CVID 0.16 (pIC50 6.87 ± 0.078 M), CVIE 0.010 (pIC50 8.37 ± 0.28 M), CVIF

0.09 (pIC50 8.12 ± 0.17 M), GVIA 0.15 µM (pIC50 6.84 ± 0.06 M), MVIIA 0.024 µM (pIC50

7.7 ± 0.13 M) and MVIIC 0.09 (pIC50 7.7 ± 0.18 M) (Figure 2-5D, Table 2.3).

60 Chapter 2

A B 100 100 NIFEDIPINE 10 M CVID 3 M 80 80 CONTROL KCl 90 mM 60 60

40 40 NIFEDIPINE + CVID 3 M RESPONSE (%) RESPONSE RESPONSE (%) 20 20

0 0 -10 -9 -8 -7 -6 -5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 LOG [NIFEDIPINE] (M) LOG [KCl] (M)

C D 100 100

80 80 CONTROL CVIA 60 NIFEDIPINE 10 M 60 CVIB CVID 3 M CVID 40 40 CVIE CVIF RESPONSE (%) RESPONSE 20 20 GVIA MVIIA 0 0 MVIIC 500 600 700 800 900 1000 -10 -9 -8 -7 -6 -5 TIME (sec) LOG -CONOTOXIN [M]

E Binding SH-SY5Y membrane Binding SH-SY5Y whole cell Functional SH-SY5Y Cell 10-12 10-11 10-10 10-9 10-8 -7

POTENCY (M) POTENCY 10 10-6 10-5 CVID GVIA MVIIA -CONOTOXIN

Figure 2-5. Functional Cavs Expressed in SH-SY5Y cells. Data obtained from fluorescent Ca2+ imaging assays of KCl-evoked Ca2+ responses in SH-

SY5Y cells. A. Cav1 and Cav2.2 activation in the presence of CVID (open ball) and nifedipine (filled ball), respectively, shifted control KCl-evoked Ca2+ responses (quadrilateral) significantly in SH-SY5Y cells (p > 0.05). B. Time course of Ca2+ responses is shown for control KCl 90 mM (black), KCl in the presence of nifedipine (blue) and KCl in the presence

of CVID (green). C. Concentration-response curve for nifedipine inhibition of Cav1 responses

D. Concentration-response curves for CVID, GVIA and MVIIA inhibition of Cav2.2 responses. The responses were normalized using controls: positive KCl and negative PSS buffer; and

61 Chapter 2 plotted across increasing concentrations of antagonists E. Comparison of ω-conotoxins CVID,

GVIA and MVIIA potencies (IC50/Kd ± SEM of n =3–4 replicates for each experiment, n = 3 experiments) in displacing 125I-GVIA from SH-SY5Y whole cell and SH-SY5Y cell membranes with the functional assays data.

A B

25 4

20 3

15 -AGATOXIN 2 SNX 482 10 MIBEFRADIL PIMOZIDE 1 FLUORESCENCE RESPONSE (%) RESPONSE 5 CONTROL + CVID 3 M + NIFEDIPINE 10 M -AGATOXIN TK 10 M 0 0 -8 -7 -6 -5 -4 500 600 700 800 900 1000 LOG LIGAND [M] TIME (s) C D

4 4

3 3

2 2 CONTROL MIBEFRADIL 30M 1 1 FLUORESCENCE FLUORESCENCE CONTROL 0 -SNX 482 30 M 0 500 600 700 800 900 1000 500 600 700 800 900 1000 TIME (s) TIME (s)

Figure 2-6. Characterization of Resistant Ca2+ Responses in SH-SY5Y Cells. Data obtained from fluorescent Ca2+ imaging of KCl-evoked Ca2+ responses in SH-SY5Y cells. A. Concentration-response curves for mibefradil, pimozide, ω-agatoxin TK and SNX 482 in inhibiting resistant KCl-evoked Ca2+ responses in SH-SY5Y cells (pre-treated with CVID 3 µM plus nifedipine 10 µM). B–D. Time course of transient Ca2+ responses activated by 90 mM KCl/5 mM CaCl2, in the presence of CVID (3 µM) and nifedipine (10 µM) and following the addition of agatoxin TK, SNX-482 and mibefradil.

These results are consistent with results from previous studies on CVIB, MVIIA, MVIIC

[82, 86, 182, 185] , CVID [82, 86, 182] and CVIE–F [86] inhibition of N-type (Cav2.2) responses in native and recombinant systems, when the α subunit was co-expressed with β and

62 Chapter 2

α2δ subunits [82, 86, 182]. While the data for CVIA does not agree with previously reported functional data [85], the potency of this toxin in native system or heterologous system co- expressing auxiliary subunits remains to be investigated, for comparison with data from the native system described here. In contrast, GVIA potency at Cav2.2 channels expressed in SH- SY5Y cells was consistently lower than previously described for heterologous expressed rat

[182, 185] and human [183] α1B co-expressed with α2δ1 and 3, but similar to data obtained using native expression systems, such as dissociated rat DRG cells [208] and chicken synaptosomes [3].

63 Chapter 2

2+ Table 2.3. Potency (IC50 ± SEM) of Cav Channel Modulators at Functional Ca Imaging Assays.

Stimulation of Ca2+ Responses Inhibition of Ca2+ Responses

Cav Channels Activator/Inhibitor EC50 (mM) IC50 (µM) KCl 17.28±0.01 - KCl + CVID 18.61±0.06 - KCl + NIFEDIPINE 20.35±0.02 - CVIA 0.018 ± 0.08 CVIB 0.219 ± 0.30 CVID 0.16 ± 0.025 CVIE 0.010 ± 0.83 CVIF 0.009 ±0.17 GVIA 0.15 ± 0.09 MVIIA 0.024 ± 0.005 MVIIC 0.009 ± 0.004 NIFEDIPINE - 0.23 ± 0.046 MIBEFRADIL - 3.0 ± 0.031 PIMOZIDE - 1.3 ± 0.097 ω-AGATOXIN TK - NDR SNX 482 - NDR

64 Chapter 2

2.5 DISCUSSION AND CONCLUSIONS

Cav2.2 channels play an essential role in neurotransmission and nociception. Cav2.2 inhibition at the spinal cord level produces analgesia in animal models of pain [46, 86] and in humans [69]; with direct (eg. Prialt) and indirect (eg. Gabapentin) inhibitors among some of the most recently developed analgesics [203]. Neuroblastoma cells, including the sympathetically derived human neuroblastoma cell line SH-SY5Y, provide excellent model systems to study Cav2.2 channels in a native context [192, 193]. However, little is known about

the Cavα and auxiliary subunits expressed, limiting interpretation of pharmacological data from

this cell line. To address this limitation, the expression and pharmacology of Cav channels in SH-SY5Y cells have characterized and mechanisms likely to influence the pharmacology of

ω-conotoxins investigated at Cav2.2 channels.

We identified mRNA expression of two L-type channel Cav1.3 isoforms, isoform 1 and 2,

in SH-SY5Y cells. In addition to Cav1.3 channel isoform, a Cav3.1 isoform was detected. In

addition, mRNA transcripts for the full-lengh Cav2.2 and the splice variants α1B2 (74 amino

acid shorter) [63, 87, 209] and α1BΔ1 (382 amino acid shorter) [87] were also detected in SH-

SY5Y cells. Functional Cav responses elicited by addition of KCl/CaCl2 were assessed in SH- SY5Y cells using a fluorescent high-throughput Ca2+ imaging assays.

KCl has been used extensively to activate Cav responses in a diversity of functional assays [182, 185, 210]. Addition of high concentrations of KCl causes changes in membrane potential 2+ in these assays, which in turn leads to the opening of Cav channels, Ca influx and a resultant increase in intracellular fluorescence. While the change in membrane potential elicited by KCl addition at the concentrations used in this study was approximately linear, the accumulation of intracellular Ca2+ is saturable and has fit a sigmoidal concentration-response curve, because a change in membrane potential leads to a finite change in channel open probability and thus Ca2+ influx.

Cav2.2 channels expressed in SH-SY5Y cells were functional and produced KCl-activated Ca2+ responses in the presence of saturating concentrations of nifedipine that were fully inhibited by the ω-conotoxins CVIA, CVID, CVIE, CVIF, GVIA, MVIIA, and MVIIC, and partially inhibited by CVIB. As expected for SH-SY5Y cells, when L-type responses were isolated by addition of saturating concentrations of CVID, nifedipine concentration-

65 Chapter 2 dependently blocked most of the remaining KCl responses. However, a small response remained (5–15%) in the presence of a combination of Cav2.2 and Cav1 inhibitors. This

resistant response was completely abolished by the Cav3 inhibitors mibefradil and pimozide, suggesting Cav3 expression in SH-SY5Y cells.

The pharmacology of Cav3 channels is complex, because many drugs block T-type currents,

however, unfortunately, none of these compounds has high selectivity for Cav3 channels.

Therefore, Cav3 channels isolation is difficult. Mibefradil inhibits T-type currents [211],

however it binds to skeletal muscle L-type calcium channels (Cav1.1) and brain voltage-gated sodium channels with dissociation constants of 2.3 and 17 nM, respectively [212]; and this drug has been taken out of the market. Anyhow, while mibefradil is not specific, it does inhibit T-type currents [213, 214], and the inhibition of the residual Ca2+ response was also observed in the presence of saturating concentrations of nifedipine and CVID, suggesting that activity of

these compounds at Cav1.3 or Cav2.2 expressed in SH-SY5Y cells did not contribute to inhibition of residual response. Based on the observations that the resistant response was not blocked by inhibitors of L-type (nifedipine), N-type (CVID), R-type (SNX 482) or P/Q-type channels (ω-agatoxin), but was completely abolished by compounds with known activity at T- type channels, it seems plausible that this response may be mediated by Cav3.1, which mRNA expression was detected in SH-SY5Y.

Alternatively, it is known that the Cav2.2 Δ1 splice variant, which mRNA expression was also detected in SH-SY5Y cells, is significantly more resistant to the blockade by MVIIA and

GVIA [215]. While inhibition by CVID of the Cav2.2 splice variants detected in SH-SY5Y

cells has not yet been characterised, it is possible that similarly to inhibition of Nav channels by the µ-conotoxin GIIIA, complete current inhibition by CVID cannot be achieved for these splice variants. Alternatively, the response remaining in the presence of nifedipine and CVID could represent another undefined resistant current, or an artefact of the KCl/Ca2+ activation buffer used in this study.

Development of non-electrophysiological HTS Cav3 channel assays has been hampered by some of the properties of this channel, including their low voltage threshold for activation and inactivation and rapid inactivation kinetics. However, although T-type currents inactive rapidly, fluorescence Ca2+ assays detect accumulation of intracellular Ca2+ rather than currents, and thus these assays are not subject to the same temporal resolution constraints. Nevertheless, compared to heterologous systems SH-SY5Y cells have a relatively hyperpolarised resting 66 Chapter 2 membrane potential [216], which would be conducive to channels being present in the resting 2+ state. Accordingly, Ca assays at Cav3 channels using the FLIPR have been successfully developed [210] and it is clearly conceivable that functional Cav3.1 responses could be elicited in SH-SY5Y cells using KCl/Ca2+ stimulation solution.

In addition to functional characterization using the FLIPR assays, Cav2.2 expression in SH- SY5Y cells was also confirmed at the protein level using 125I-GVIA in binding assays. The ω- conotoxins CVIA, CVIB, CVID, CVIE, CVIF, GVIA, MVIIA and MVIIC each fully displaced 125I-GVIA binding to SH-SY5Y cell membranes with high affinities. Interestingly, while the affinity of almost all the ω-conotoxins was not significantly different between human and rat, CVID and MVIIA affinities were ~ 10-fold higher in human SH-SY5Y membranes compared to rat brain membranes. These results engage with the hypothesis that MVIIA and CVID interact with human Cav2.2 channels through a different pharmacophore, as that proposed for GVIA [217]. Although all the -conotoxins backbone structures belong to the well-known inhibitory cystine knot (ICK) motif [218], which is highly conserved [219], sequence hypervariability is observed for both intra and inter ω-conotoxin species, with the six Cys and one Gly being the only residues conserved throughout this set of peptides (for review see ref [220]).

Variation in the affinity of -conotoxins between species is also likely influenced by the

binding to Cavα splice variants, with differences in toxin sensitivity, time course and voltage- dependence of inactivation, single channels conductance, gating behaviour and sensitivity to G-protein-mediated modulation reported for splice isoforms endogenously expressed in neuronal cells of rat, mouse, rabbit and humans [63, 87, 209, 215, 221-224] (for review see ref

[222]). In pain, the Cav2.2 splice variant 37a replaces the usual variant 37b in a specific subset of nociceptive neurons, and thus may represent a potential therapeutic target [215, 222, 225]. However, this variant has to date only been described in rat dorsal root ganglion neurons, and is not known to be present in human tissue. Additional human splice variants include two α1B isoforms that have long or short C-termini [63], and two human forms that lack large parts of the domain II-III linker region, including the synaptic protein interaction site. These splice variants, termed Δ1 and Δ2, have been previously isolated from IMR32 human neuroblastoma cell line and human brain cDNA libraries [87]. I have identified mRNA transcripts for the full- length α1B1, α1B2 (74 amino acid shorter) [63, 87, 209] and the splice variant Δ1 (382 amino

acid shorter) [87] in SH-SY5Y cells. The α1B1 is an axonal/synaptic isoform, while α1B2 is

67 Chapter 2 restricted to neuronal soma and dendrites [209, 226], however, apart from differential susceptibility to Gαi/Gαo-versus Gαq-mediated inhibition, little is known regarding its biophysical and pharmacological properties.

On the other hand, the Δ1 splice variant has lost part of the synaptic protein interaction (synprint) site and is thus unlikely to play a role in fast synaptic transmission, with shifts in the voltage dependence of steady-state inactivation and a more rapid recovery from inactivation compared to full length α1B1 [87]. Importantly and clinically relevant, the Δ1 variant was significantly more resistant to the blockade by MVIIA and GVIA; however the degree of effect varied for each toxin [87]. Thus, expression of the Δ1 variant in SH-SY5Y cells may contribute to the reduced -conotoxin affinity observed. While expression of these splice variants in SH- SY5Y cells was detected using gene specific primers, which have been extensively validated in the literature [17], further confirmation of expression at the protein level is warranted.

Cav channel auxiliary subunits can also influence the pharmacology of Cav inhibitors, with

-conotoxins displaying reduced affinity in the presence of the 2 subunit [81, 82, 86, 182, 206, 211]. Specifically -conotoxins GVIA, MVIIA and CVID had reduced affinity when the

α2δ1 subunit was co-expressed with the Cavα1B [82, 86]. α2δ up-regulation has been associated

with chronic pain and epilepsy, with gabapentin and pregabalin binding to 2 reducing Cav2.2 trafficking and the symptoms of pain [81]. The α2δ1–3, β1, 3 and 4, γ1, γ4–5 and γ7 subunits were detected in SH-SY5Y cells and potentially contribute to the differences in -conotoxins potency in whole cell vs. membrane assays.

The γ1 subunit was originally identified in skeletal muscle in complex with Cav1 channels

[109], but effects of this subunit on the -conotoxins affinity at Cav2.2 have not been

determined. In contrast, co-expression of the γ7 subunit almost abolished the functional

expression of Cav2.2 in both Xenopus oocytes and COS-7 cells [110, 111]. The neuronal 2 is associated with epileptic and ataxic phenotypes of the stargazer mouse [53], but was not

detected in SH-SY5Y cells. The γ5 and γ7 subunits represent a distinct subdivision of the γ subunit family of proteins identified by structural and sequence homology to stargazing. The

γ4 subunit affected only the Cav2.1 channel [53, 112], while the γ5 subunit may be a regulatory

subunit of Cav3.1 channels (for review see: [113]). These subunits may also potentially contribute to differences in -conotoxins binding affinities observed in whole cell vs. membrane assays.

68 Chapter 2

While auxiliary subunits affect ω-conotoxin affinity in functional studies, this quaternary complex is likely to be disrupted upon preparation of homogenized membranes for the binding assays [227]. To examine this possibility, I studied the ability of GVIA to displace 125I-GVIA from whole SH-SY5Y cells compared to homogenized membranes. Interestingly, ω- conotoxins CVID, MVIIA and GVIA had higher affinity to displace 125I-GVIA from the homogenized membranes compared to the whole cells, an effect that was most pronounced for CVID and MVIIA (~ 100-fold) compared to GVIA (~ 10-fold). A similar trend for both CVID

and MVIIA in heterologous expression system with and without the α2δ subunit has been previously reported [82].

Potency estimates obtained with the functional assays were significantly lower than estimates obtained in whole cell radioligand binding assays for the ω-conotoxins CVID, GVIA and MVIIA. The relatively high level of Ca2+ in the physiological saline used traditionally for functional assays compared to binding assays could contribute to these differences, since Ca2+ non-competitively inhibits ω-conotoxin binding [85]. However, our whole cell data was also obtained by incubating -conotoxins in a Ca2+-free physiological saline solution and the origin of these differences is unclear. Interestingly, this effect was most marked for GVIA, intermediate for CVID and insignificant for MVIIA.

In summary, I have characterized functional Cav channels expressed in the SH-SY5Y human

neuroblastoma cell line. Our studies have shown that expression of different Cavα splice

variants, in conjunction with auxiliary subunits in a native context, can modulate the

pharmacology of Cav2.2 channel inhibitors. The SH-SY5Y cell line provides a useful model

for the investigation of novel human Cav2.2 inhibitors and is amenable to the establishment of high-throughput assays [195], which can be adapted to detect endogenously expressed human

Cav1.3, Cav2.2 and possibly Cav3.1, in the presence of appropriate inhibitors. These assays are

expected to prove useful for the discovery and pharmacological characterization of novel Cav channel modulators targeting human Cav related diseases.

69 Chapter 3

Chapter 3

70 Chapter 3 Chapter 3 Novel Analgesic ω-Conotoxins Discovered from the venom of Conus moncuri. Functional, Molecular and Structural Studies.

3.1 FOREWORD

Most parts of this Chapter are intended for submission to The Journal of Biological Science. Most of the work presented in this chapter is my own; however this chapter also contains work contributed by others, as indicated in the statement of contributions by other, at the beginning of this thesis.The cone snail Conus moncuri was collected on a field trip by members of Prof. Richard Lewis’ group, University of Queensland. Through collaboration with Prof. Paul Alewoods’ laboratory, University of Queensland, Dr Andreas Brust synthesized the conopeptides. Dr Sebastien Dutertre and Dr Lotten Ragnarsson from Prof. Richard Lewis’ laboratory, assisted with LC/MS and PCR cloning, respectively. The DRG neurons extraction, culture, and electrophysiological assays were performed by Dr Jeffrey Mc Arthur and me, through collaboration with Prof. David Adams’ laboratory, at the RMIT University (Melbourne). Dr Johan Rosengren from the School of Biomedical Sciences, at the University of Queensland, calculated the NMR structure and assisted with the NMR structure analysis. The animal in vivo studies were performed by Rebecca Smith and myself, in collaboration with Prof. Macdonald Christie’s laboratory, at the Kolling Institute of Medical Research, University of Sydney. Prof. Richard Lewis, Dr Lotten Ragnarsson and Dr Irina Vetter contributed with experiment guidance and proof reading/editing the chapter.

3.2 INTRODUCTION

Cone snails are predatory marine gastropod molluscs found in tropical waters around the world. Cone snails are particularly abundant in the Indian-Pacific oceans, including the Philippines, Papua New Guinea and Australia [140, 228]. Based on their prey type, cone snail are classified as piscivorous, molluscivorous or vermivorous.

Cone snails have evolved a highly complex toxins which are mixtures of venom peptides (conopeptides or conotoxins), most probably to facilitate prey capture as well as for interactions with competitors and predators [141, 229-232]. The venom from a single cone snail can

71 Chapter 3 paralyse a man. However, the venom from C. geographus is the only venom that has proved lethal to humans. With 500–700 Conus species, each expressing ~ 100 peptides, the Conus genus is amongst the richest natural peptide library, consisting of over 50,000 pharmacologically active peptides [80, 140, 141, 228].

Most conus peptides target receptors and ion channels of the central and peripheral nervous system, and are therefore neurologically active peptides or neurotoxins. ω-Conotoxins display high specificity targeting Cav2.2 channels. Due to Cav2.2 essential role in controlling neurotransmitter release, cardiac and skeletal muscle function, in addition to Cav2.2 essential role in transmission of pain sensation, ω-conotoxins have been widely used as tools in

neuroscience. Importantly Cav2.2 antagonists are anti-nociceptive in animals and humans [43, 44, 180, 233], and thus, ω-conotoxins are potential analgesic leads which can be used for the development of neuropathic pain drugs [44].

Conotoxins belong to the O-superfamily of structurally related four-loop, six cysteines conopeptides that are first translated into larger precursor sequences consisting of three regions: the signal sequence or pre-region, an intervening or pro-region and at the C-terminus end the mature peptide region [141]. A superfamily retains the same conserved signal sequence at the N-terminus throughout the genus, in contrast with a much more hyper variable mature peptide region at the C-terminus (see figure 3.4) [229]. With some exceptions, the number and arrangement of cysteine residues (C) and disulfide bond patterns are conserved features of all peptides within the same gene superfamily (for review see ref [80, 141, 145]).

Ziconotide (MVIIA, a synthetic peptide conotoxin from Conus magus [234]) is currently marketed as Prialt, and has shown effectiveness on treating complex neuropathic conditions generated by HIV/AIDS and cancer, for example. The analgesic efficacy of ziconotide likely results from its ability to block Cav2.2 channels and interrupt pain signalling at the level of the spinal cord (see chapter 1). As ziconotide is a peptidic drug, it has only limited ability to cross the blood brain barrier. Accordingly, in order to achieve optimal analgesic efficacy, with reduced potential for serious side-effects, ziconotide must be administered intrathecally to patients. Ziconotide is a potent analgesic with a narrow therapeutic window, thus the drug requires a slow titration in order to achieve analgesia while avoiding dose-limiting side effects. Spinal route of administration permits the drug to reach its maximum local concentration

quickly, producing a rapid onset of analgesia [74]. Cav2.2 inhibition reduces the release of pro- nociceptive neurotransmitters in the dorsal horn of the spinal cord,and thus inhibit pain signal transmission (revised in chapter 1 and by ref [44, 45, 235, 236]).

72 Chapter 3 Opioids, arguably the most used analgesic used to treat neuropathic pain conditions, fail to produce pain relief in certain individuals even at high doses due to the development of tolerance, a consequence in chronic pain that complicates the utility of opioids for long term therapy [237]. An improved therapeutic window was seen when ziconotide when associated with opiates [44, 237], reducing dose limiting side effects of both drugs.

A number of other Cav2.2 antagonists have been isolated from predatory fish hunting cone snails with improved characteristics, including the ω-conotoxins CVIA-F from Conus catus [85, 86] which seem to be more reversible versions of the ω-conotoxins. Intravenous injection of CVID (Leconotide) had synergistic anti-hyperalgesic effects with morphine in a rat model

of bone cancer pain [233], and exhibited the highest selectivity for Cav2.2 over Cav2.1 in

radioligand binding assays using rat brain cells [85]. Avoiding Cav2.1 is important for avoiding

motor side effects, as Cav2.1 is the major Cav channel mediating synaptic neurotransmission at neuromuscular synapses [49, 67]; and thus important for normal physiology at these synapses.

Importantly, although Cav2.1 and Cav2.2 in humans are highly conserved, subtype specificity of some ω-conotoxins have been described [85]. Accordingly, additional ω-conotoxins are currently undergoing pre-clinical and clinical trials (see chapter 1 and http://www.clinicaltrials.gov/) and hopefully a novel ω-conotoxin with improved safety margin will reach the clinic in the near future.

This chapter describes the discovery and characterization of two novel analgesic leads, ω- conotoxins, identified from the venom of Conus moncuri, a worm hunter cone snail found in Indo pacific waters.

3.3 MATERIALS AND METHODS

3.3.1 Drugs and Chemicals

Drugs and chemicals were obtained from different sources, as detailed in the appendix 1 and throughout the text.

3.3.2 Crude Venom Extraction

In 2011 a group went on a trip to Papua New Guinea to collect cone snails. A range of cone snail species were collected and identified, and their crude venom and venom ducts extracted on ice. Venom ducts were carefully removed using forceps and scalpel. Venoms were extracted

73 Chapter 3 from the isolated ducts and pooled into micro centrifuge tubes, as previously described [85], then resuspended in 30% ACN/H2O (acetonitrile/water) acidified with 0.1% TFA (trifluoroacetic acid) and centrifuged at 10,000 g for 5 min. The soluble material was

lyophilised and re-dissolved in 5% ACN/H2O (acetonitrile/water). Protein content was estimated by measuring the absorbance at 280 nm (E280), using a Nanodrop (Thermo Scientific, Australia). The venoms were stored at 4°C for a week prior to use, or –20 °C for longer-term storage. Empty venom ducts were placed in RNAlater® (Ambion), a tissue storage reagent that protects and stabilizes RNA, and kept at –20 °C for later RNA extraction and cDNA analysis.

3.3.3 Cell Culture

SH-SY5Y human neuroblastoma cells (Victor Diaz, Goettingen, Germany) were cultured and routinely maintained as described in section 2.4.5, chapter 2 and in ref 3 [3].

3.3.4 Rat Dorsal Root Ganglia Neurons (rDRG)

Rat dorsal root ganglion (rDRG) neurons were enzymatically dissociated from 10–16 day old Wistar rats, as previously described [22]. Rats were euthanized by cervical dislocation, as approved by the RMIT University Animal Ethics Committee. The spinal column was hemi- segmented and dorsal root ganglia was removed from the spinal cord, rinsed in cold Hank's Balanced Salt Solution (HBSS) (MultiCell), minced and incubated in 1 mg/ml collagenase (type 2; 405 U/mg) (Worthington Biochemical) and kept at 37°C for ∼ 30 min. After incubation, ganglia were rinsed three times with warm (37°C) Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, CA, USA), supplemented with 10% fetal calf serum (FCA) and 1% penicillin/streptomycin and gently triturated with a fire-polished Pasteur pipette. rDRG cells were plated on glass coverslips, incubated at 37°C in 95% O2/5% CO2 and used within 4– 48 h.

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3.3.5 Discovery of MoVIA and MoVIB

Extracted crude venoms were diluted in Phosphate Salt Solution (PSS, protocol described in Appendix 1). Estimated 20 µg crude venom/well was tested for biological activity using the FLIPR high throughput screening (HTS) assays and the human SH-SY5Y cells. SH-SY5Y

cells express endogenous Cav1.3 and Cav2.2 channels [3]. Saturating concentrations of

nifedipine (10 µM) were used to inhibit endogenous Cav1.3 channels in SH-SY5Y cells and to Tetra screen for Cav2.2 blockers [3, 192]. Briefly, the crude venoms were placed in the FLIPR (Fluorescent Plate Image Reader, Molecular Devices) and kinetic readings taken for 10 min before the stimulation buffer (KCl 90 mM/CaCl2 5 mM) was added and responses read for

further 5 min. Crude venoms that inhibited more than 50% of the functional hCav2.2-mediated Ca2+ responses were screened for a second time (in duplicates) to confirm activity.

HTS FLIPR assays were used additionally for characterization of isolated conotoxins in further experimentation. Briefly, SH-SY5Y cells were incubated with the Ca2+ dye Fluo 4, containing in addition saturating concentrations of nifedipine (10 µM), for 30 min before MoVIA, MoVIB, [R13Y]-MoVIB or any control ω-conotoxin (CVID, MVIIA or GVIA [85, 234, 238]) were added by the FLIPR. Plates were incubated for additional 10 min with the toxins, before KCl stimulation buffer solution was added and responses observed for 5 extra minutes.

3.3.6 HPLC Isolation of Active Peptides

Analytical RP-HPLC (Reverse Phase High Performance Liquid Chromatography) was performed using a Vydac 218TP C18 column (250 × 4.6 mm, 5 μm), eluted at 0.7 ml/min with a linear gradient of 0–100% solvent B over 60 min (solvent A: 90% water, 0.1% formic acid; solvent B, 90% acetonitrile, 10% water, 0.09% formic acid), using a Dionex UltiMate® 3000 LCi solvent delivery system. The fractions were collected on a Gilson FC204 fraction collector and aliquots (estimated quantity 10 µg crude/well) freeze dried and re-dissolved in PSS buffer containing nifedipine (10 µM) prior to testing for biological activity. Two to three rounds of fractionation/activity testing were performed until the two active toxins were isolated. Active fractions were analysed for purity with a combination of RP-HPLC and MALDI-TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Mulgrave, VIC) analysis. The MALDI-TOF matrix used was α-cyano-4-hydroxycinnamic acid (CHCA; Sigma Aldrich) (5 mg/mL). The primary structure of MoVIA and MoVIB mature toxin were obtained using outsourced Edman Degradation [239].

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3.3.7 Edman Sequencing Analysis

Outsourced Edman degradation (Australian Proteome Research Facility) was used for determination of the N-terminal sequences of MoVIA and MoVIB. As a brief description of the methods used, the samples were dissolved in urea (4 M) and ammonium bicarbonate (50 mM) and reduced using dithiothreitol (100 mM) at 56 οC for 1 h under argon. Subsequently, the samples were alkylated with acrylamide (220 mM) for 30 min in the dark, and reaction quenched by the addition of an excess of dithiothreitol. Next, the peptide samples were desalted by reverse RP-HPLC and fractions collected and dried, using a speed vac dryer. Subsequently, samples were loaded onto pre-cycled bioprene discs and subjected to 35 cycles of Edman degradation for N-terminal sequencing. Automated Edman degradation was carried out using an Applied Byosystems 494 Procise Protein Sequencing System.

3.3.8 Preparation of C. moncuri RNA

Conus moncuri venom duct tissue (~ 1 mg) was placed in RNA later (Ambion) and kept in 4°C until RNA extraction. On the day of the extraction the tissue was grounded and homogenized. Total RNA extraction was carried out accordingly to instructions of the TRIZOL® Reagent kit (Invitrogen). The isolated RNA was subsequently treated with RNase/DNase-free (Qiagen), to remove any genomic DNA contamination. RNA concentration was determined by absorbance measurements at 260 nm and its purity/integrity was accessed by analysing the ratio 260/280 nm with a Nanodrop® (Thermo Scientific).

3.3.9 Preparation of cDNA and Polymerase Chain Reaction (PCR)

The 3’ RACE first strand cDNA was synthesized from 1 g total RNA using the FirstChoice RLM-RACE kit (Ambion), following the manufacturer’s instructions. The resulting cDNA was used as template in a polymerase chain reaction (PCR) using specific primers, which were based on published literature [5] to detect members of the O-superfamily of conotoxins, discovered from vermivorous Conus and identified by cDNA cloning. Primer sequences were F1=5’-CATCGTCAAGATGAAACTGACGTG-3’ and R1=5’- CACAGGTATGGATGACTCAGG-3’.

The PCR reaction was performed using FastStart Taq DNA polymerase (Roche) with 500 ng of cDNA as template, under the following cycling conditions: 95°C for 4 min, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min and a final elongation step at

76 Chapter 3 72°C for 7 min. PCR products were analysed and purified after separation on a 1% agarose gel using a QIAquick Gel Extraction kit (Qiagen). Gel extracted PCR products were sequenced at the Australian Genome Research Facility (AGRF), using the forward and reverse primers F1 and R1, respectively. Sequencing data was transferred to Expasy Tools [240] for translation of the cDNA sequence and prediction of the amino acid sequences. Peptide sequences of other conotoxins belonging to the O-superfamily were retrieved either from GenBank [241] or Conoserver [205] and compared with MoVIA sequence. The sequence alignment and construction of phylogenetic tree was performed using Clustal W [242] and Jalview version 2.8 [243], respectively.

3.3.10 Chemical Synthesis of MoVIA and MoVIB

The peptides from C. moncuri were synthesized using standard method in situ neutralization Boc-SPPS on a Boc-Asn-PAM/Boc-Tyr-PAM resin, respectively, employing HBTU/DIEA activation, as previously described [85, 244]. The obtained peptidyl-resin was cleaved with hydrogen fluoride for 1 hour employing p-Cresol/p-thio-cresol scavenger (10%). The obtained crude peptides were precipitated from ether, filtered and lyophilized from acetonitrile/H2O. After HPLC fractionation, the pure reduced peptides (20 mg each) at a concentration of 0.2 mg/mL were oxidized at pH 7.8 in a solution of 0.3 M NH4OAc/0.3 M guanidine-HCl, in the presence of GSH/GSSG (100:10 mol eq). Two main peptide isomers were obtained after RP- HPLC in quantities of 1–2 mg corresponding to MoVIA and MoVIB. The masses of both peptides were confirmed by MALDI-TOF MS and, LC-MS/MS and HPLC co-elution of the synthetic peptides with a sample of native peptides (Figure 3.3).

3.3.11 NMR Spectroscopy

For solution NMR spectroscopy studies MoVIB and [R13Y]-MoVIB samples were prepared in 0.5 ml of 2 mg/ml in 90% H2O, 10% D2O (pH 5.0) or 100% D2O. Two dimensional (2D) homonuclear 1H-1H TOCSY, NOESY and ECOSY datasets and a 2D heteronuclear 1H- 13C HSQC were recorded at 900 MHz on a Bruker Avance II spectrometer equipped with a cryogenically cooled probe. All data were recorded and processed using Topsin 3.0 (Bruker). Homonuclear data were recorded with 2048 data points in the direct dimension and 512 increments in the indirect dimension over sweep-widths of 12 ppm and 106 ppm, respectively.

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3.3.12 3D Structure Calculations

All NMR data were analysed using the program Computer Aided Resonance Assignment (CARA, ref (36)). Structural restraints derived from the NMR data included (i) Inter-proton

distances derived from NOESY cross-peak intensities in spectra recorded in either H2O or D2O with a mixing time of 100 ms. (ii) Backbone dihedral angles (Phi and Psi) derived from a TALOS+ (37,38) analysis of Cα, Cβ, Hα and HN chemical shifts. (iii) Side chain dihedral angles (χ1) derived from analysis of 3JHαHα coupling constants and intra residual NOE patterns (39). (iv) Hydrogen-bond derived from amide exchange rates and analysis of preliminary structures.

NOE cross peaks were manually picked and subsequently calibrated and assigned automatically using the automatic assignment and structure calculation module of CYANA 3.0 (40). For the final structures distance restraint lists from CYANA were used as input for simulated annealing and water minimization within CNS (41), using protocols from the RECOORD database (42), modified as described previously (43). In the final round 50 structures were calculated, and the best 20 based on energies and quality of packing and geometry as judged by MOLPROBITY (44) scores were chosen as representative of the solution structure of MoVIB. Figures were prepared using MOLMOL (45).

3.3.13 Electrophysiology Recording of HVA Ca2+ Currents from rDRG

Whole-cell patch clamp recording was performed using a MultiClamp 700B Amplifier (Molecular Devices). Data was digitalized with a Digidata 1322A (Molecular Devices), filtered at 10 kHz and sampled at 100 kHz using a pClamp 9.2 software and MultiClamp 700B Commander (Molecular Devices). External recording solution contained (in mM): 150 TEA-

Cl, 2 BaCl2, 10 D-Glucose and 10 HEPES, adjusted to pH 7.4 with TEA-OH. The pipette electrodes had a final resistance of (1–3 MΩ), with an intracellular solution containing (mM):

140 CsCl, 1 MgCl2, 5 BAPTA and 10 HEPES adjusted to pH 7.2 with CsOH. High voltage- activated (HVA) calcium channel currents were recorded by measuring peak inward Ba2+ currents elicited by 75 ms voltage steps to 0 mV from a holding potential of –80 mV. After current achieved steady state, ω-conotoxins MoVIA, MoVIB or the analogue [R13Y]-MoVIB were added to the physiological solution and the peak inward current values were plotted every 10 seconds. Series resistance was typically compensated at 70–80%, while leak and capacitance currents were subtracted using a –P/4 pulse protocol. Agatoxin-IVA (Abcam) a

78 Chapter 3 selective Cav2.1 channel (P/Q-type current) inhibitor and CVIE, a selective Cav2.2 channel (N- type current) inhibitor, were used to examine the selectivity profile of MoVIA and MoVIB.

3.3.14 Preparation of Membrane for Radioligand Binding Assays

The protocol used to prepare rat brain and SH-SY5Y cell membranes was previously described by Wagner, et al., 1988 (46), with slight modifications when SH-SY5Y cell membranes were prepared. Establishment of this modified protocol is described in detail in chapter 2, section 2.4.7.

3.3.15 Radioligand Binding Assays

The detailed protocol for radioligand binding assays was described in chapter 2, section 46 and in ref (24). Radiolabelled peptide Tyr22[125I]-GVIA (Perkin Elmer) stock was diluted to 20.000 cpm/50 µL (activity equivalent to 30 pM) in binding buffer (as described in chapter 2, and in the appendix 1). Radiolabelled GVIA was incubated with SH-SY5Y membranes, rat or fish brain membranes, and increasing concentrations of the competing ligand (MoVIA, MoVIB, the analogue [R13Y]-MoVIB, or other ω-conotoxins used as control) in triplicates, on 96 well-plates. The plates were incubated with shaking for 1 h at room temperature and vacuum filtered through a glass fibre filter previously soaked in 0.6% polyethyleneimine (PEI, used to reduce non-specific binding) and binding buffer, using a vacuum system (Tomtec harvester). The filters were dried at 37°C, soaked in liquid scintillant and placed in sample bags. Radioactivity was counted using a 1450 Microbeta Wallac Jet (Wallac, Finland) and non- specific binding determined in the presence of 50 µL of unlabeled peptides.

3.3.16 Intrathecal MoVIB in Rats with PNL-Induced Neuropathy

Rats 8–10 weeks old male Sprague-Dawley (200–260 g) were housed four per enclosure and maintained on a standard 12-h light/dark cycle with free access to food and water; following the guidelines of the NH&MRC Code of Practice for the Care and Use of Animals in Research in Australia, and with the approval of the Royal North Shore Hospital Animal Care and Ethics Committee. Production of a unilateral hind-limb neuropathy was achieved with a partial tight sciatic nerve ligation (PNL), as previously described (21,47). Briefly, following gaseous induction and maintenance of anaesthesia with isoflurane (1–3 % in O2, Abbott Australia), the left sciatic nerve was exposed at mid-thigh level, approximately 3 mm proximal

79 Chapter 3 to the trifurcation of the sciatic nerve at the popliteal fossa. A 4-0 silk suture was inserted into the nerve to tightly ligate the dorsal 1/3–1/2 of the nerve trunk. The muscle (4-0) and skin (3- 0) were closed with silk sutures and the animals were left in warm bedding to recover. Animals were given a week to recover from surgery before testing to confirm development of neuropathy. The rats that presented significant mechanical allodynia (as shown in the von Frey test) received long-term polyethylene lumbar intrathecal catheters, inserted between vertebrae L5 and L6, advanced 3 cm rostrally and occipital region. All of these procedures were carried out under isoflurane anaesthesia exteriorized via the (1–3 % in O2).

3.3.17 Behavioral Tests in Rats PNL treated

Rats were placed individually in plastic enclosures with a wire mesh bottom 2–3 days to allow for acclimation before the procedures were carried out. At the day of the experiment, the acclimation was carried out twice more over a 30 min period before animal behavioural tests were performed. Behaviour was observed at set time points over a 4-hour period following intrathecal injection. Intrathecal injections were performed via the exteriorized catheter, using gentle restraint. MoVIB was dissolved to desired concentration (0.01–3 nmol) in 0.9% saline just before the experiment. MoVIB 10µl was injected through the catheter, followed by 15 µl of 0.9% saline to wash the drug from the catheter dead space. Control animals received 0.9% saline injections of equal volume.

To assess the mechanical allodynia, the paw withdrawal threshold (PWT) was measured with a series of von Frey hairs (range 0.4–15 g) and calculated using the up-down paradigm [245]. Briefly, von Frey filaments with bending force of 0.4–15 g were pressed perpendicularly against the medial part of the plantar skin and held for 2 s. Responses to these stimuli (flinching or licking of the hind paw) were observed from time 0–4 hs [245]. Stimulation of the same intensity was applied six times to each hind paw at intervals of several seconds, and an average of six values per animal served as the pain-related score (n = 4 animals/group of treatment dose). The maximum possible score (MPE) was recorded when animals failed to respond to the 15-g von Frey hair. The experimenter was blinded to all drug treatments. Catheter placement was checked after all experiments by injection of lignocaine (2%) and observation of rapid bilateral hind limb paralysis. Animals that had no paralysing symptoms after lignocaine and animals that had lost weight after the surgery for placement of the catheter were excluded from the analysis.

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3.3.18 Side Effect Score

Presence of side effects was assessed by blinded behavioural observations and measured using a qualitative side effect score rated as 0–3 (0 = nil, 1 = mild, 2 = moderate or 3 = severe). Mild toxicity manifests as occasional spontaneous tail twitching or lower back hunching. Moderate toxicity exhibits frequent tail flicks or occasional writhing of the tail and spontaneous hind-limb twitching, accompanied by intermittent ataxia and frequent urination. Severe toxicity was described when marked tail writhing occurred, along with frequent hind-limb twitching or hind-limb splaying and severe ataxia causing inability to walk normally [246, 247].

3.3.19 Statistical Analysis

Results presented in this chapter were expressed as the mean ± standard error of the mean (SEM), determined from at least 3 replicates, and are representative of at least 3 independent experiments. Microsoft Excel (version 12.2.0) was used to calculate average peak current values, as well as standard deviations and SEM. Sigmoidal concentration-response curves were generated and fitted using GraphPad Prism, Version 5.00, San Diego, California. IC50 values were calculated following nonlinear regression analysis of a four-parameter Hill equation, with variable Hill slope, fitted to the functional FLIPR data, and a fitted Hill slope of –1 fitted to the radioligand binding assays for all ω-conotoxins. Statistical significance was determined using analysis of variance (ANOVA) or student’s t-test, with statistical significance defined as p < 0.05, unless otherwise stated.

Dose response profiles in behavioural experiments were calculated as an average over the 1 to 4 hour period post-drug injection (time which maximal effect was observed) and normalized to a maximum possible effect (% MPE). MPE values were applied to the maximum pain

threshold and maximum side effect score. ED50 values were calculated for % MPE data using a four parameter Hill slope with variable slope, and compared using one-way ANOVA. When ANOVA tests were significant post-hoc comparisons between drug/treatment groups and vehicle at individual time points were made using the Bonferroni adjustment for multiple comparisons (GraphPad Prism 5 Software).

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3.3.20 Calculating peptide net charge

The net charge (Z) of a peptide at a certain pH can be estimated by calculating:

Where Ni is the number, and pKai the pKa values, of the N-terminus and the side chains of Arginine, Lysine, and Histidine. The j-index pertains to the C-terminus and the Aspartic Acid, Glutamic Acid, Cysteine, Tyrosine amino acids [248].

3.4 RESULTS

3.4.1 Discovery and Assay-Guided Isolation of MoVIA and MoVIB

A total of 30 cone snail species collected from Bismarck Sea, Papua New Guinea, and 18 other crude venom species collected in the Australia Sea, had the venoms extracted and screened for bioactivity at the human Cav2.2 channels endogenously expressed in SH-SY5Y 2+ cells. Cav2.2-mediated endogenous Ca responses were inhibited by a range of crude cone snail venoms as shown in Figure 3-1 (see below). We have for the first time identified a ω- conotoxins from a vermivorous (a worm hunting) cone snail. Previous to our discovery all

Cav2.2 inhibitors were identified from piscivorous cone snail species (for review see Chapter 1 and [1, 80, 231]. Successive rounds of HTS assay-guided fractionation lead to the isolation of two active peptides (Figure 4–2) eluting as early as ~ 10% solvent B, indicating active venom components were highly hydrophilic.

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Figure 3-1. FLIPR Cav2.2 HTS Assay. Plate layout.

The crude venoms from 48 cone snail species were tested for bioactivity at Cav2.2 channels using the FLIPRTetra imaging reader and the human neuroblastoma SH-SY5Y cells endogenously expressing Cav2.2 channels. From these crude venoms, 35 were from vermivorous species. Inhibition was evident for crude C. moncuri venom (well A2, pink colour), a vermivorous Conus. Other active crude venoms highlighted in colour are: C. canonicus (well 4A, dark green), C. auliculus (well 6A, dark blue), C. tulipa (well D5, orange) and C. catus (green). Showing in row E, PSS buffer (negative control, light green colour) and in row H KCl buffer (positive activation control, in light blue colour).

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* *

Figure 3-2. Assay-guided Fractionation. Active Cone Snail Toxins. Crude C. moncuri venom was injected onto HPLC coupled to a 5600 mass spectrometer. Two 2+ peaks were active inhibiting the Cav2.2-mediated Ca responses in SH-SY5Y cells. Peaks eluting at minutes 19.5–20 and 24.4–25, highlighted in pink, corresponded to two novel peptide toxins, named MoVIA and MoVIB, respectively. A figure on top of the HPLC trace shows the picture taken from Conus moncuri cone snail shell, collected in the Bismarck Sea, Papua New Guinea.

3.4.2 MoVIA and MoVIB Chemical Synthesis and Mass Spectrometry

Mass spectrometry indicated the two active peaks were each dominated by a single mass (data not shown). Edman degradation revealed N-terminal sequences of the two peptides, named MoVIA and MoVIB, with the cysteine framework VI/VII (-C-C-CC-C-C-), typical of four-loop conotoxins. The peptides primary structures comprised of 30 (MoVIA, CKPOGSKCSOSMRDCCTTCISYTKRCRKYYN) and 31 (MoVIB, CKPOGSKCSOSMRDCCTTCISYTKRCRKYY) amino acid residues (Table 3.1).

84 Chapter 3 A de-amidation of the C-terminus has caused a drop in GVIA potency in previous studies [248], indicating that this PTM (post-translational modification) was important for activity ω- conotoxins. However, interestingly, while most active ω-conotoxins have an amidated C- terminus, MoVIA and MoVIB have a free C-terminus, as indicated by its calculated mass of [M+H]+ 3605 Da and 3492 Da.

MoVIA and MoVIB were chemically synthesised with a free C-terminus. Synthetic peptides were analised by a mass spectrometer coupled to an HPLC. Masses and elution times (19.5–20 and 24.4–25 min) of MoVIA and MoVIB were identical to the masses of corresponding native peptides (Figure 3.3 A–D).

Table 3.1. ω-Conotoxins MoVIA and MoVIB AA Sequences and Alignment. Conotoxin Amino Acid Sequence MoVIA --CKPOGSKCSOSMRDCCTTCISYTKRCRKYYN MoVIB --CKPOGSKCSOSMRDCCTTCISYTKRCRKYY- GVIA --CKSOGSSCSOTSYNCCRSCNOYTKRC--Y-- GVIIA --CKSOGTOCSRGMRDCCTSCLLYSNKCRRY--

GVIIB --CKSOGTOCSRGMRDCCTSCLSYSNKCRRY-- CVID --CKSKGAKCSKLMYDCCSGSCSGTVGRC----- MVIIA --CKGKGAKCSRLMYDCCTGSC--RSGKC----- MVIIC CCCKGKGAPCRKTMYDCCSGSCG-RRGKC----- The software Clustal W was used to generate the alignment. The colouring scheme was done according to physicochemical criteria, as folow: Small + Hydrophobic aminoacids in red (including AVFPMILW and aromatic Y), acidic in blue (including DE), basic in pink (including H), Hydroxyl + sulfhydryl + amine + G in green (including STHCNGQ) and unusual PTM in grey (including O for the hydroxyproline).

Other groups have performed structure-function studies on synthetic ω-conotoxins by making single or multiple amino acid substitutions. These studies have determined the effect of individual aminoacids on the binding properties of some ω-conotoxins. The lysine in position 2 (K2), tyrosine in position 13 (Y13), and arginines in positions 10 and 21, and the amino terminus were found to be all important for the interaction of ω-conotoxins with the

Cav2.2 channel [217,218, 249-251].

85 Chapter 3 While aminoacid tyrosine (Y13) is conserved in most fish hunting cone snail species, MoVIA and MoVIB have an arginine substitution at this position (R13). This tyrosine in most fish hunting cone snails has proved to be highly important for high affinity binding to the channels [217,218, 249-251]. Thus, to investigate if an analogue of MoVIB with a tyrosine substituting the arginine at position 13 (Y13) would have higher affinity for Cav2.2 channels,

and to try making more potent Cav2.2 channel inhibitors, an analogue version of MoVIB with a tyrosine in the position 13 (R13Y) was synthesized and pharmacologically characterized.

A B

100 100

80 80

60 60 Synthetic MoVIA Synthetic + Native 40 40

Relative intensity 20 20

0 0 10 15 20 25 30 10 15 20 25 30 Time (min) Time (min) C D

100 100

80 80

60 60 Synthetic MoVIB Synthetic + Native 40 40

Relative intensity Relative 20 20

0 0 15 20 25 30 35 15 20 25 30 35 Time (min) Time (min)

Figure 3-3. LC/MS Analysis of Synthetic and Native MoVIA and MoVIB. The Insets represent the LC/MS (Liquid Chromatography/Mass Spectrometry) reconstructed spectra of MoVIA and MoVIB. A–B. Extracted ion chromatograms of synthetic (black trace) and native (red coloured trace) MoVIA forms C–D. Extracted ion chromatogram of synthetic (black coloured trace) and native (red coloured trace) forms of MoVIB overlapped. Retention times (~19 and 26 min, MoVIA and MoVIB, respectively) and masses for native and synthetic

86 Chapter 3 peptide toxins MoVIA and MOVIB (3605 Da and 3492 Da, respectively) overlapped, indicating the synthetic peptide forms was identical to native. Conotoxins MoVIA and MoVIB Belong to the O-Superfamily.

While the Edman degradation revealed the sequences of mature peptides MoVIA and MoVIB, PCR amplifications of cDNA cloning and sequencing revealed the MoVIA precursor sequence. cDNA sequence was translated to AA sequence using the Expasy Tools [240] and results indicated both peptides consist of 76 AA residues, including the signal, pre-pro and mature peptide regions (Figure 4–4). The AA sequence identified through cDNA amplification was identical to the aa sequence identified through activity-guided isolation and subsequent Edman degradation analysis. Phylogenetic reconstruction of the untranslated and coding regions allowed the calculation of a cladogram tree (Figure 3.5), which indicated that MoVIA is most closely related to ω-conotoxins from piscivorous Conus. Despite our suspect that C. moncuri is a worm hunting cone snail, it was not surprising that MoVIA cluster more closely to piscivorous Conus, as MoVIA belongs to the O-superfamily, which includes peptides from molluscivorous, piscivorous and vermivorous Conus [5, 73, 85, 205, 252, 253].

The subsequent pre-pro-region of MoVIA has an intermediate level of conservation amongst ω-conotoxins, followed by highly divergent mature toxin region, similarly to other ω- conotoxins. MoVIA sequence is most homologous to sequences from conotoxins of the fish hunting Conus geographus venom (58–62% identity with GVIA, GVIIA and GVIIB). The residues in MoVIA that are conserved amongst ω-conotoxins include a lysine at position 2 (K2), a glycine at position 5 (G5) and an arginine at position 25 (R25). Moreover, many of the aa residues in MoVIA and MoVIB contain hydroxyl moieties, in addition to two hydroxyprolines (O) at positions 4 and 10, similarly to GVIA; and seven basic groups (K2, K7, R13, k24, R25, R27 and K28). Both MoVIA and MoVIB have a single acidic amino acid (aspartic acid, D14) and are thus, highly positively charged (net charge +5.7). Interestingly, while most active ω-conotoxins have an amidated C-terminus, MoVIA and MoVIB have free C-termini. A de- amidation of the C-terminus caused a drop in the potency of GVIA in previous study [254], indicating this PTM is important for the activity of other ω-conopeptides from piscivorous Conus. Comparison of MoVIA and MoVIB aa sequences to other ω-conotoxins (3-4) identified several additional unusual features that will be discussed in the following sections of this chapter.

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Figure 3-4. AA precursor sequences of MoVIA Predicted from cDNA Sequences and Aligned with Sequences of Other Conotoxins Belonging to the O-Superfamily. PCR was performed to identify MoVIA precursor sequence. Primers were designed based on published sequences of vermivorous cone snails, belonging to the O-superfamily (18). MoVIA precursor peptide sequence was predicted from cDNA sequences using Expasy tools, as described in the methods section. Aminoacid sequences of conotoxins belonging to the O-superfamily were retrieved from Conoserver [205] for comparison to MoVIA sequence. Jalview version 2.8 [243] was used to generate this figure, and to calculate the cladogram trees (Figure 3.5). The scheme of colour was based on the Clustal W. Conotoxins from cone snails: C. magus, C. geographus, C. catus, C. caracteristicus, C. achatinus, C. vexillum, C. emaciatus, C. imperialis, C. ventricosus, C. pennaceus, C. arenatus, C. lividus, C. tessulatus, C. virgo, C. caracteristicus and C. litteratus.

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* O1

O2

O3

Figure 3-5. Cladogram Tree of the O-Superfamily. Cladogram tree was generated using the Jalview version 2.8 tool Average Distance Tree BLOSSUM 62. Average distances numbers are shown by the tree roots. Highlighted are the O1- superfamily (black coloured asterisks) and the localization of MoVIA in the tree (asterisks in black colour). Results indicate ω-conotoxin MoVIA and MoVIB are closer related to ω-conotoxins from fish hunting cone snails.

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3.4.3 MoVIA and MoVIB Displace 125I-GVIA from Cell Membranes

SH-SY5Y human neuroblastoma cells have 125I-GVIA binding sites that can be fully

displaced by the Cav2.2 selective inhibitors -conotoxins CVID, GVIA and MVIIA (see Chapter 2 and [3, 85, 200, 255]. Hence, we used radiolabelled 125I-GVIA to confirm that the

MoVIA and MoVIB binding sites at the human Cav2.2 in SH-SY5Y cell membranes is the same as that of the other ω-conotoxins from fish hunting snails. This was suggested earlier in 2+ this chapter from full inhibition of the Cav2.2-mediated Ca responses by the crude Conus moncuri venom in the presence of saturating concentrations of nifedipine (see chapter 2, and ref [3]). Figure 3-6A shows that both peptides MoVIA and MoVIB fully displaced 125I-GVIA binding from SH-SY5Y cell membranes, with high affinity. MoVIA was more potent than MoVIB (pIC50 ± SEM [M], MoVIA 10.30 ± 0.13 and MoVIB 9.01 ± 0.37, respectively, Figure 2-2, Table 3.2).A Hill slope of ~ –1 suggests MoVIA and MoVIB occupy a single class of receptor. Altogether, these results indicate MoVIA and MoVIB share a common binding site in the human SH-SY5Y cell membranes with ω-conotoxins isolated from piscivorous cone snails.

Since MoVIA and MoVIB were discovered from a putative vermivorous Conus species, we determined if these peptides were able to displace 125I-GVIA from fresh fish brain membranes. A surprising pattern was revealed with these assays, with MoVIA and MoVIB being highly potent at displacing 125I-GVIA from the fish brain membrane preparation, indicating these

toxins have high affinity for fish brain Cav2.2 channels. In fact, these toxins were the most

potent Cav2.2 inhibitors in fish brain membranes, when compared with other high affinity ω- conotoxins from piscivorous, including, GVIA, CVID and MVIIA. MoVIA potency was

similar to that of MoVIB (pIC50 ± SEM values [M]), MoVIA 11.49 ± 0.19, MoVIB 11.23 ± 0.05, GVIA 10.09 ± 007, MVIIA 10.58 ± 0.09 and CVID, 10.16 ± 02. (Table 3.2B and D; see

IC50 in Table 3.2). MoVIA was more potent at the fish membrane than at the human membrane (Two way-Anova, p<0.05), similarly to MoVIB (p<0.001).

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A B SH-SY5Y Membranes 100 Fish Brain Membranes 100 80 80

60 CVID 60 CVID MoVIA GVIA 40 MoVIB 40 MVIIA MoVIB-[R13Y] MoVIA 20 20 I-GVIA BINDING (%CPM) MoVIB

125 MoVIB-[R13] 0 0 -14 -13 -12 -11 -10 -9 -8 -7 -14 -13 -12 -11 -10 -9 -8 -7 LOG -CONOTOXIN [M] LOG -CONOTOXIN [M]

C D Fish brain membrane ) -11 Human SH-SY5Y membrane 10 -12 50 10

10-11 10-10

10-10 10-9 10-9 I-GVIA BINDING (PIC BINDING I-GVIA

125 10-8 10-8 ] A Y 3Y] oVIB 1 oVI 13 MoVIA M [R M MoVIB [R

Figure 3-6. 125I-GVIA Radioligand Binding Assays. MoVIA and MoVIB fully displaced 125I-GVIA binding from fish brain and SH-SY5Y membranes. A. MoVIA and MoVIB displaced 125I-GVIA. The affinity of analogue [R13Y]- MoVIB was 10-fold shifted to the left of the curve, compared to the wild type B. Fish brain 125 membranes. MoVIA and MoVIB were the most potent toxins in displacing I-GVIA from fish brain membranes. C–D. In SH-SY5Y cells the affinity of the analogue [R13Y]-MoVIB was significantly lower than the wild type (~ 100-fold) (IC50 in Table 3.2). The potency of

MoVIA was (pIC50 ± SEM values) (M) 11.49 ± 0.19, MoVIB 11.23 ± 0.05, GVIA, MVIIA and

CVID 10.09 ± 007, 10.58 ± 0.09 and 10.16 ± 02, respectively (see IC50 in Table 3.2). MoVIA was more potent on fish membrane than human membrane (Two way-Anova, p<0.05) similarly

91 Chapter 3 to MoVIB (p<0.001), while the analogue [R13Y] affinity was similar in both human and fish (p>0.05). Data was calculated as mean ± SEM, n = 3.

125 Table 3.2. ω-Conotoxin Affinity (IC50 ± SEM, [nM]) at Displacing I-GVIA Binding. SH-SY5Y Fish brain Rat brain ω-Conotoxin human cell membranes membranes membranes

MoVIA 0.055 ± 0.017 0.004 ± 0.0026 ND

MoVIB 1.60 ± 0.204 0.006 ± 0.001 ND

MoVIB-[R13Y] 0.84 ± 0.003 0.212 ± 0.045 ND

GVIA 0.032 ± 0.012 0.080 ± 0.051 0.043

GVIIA ND ND 3.7 [256]

GVIIB ND ND ND

CVID 0.0034 ± 0.009 0.069 ± 0.052 0.035 [3]

MVIIA 0.0065 ± 0.001 0.026 ± 0.074 0.064 [3]

ND: not determined.

3.4.4 MoVIA and MoVIB Selectively Inhibit Functional hCav2.2 Channels

In chapter 2 of this thesis and in ref [3], I showed that the maximal activation of the human 2+ Cav2.2-mediated Ca responses in SH-SY5Y cells is achieved when using a KCl stimulation 2+ solution (90 mM KCl + 5 mM CaCl2). Ca responses were quantified using a fluorescent dye and the fluorescence read using the HTS machine FLIPRTetra. The potency and selectivity of 2+ MoVIA and MoVIB to inhibit Cav1.3 and Cav2.2-mediated Ca responses was assessed using adequate inhibitor.

92 Chapter 3

A B 100 100

80 80 60 MoVIA 60 MoVIA MoVIB 40 MoVIB [R13Y] 40 [R13Y]

Response (%) Response 20 CVID 20 GVIA 0 0 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4

Log -Conotoxin (M) LOG -Conotoxin (M)

C 10-7 ) Human SH-SY5Y cells 50

2.2 (PIC v -6 10

Functional Ca Functional 10-5

IA IB oV oV 13Y] M M [R

Figure 3-7. Functional Cav2.2 Assays. 2+ MoVIA and MoVIB inhibited KCl-activated Ca responses from Cav2.2, but not Cav1.3 in the

human SH-SY5Y cells. A. Functional Cav2.2. Cav2.2 inhibition by MoVIA, MoVIB and

[R13Y]-MoVIB and comparison with CVID, GVIA and MVIIA B. Functional Cav1 assay; 2+ MoVIA, MoVIB and [R13Y]-MoVIB (up to [10 µM] did not affect the Cav1.3-mediated Ca responses in SH-SY5Y cells. Data was calculated as mean ± SEM, n = 3 independent experiments (4 replicates/experiment). C. The affinity of MoVIA was (pIC50 ± SEM)

6.45±0.04 M and MoVIB 6.13 ± 0.15 M (see IC50 in Table 3.2). The potency of [R13Y]- MoVIB was lower compared to the wild type MoVIB (~ 10-fold) (pIC50 ± SEM of 5.7 ± 0.21; p< 0.05 using student’s t-test).

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Saturating concentrations of adequate pharmacological inhibitor of Cav1 (nifedipine 10

µM), Cav2.2 (CVID 3 µM) or Cav3.1 inhibitor (mibefradil 30 µM). MoVIA and MoVIB 2+ inhibited functional Cav2.2-mediated Ca responses with nanomolar concentrations (pIC50 ±

SEM of MoVIA 6.45 ± 0.04 M) and MoVIB (6.13 ± 0.15 M) (Figure 2-5A; see IC50 in Table 3.3). Unexpectedly, the potency of the [R13Y]-MoVIB was significantly lower compared to

the wild type (~ 10-fold) (pIC50 ± SEM of 5.7 ± 0.21; p< 0.05 using student’s t-test (Figure 3.9 C), despite no significant difference in binding affinity in SH-SY5Y membranes (see Table

3.2). The selectivity of MoVIA and MoVIB (30 µM) were also tested against native Cav1.3 and Cav3.1 channels endogenously expressed in the human SH-SY5Y cells [3]. These responses were unaffected by MoVIA and MoVIB (Table 3.3), suggesting these -conotoxins have a strong preference for Cav2.2 channels over other subtypes expressed in this cell line.

3.4.5 MoVIA and MoVIB Selectively Inhibited HVA N-type in Rat DRG

ω-Conotoxins MoVIA and MoVIB (0.1–3 µM) potently and selectively inhibited depolarization-activated Ba2+currents in rat DRG cells (Figure 3-8A–C). Inhibition by MoVIA and MoVIB (~ 33 % of the total current) appeared to be irreversible following > 5 min washout;

while CVIE, a reversible Cav2.2 blocker, exhibited rapid recovery from block. N-and P/Q-type

calcium currents (from Cav2.2 and Cav2.1 channels, respectively) are the main currents in rat DRG [257, 258]. Co-addition of saturating concentrations of the selective N-type calcium channel inhibitor ω-conotoxin CVIE after saturating concentrations of MoVIA (3 µM did not further affect Ba2+currents (Figure 3-8B), indicating MoVIA and MoVIB fully inhibited HVA

(high voltage-activated) N-Type currents through native Cav2.2 channels in rat DRG neurons. To further confirm this selectivity of MoVIA at the N-type currents we isolated this current type using saturating concentrations of MoVIA (3 µM) and after full inhibition of the N-type currents by MoVIA (~34% of the responses in DRG), saturating concentrations ω-Agatoxin (10 µM) caused a further 40%inhibition of the residual current (data not shown). This

percentage of current corresponds to P/Q-type-type currents from Cav2.1 channels, as shown previously by other studies [86, 257].

These results confirmed MoVIA or MoVIB had little effect on the P/Q-type currents endogenously expressed and suggested that MoVIA and MoVIB are selective inhibitors of N- type currents in rat DRG neurons, since Cav1 and Cav2.3 are minor components of the rat DRG neurons currents (~2 and 5%, respectively) [257]. However, to confirm selectivity, future

94 Chapter 3

studies should investigate the effects of MoVIA and MoVIB on all Cav subtypes using a heterologous system. If selectivity for N-type current is confirmed, MoVIA and MoVIB may have potential as lead molecules for the treatment of pain conditions, as well as they may be useful as template molecules for drug design of small molecules peptidomimetic research tools.

Figure 3-8. High Voltage-Activated (HVA) Cav Currents Rat DRG Neurons.

A. Superimposed depolarization-activated whole-cell Ba2+ currents elicited by a 75 ms voltage step to 0 mV from a holding potential of -70 mV. B. Time course of experiment in A, plotting peak inward current values every 10 seconds. C. Concentration-response curves for MoVIA, MoVIB and MoVIB (R13Y). Data points are mean ± SEM, n=3 independent experiments.

Table 3.3. Potency of ω-Conotoxins Inhibiting Functional Cav2.2 Channels.

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Functional Functional Selective

2+ ω-conotoxin Ca assay N-type currents in Cav2.2 blocker

hCav2.2 (µM) rDRG neurons (µM) (µM)

MoVIA 0.33 ± 0.018 0.083 ± 02 Y*

MoVIB 0.60 ± 0.121 0.18 ± 03 Y*

[R13Y]-MoVIB 3.47 ± 0.55 0.90 ± 12 Y*

GVIA 0.17 ± 0.025 0.10 (43) Y

GVIIA NK ND ND

GVIIB NK ND ND

CVID 0.16 ± 0.025 0.078 [257] Y

MVIIA 0.024 ± 0.005 0.052 [257] Y

The asterisks (*) indicates potencies of the marked toxins were > than 30 µM. Y= yes. ND = not determined. (IC50 ± SEM, [µM]).

3.4.6 1H NMR Solution Structure of MoVIB

To investigate if MoVIB had any unique structural feature, that could explain differences in pharmacophore, we determined its three-dimensional (3D) structure. The peptide was subjected to detailed characterization by solution NMR spectroscopy at ultra-high field (900 MHz). NMR data were of excellent quality with generally sharp line and distinct signal dispersion, consistent with a well-defined structure in solution. Resonance assignments were achieved through standard homonuclear sequential assignment strategies [254], which allowed complete assignments of both backbone and side chain resonances.

The 20 superimposed NMR structures of MoVIB are shown in Figure 3-9, and structure calculations in Table 3.4. From these studies it was clear that the structure of MoVIB is well defined and of high quality in terms of geometry. The structure is dominated by the cysteine knot framework [217], which in MoVIB stabilise a β-hairpin comprising residues 19-26. The S6 and T17 hydroxyl side chains, which were observed in the NMR spectra, form hydrogen

96 Chapter 3 bonds to the K7 and R27 backbone carbonyls respectively. Structural information including inter-proton distance restraints, backbone and side-chain dihedral angles and hydrogen bonds were derived from the NMR data and included as input restraints for simulated annealing calculations. In the final round a structural family of 50 structures were calculated, refined and energy minimized in explicit water, and the twenty best structures judged on energies, consistency with the experimental data and covalent geometry were chosen to represent the solution structure of MoVIB.

Figure 3-9. The 20 superimposed 3D NMR Structures of MoVIB in Stereoview The 20 superimposed NMR structure of MoVIB were calculated and are represented in a 3D stereoview.

Peptide MoVIA has two hydroxy-proline and one proline aminoacid residue, all of which were confirmed to be in a trans- conformation based on 13C chemical shifts and sequential NOE patterns. Hydroxyl protons are generally not observed in NMR spectra recorded in aqueous solution do to the fast exchange with solvent; however, in the data of MoVIB the side chain hydroxyl protons of both S6 and T17 were clearly visible, suggesting these are protected from the solvent through hydrogen bonding.

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Table 3.4. Energies and structural statistics for the family of 20 MoVIB structures with highest overall MolProbity score. Energies (kcal/mol) Overall -986.6 ± 31.6 Bonds 18.37 ± 1.40 Angles 56.18 ± 6.55 Improper 20.93 ± 2.44

Van der Waals -104.3 ± 5.39 NOE 0.265 ± 0.019 cDih 0.179 ± 0.130 Dihedral 138.4 ± 1.75 Electrostatic -1116.7 ± 33.8 MolProbity Statistics Clashes (>0.4 Å / 1000 atoms) 8.28 ± 3.44 Poor rotamers 0.37 ± 1.14 Ramachandran Outliers (%) 0.19± 0.86 Ramachandran Favoured (%) 92.31 ± 1.25 MolProbity score 1.93 ± 0.14 MolProbity score percentilea 78.6 ± 6.86 Residues with bad bonds 1.43 ± 1.79 Residues with bad angles 0.00 ± 0.00 Atomic RMSD (Å) Mean global backbone (residues 1-28) 0.59 ± 0.19 Mean global heavy (residues 1-28) 1.26 ± 0.23 Distance Restraints Intraresidue (i-j = 0) 101 Sequential (/i-j/ = 1) 105 Medium range (/i-j/ < 5) 40 Long range (/i-j/ > 5) 97 Hydrogen bonds 6 (for 3 h-bonds) Total 349 Dihedral angle restraints  9 Ψ 8  15 Total 32 Violations from experimental restraints Total NOE violations exceeding 0.3 Å 0 (highest 0.275) Total Dihedral violations exceeding 3.0° 0 (highest 1.97)

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3.4.7 Secondary Chemical-Shifts

To verify that the structure was not affected by the replacement of R13 with a Y13, NMR data were also recorded for the variant MoVIB-[R13Y], and again the data were of high quality and full assignments was achieved. In addition the recording of a 1H-13C HSQC at natural abundance allowed determination of 13C carbon chemical shifts for the majority of residues. The H secondary shifts, i.e. the difference between the observed resonance frequencies and the frequencies reported for random coil conformations, are sensitive indicators of secondary structure. A comparison of the secondary shifts for MoVIB and MoVIB-[R13Y], shown in Figure 3-11, reveals minimal difference throughout the sequence, confirming that the replacement had no effect on the overall fold of the peptide.

Shifts (ppm) α Secondary H

MoVIB AA Sequence

Figure 3-10 Hα and NH Chemical Shifts of MoVIB and [R13Y]-MoVIB.

Samples of the native and mutant MoVIB were diluted in 95% H2O, 5% D2O and pH 5.1 for the chemical shift analysis. Secondary Hα chemical shifts were analysed and 2D NMR identified similar molecular conformations for both native MoVIB and mutant [R13Y]. Overall, the structure of MoVIB and the mutant [R13Y]-MoVIB were similar, indicating the structure hasn’t been altered significantly with the mutation.

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3.4.8 MoVIB produces dose dependent analgesia in the PNL model.

The PNL (partial nerve ligation) model was developed and described for the first time by Seltzer, et al in 1990 [259]. In this model rats are unilaterally ligated about half of the sciatic nerve high in the thigh. The model is characterized by rapid onset of behaviours suggesting spontaneous pain and disordered responses to non-noxious and noxious stimuli [260]. These responses include reduced withdrawal thresholds to repetitive touch (touched-evoked hyperesthesia), touch-evoked allodynia, reduced withdrawal thresholds to noxious thermal stimuli and exaggerated responses to noxious heat and mechanical stimuli (thermal hyperalgesia) [260]. Some of these disorders are seen at mirror image sites on the hind limb opposite the lesion. These disorder may start within hours after partial nerve ligation and last many months [260]. Based on the immediate onset and long-lasting perpetuation of similar symptoms such as, touch-evoked allodynia and hyperalgesia, pain models mimic causalgiform pain syndromes in humans (for review see: [261]). In humans partial nerve injury is the main form of causalgiform pain syndromes triggered and maintained by sympathetic activity [259, 260].

In our studies, the pre-surgery (PNL) baseline for mechanical PWT in animals was 14.9 ± 0.1 g; at 9 days post-PNL surgery (prior to intrathecal catheter placement) 0.7 ± 0.1 g, and 0.6 ± 0.3 g at 14 days post-PNL surgery (4 days after intrathecal catheter, n = 24), indicating neuropathic pain had been induced. The intrathecal actions of MoVIB at 0.1–1 nmol doses were first examined at 14 days post-PNL surgery (4 days post-intrathecal catheter placement). MoVIB dose-dependently produced a highly significant increase in mechanical PWT compared to vehicle (n = 4 rats/group of dose) at 1–4 hours post-injection (significant (p >0.05) and the PWT and side effect scores were similar to that of the vehicle (Figure 3.11, p < 0.001).

A dose response was plotted for the pain threshold relative to side effect score to calculate MoVIB therapeutic index (TI). This measurement was used to understand the margin of safety between the dose needed for the desired effect and the dose that produces unwanted and possibly dangerous side effects. MoVIB TI was calculated as the ratio between half-maximum effects (ED50) for the mechanical PWT relative to side-effect score. The %MPE data was averaged for times 1–4 h for all the group of doses (mean ± SEM of the maximal possible effect, n = 4 rats/group of dose). The intrathecal treatment with MoVIB caused significant dose-

dependent analgesia. However, it showed increasing side effects at doses ≥0.1 nmol (EC50 0.05 ± 0.03 and 0.09 ± 0.05 nmol, respectively) (p ≥ 0.001, one-way ANOVA), indicating MoVIB

100 Chapter 3 has a small TI of 1.8 (Figure 3.11), higher than the marketed ω-conotoxin MVIIA and similar to CVID [42].

Mechanical allodynia was measured using von Frey hair filaments with bending force of 0.4–15 g applied to the left hind paw (operated paw) before and after treatment with MoVIB or vehicle. Behaviour was observed from time 0–4 h. In rats that went PNL surgery (partial nerve ligation) the PWT (paw withdrawal threshold) decreased significantly compared to the pre-surgery baseline (p<0.001), confirming PNL had been induced. The experiment was blinded to all groups of treatments.

A B MoVIB x Pain Threshold MoVIB Side Effect 3 15

saline 2 saline 10 1 nmol 0.3 nmol 1 nmol 0.1 nmol 0.3 nmol 0.1 nmol 5 0.03 nmol 1 0.01 nmol 0.03 nmol 0.01 nmol SideEffect Score (0-3) von Frey Threshold (g) Threshold Frey von 0 0 0 1 2 3 4 0 1 2 3 4 Time (h) Time (h)

C Pain Threshold/Side Effect 100

80

60

40 MPE (%) MPE

20 Pain Threshold Side Effect 0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 LOG [MoVIB] (nmol)

Figure 3-11. Effects of Intrathecal MoVIB on Pain of PNL-Induced Rats. Mechanical allodynia induced in rats. A. Intrathecal MoVIB (0.001–1nmol), but not vehicle (saline), dose dependently relieved mechanical allodynia in rats with PNL. Data was expressed as percentage of maximal possible effect (%MPE) and plot against time. B. The toxicity caused by intrathecal doses of MoVIB was assessed using a qualitative side effect score0 –3: 0 = nil, 1 = mild, 2 = moderate or 3 = severe, rated by behavioural observation (detailed in the methods

101 Chapter 3 section). C. Dose response curves were plotted from intrathecal doses of MoVIB to identify the therapeutic index (TI) of MoVIB. The ED50 values of (mean ± SEM) 0.05 ± 0.03 and 0.09

± 0.05 nmol, respectively was calculated as 50% MPE for MoVIB on PWT and side effect score. MoVIB TI was 1.8.

3.5 DISCUSSION AND CONCLUSIONS

This chapter reports the discovery of the ω-conotoxins MoVIA and MoVIB from the venom of Conus moncuri. We collected this recently described species in Papua New Guinea and its shell features and habitat closely matched the description of other C. moncuri specimens found in the pacific sea, surrounding the Philippines [262]. C. moncuri has a pronounced purple- brown basal stain, a sutural ridge on the spire whorls and a dome like apex and lives amongst rocks and corals in strong current at a depth of 20–50 m [262]. Questions whether the specimens found in the Philippines were indeed a new species or merely a variety of the Conus litteratus Linnaeus, a closely related Conus, still remain [262, 263]. However, while the feeding behaviour has not been confirmed in the native habitat, based on the characteristics of the shells, and comparison with other worm hunters including C. litteratus and C. leopardus [262], Conus moncuri is suspected to be vermivorous.

The venom of piscivorous snails consists of a cocktail of various pharmacologically active peptides. This has been associated with the “lightning-strike cabal”(δ and κ- conotoxins block

Kv and delays Nav channels’ inactivation, respectively, thus acting by a rigid immobilization characteristic of excitotoxic shock and sudden tetanus) and “motor cabal” (α, ω, µ-conotoxins act by blocking neuromuscular transmission and paralysing the prey by targeting nicotinic

acetylcholine receptor, blocking Cav channels pre-synaptically and blocking skeletal muscle

Nav channels, respectively) effects that rapidly immobilize more mobile prey [28, 230-232].

Because of the slow movements of the snails, in contrast to fish, the expression of paralytic toxins, such as the ω-conotoxins, in piscivorous Conus have only been associated with their ability to capture prey [232]. Following this line, the venoms from Conus species that specifically prey on fish have no effect on molluscs or worms and vice versa (for review see ref. [232]). Accordingly, the worm-hunting species would likely produce conopeptides specific to annelids, snail-hunting Conus would likely produce mollusc-specific conopeptides and fish hunting snails would produce vertebrate-specific conopeptides; and indeed, all previously

102 Chapter 3 discovered ω-conotoxins have been identified only from piscivorous Conus. However, a δ- conotoxin which is also part of the “lightning-strike cabal” used for immobilization of prey [230, 232, 264] has been recently identified in molluscivorous and vermivorous Conus venoms [264, 265]. Thus, it seems plausible that active ω-conotoxins are also found in the venom of vermivorous Conus. This chapter shows evidences for the activity of two novel ω-conotoxins from vermivorous Conus. While the exact reason why a vermivorous Conus expresses paralytic toxins with high affinity for fish Cav channels in their venom is unknown, it is possible that paralytic toxins in the venom of vermivorous allow the capture of a wider diversity of prey, as seen for C. californicus, which feeds on at least four different phyla [5, 73]. Alternatively, these toxins may be used in defence strategies such as protection from fish predation. Finally, MoVIA and MoVIB may also be active on worm ion channels and thus, involved in prey capture.

The MoVIA precursor sequence we identified by PCR cloning is highly homologous to the precursor sequence of ω-conotoxins from piscivorous cone snails [85, 86, 238]. Accordingly, it was not surprising that the cladogram tree showed MoVIA clustered more closely to fish

hunting cone snails in Superfamily O1 [266]. The O-Superfamily is found across the entire genus Conus [267], including molluscs [253], fish [85, 86] and worm hunters (this work and ref [5, 73]). The high homology of the MoVIA signal sequence with the signal sequences of ω- conotoxins from fish hunting cone snails reflects the high level of conservation observed within the O-superfamily, with the hypervariability observed on the cladogram suggesting selective rapid evolution of the genes that encode the mature toxins [267].

The NMR structure of MoVIB revealed strong structural similarities to the ω-conotoxins from piscivorous Conus, mainly due to the six cysteines cross-linked to generate a conserved cysteine knot [218]. A direct comparison of the tertiary structure of MoVIB with published structures of homologous ω-conotoxins (PDB ID code: GVIA, 1TTL, MVIIA, 1TTK and MVIIC, 1CNN) is given in The side chains important for the pharmacophore of GVIA, MVIIA and MVIIC (for review see chapter 1 and ref [1]), including a high number of positive and hydrophobic residues are represented on the Figure 3-12.

103 Chapter 3 A B

II II IV

IV

C D

II IV II IV

Figure 3-12. NMR Solution Structure and Side Chains of MoVIB. NMR structure and side chains of MoVIB were labelled for comparison with GVIA, MVIIA and MVIIC. A. MoVIB (top left, grey coloured backbone) B. GVIA (top right, light blue backbone). C MVIIA (bottom left, green backbone) and D MVIIC (bottom right, red backbone). The amino acid residues in the side chains that were important for the pharmacophore of GVIA, MVIIA and MVIIC, including Y13 (tyrosine13) and K2 (lysine2) [268, 269] and several positively charged (blue) and hydrophobic residues (green) are labelled in the side chains, including R13 in MoVIB and the longer hydrophobic tyrosine tail. Disulphide bonds are shown in yellow, ball-and-stick representation. Indicated with Roman letters are the Loops 2 and 4.

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A MoVIB

B GVIA

C MVIIA

D MVIIC

Figure 3-13. Electrostatic Surface Mappings. Represented are the electrostatic surface mappings of (A) MoVIB (B) GVIA, (C) MVIIA and (D) MVIIC in two different orientations. The differences in charge states for MoVIB are highlighted (positively charged residues in blue, negative in red and hydrophobic in grey). and compared with GVIA, MVIIA and MVIIC (B– D).

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Overall, MoVIB tertiary structure was highly similar to those of ω-conotoxins from fish hunting snails, despite considerable variability in primary structure within the inter-cysteine residues. The most notable difference in structure between all the peptides compared is seen for the conformation of the loop 2, which in GVIA is substantially different (Figures 3-12 and 3-13).

Interestingly in MoVIB the loop 2 conformation is similar to the other ω-conotoxins, and the R13 positions itself in a pocket normally filled by the Y13 (Figure 3-12B). The fact that different conformations are observed for this loop may suggest it may be altered upon binding to the receptor. MoVIB has seven positively charged amino acid residues (K2, K7, R13, K24, R25, R27, K28) and a single acidic group (aspartic acid, D), plus a free C-terminus not seen in other ω-conotoxins (for review see ref [80]. Additionally, MoVIA has an elongated C-terminus with a tyrosine and asparagine in the last two positions (Y30, N31), while MoVIB finishes at Y30. The fact that MoVIA was more potent than MoVIB having a single aa residue different, suggest an interaction of this position (N31) with the Cav channel, as position 31 is located in loop 4, which has been involved in binding interactions [269]. Because amidation has a positive effect on the potency of other -conotoxins [85, 86, 257], the higher potency of MoVIA in the functional assays compared to MoVIB, suggest a possible interaction of the amide group present in the amino acid placed at the last position in MoVIA (N31) with the channel.

Similar to other ω-conotoxins isolated from fish-hunting cone snails, MoVIA and MoVIB were found to be highly selective Cav2.2 inhibitors. In native rat DRG neurons, N- and P/Q- type are the main HVA calcium currents ([258]). MoVIA and MoVIB at concentrations up to

30 µM were found to selectively block the N-type current (Cav2.2) without affecting the P/Q-

type currents in rat DRG (Cav2.1). Moreover, MoVIA and MoVIB (30 µM) failed to inhibit 2+ Ca influx associated with Cav1.3 and Cav3.1 endogenously expressed in the human SH-SY5Y cells [3]. These findings suggest that MoVIA and MoVIB block Cav2.2 channels preferentially

over other Cav channel subtypes in rat and human neuronal cells. Thus, these new δ-conotoxins are likely to avoid unwanted side effects associated with inhibition of these physiologically

significant Cav channels (for review see ref [80]). Future studies should confirm MoVIA and

MoVIB selectivity at Cav2.2, studying the Cav subtypes that were not included in this study

(Cav1.1 and Cav2.3) in recombinant systems, as they are not expressed in the native systems used in this study

106 Chapter 3

Although the pharmacology of MoVIA and MoVIB was similar to other -conotoxins from fish hunting cone snails, their sequences were unusual because an arginine was present at position 13, where other -conotoxins have a crucial tyrosine. Although two other ω- conotoxins from C. geographus venom (GVIIA and GVIIB) also have an R13, the pharmacology of these toxins has not been extensively investigated. Early binding studies indicate that the affinity of GVIIA is ~ 100-fold lower than that of the highly homologous GVIA, also found from C. geographus venom (see Table 3.2) [231, 238, 256]. Thus, it would be expected that a replacement of the arginine for a tyrosine at position 13 would give higher affinity to an analogue. However, unexpectedly, the potency of the analogue [R13Y] was not significantly increased in binding experiments using human Cav2.2 endogenously expressed in SH-SY5Y cells, while in functional assays using the same human cells or rat DRG neurons the potency was reduced ~ 10-fold.

Since R13Y-[MoVIB] analogue had a similar structural conformation to that of the wild type MoVIB; it appears that R13 can replace Y13 in these new -conotoxins. While MoVIA

and MoVIB might have a different mode of interaction with Cav2.2 compared to other ω- conotoxins, R13 in MoVIB overlays the Y13 in the other ω-conotoxins (Figure 3-12). Thus we speculate that ionic interactions between the channel and the R13 contributed to the activity of the new ω-conotoxins, much like the OH group of Y13 contributes to the affinity of piscivorous -conotoxins [250].

As reviewed in chapter 1, the large putative extracellular loop between IIIS5 and IIIH5 of

the Cav channel has been described as critical for Cav2.2 channels’ block by the ω-conotoxin GVIA [142, 144]. In particular, negative residues G1326 and Q1332 [144], G1327, Q1334, Q1337, and G1339 of this region seem to interact with positive residues of the ω-conotoxins [142]. Electrostatic surface mapping of MoVIB revealed a range of basic residues and several possibilities of hydrogen bonds and salt bridges with the corresponding residues located in the pore region of the channel [270]. The residues localized in the loop 2, mainly Y13 were important to the activity of most fish hunting ω-conotoxins but the specific interaction contributing to Y13 effect has not been experimentally confirmed. Additionally, residues in loop 4 including R17, Y22, K24 and R25 have smaller effect on GVIA and MVIIA affinity [249, 250]. The MoVIB 1H NMR solution structure and comparison with the side chains of other high affinity ω-conotoxins revealed that the interacting surface may be possibly formed

107 Chapter 3 by the residue R13 (R13), surrounded by a range of positive residues in loops 2 and 4 (K2, K24 and R25). However, this remains to be confirmed through additional structure-activity studies.

Interestingly, while Y13 is conserved and was crucial for the activity of fish hunting ω- conotoxins [249, 250, 258, 271], fish hunting µ-conotoxins (muscle Nav channel blockers) have

a conserved R13 that is essential for activity at Nav channels [205]. To rule out the possibility that MoVIA and MoVIB act on endogenous Nav channels expressed in SH-SY5Y cells,

MoVIA and MoVIB were tested on the Nav channels using membrane potential assays, as previously described [195]. Saturating concentrations (30 µM) of MoVIA or MoVIB did not affect veratridine-activated Nav1.2, Nav1.3 or Nav1.7 responses (data not shown) from endogenous channels expressed in SH-SY5Y cells. These findings support the data showing that despite having an R13, MoVIA and MoVIB are selective Cav2.2 inhibitors and are pharmacologically similar to other ω-conotoxins.

Consistent with Cav2.2 inhibition being a validated analgesic strategy, behavioural studies using the PNL model of neuropathic pain in rats confirmed the analgesic potential of MoVIB. Intrathecal delivery of the peptide MoVIB produced an increase in mechanical PWT in animals which had undergone partial nerve ligation of the sciatic nerve that lasted for up to four hours. PWT levels after MoVIB injection approached the levels prior to nerve ligation. Generally, ω- conotoxins have demonstrated efficacy but have a relatively low therapeutic index. Unfortunately, reduction in allodynia by MoVIB was accompanied of side effects common for ω-conotoxins, including shaking, tail twitching and serpentine tail movements. These side effects appeared in parallel with efficacy, contributing to the small safety window observed for MoVIB following intrathecal administration. This means MoVIB would likely need to be carefully titrated intrathecally to reach an effect in a clinical setting to limit the extent of unwanted side effects, as is required for Y13-containing ω-conotoxins MVIIA and CVID from fish hunter cone snails [86, 247]. Future studies should investigate if MoVIA activity and side effect profile has any advantage over MoVIB in animal models of neuropathic pain.

Cav2.2 is known to be expressed at projection fibres and in motor-neurons [19], which consist of efferent and afferent fibres uniting the cortex with the lower parts of the brain and with the spinal cord [46]. The motor tract, occupying the genu and anterior two-thirds of the occipital part of the internal capsule, and consisting of (a) the geniculate fibres, which decussate and end in the motor nuclei of the cranial nerves of the opposite side; and (b) the cerebrospinal

108 Chapter 3 fibres, which are prolonged through the pyramid of the medulla oblongata into the spinal medulla.

Ataxia and other supra spinal neurological side-effects, such as dizziness, confusion, abnormal gait, memory impairment, nystagmus, or hallucinations, that develop after administration of -conotoxins given intrathecally might be supra spinal effects, as these peptides move rostrally up the neuraxis and reach the brain [46]. However, Cav2.2 KO animals do not display motor incoordination [272], suggesting that these effects may relate to off-target effects such as P/Q-type block, or that there is significant developmental compensation in these KO animals, not observed during short term drug administration.

3.5.1 Other Factors that May Influence ω-Conotoxins Efficacy

Regardless of the similar structural conformations between MoVIB and the analogue [R13Y]-MoVIB, we observed interesting differences in potency across the binding and functional assays. Specifically, we found that MoVIB and the analogue [R13Y] had similar potency in binding assays, while in functional assays [R13Y]-MoVIB was 10-fold more potent than the wild type MoVIB. This difference may arise from the non-physiological conditions used for the binding assays, which are Ca2+ free, while the functional assays use solutions with much higher concentrations of positive ions, approximating physiological conditions. High concentration of ions, such as Ca2+ and Ba2+ used in the buffer solutions are known to non- competitively inhibit -conotoxin affinity [3, 85] and may differently affect the more polar

native peptides. Alternatively, inhibition of Cav2.2 by MoVIA and MoVIB may be voltage and/or state-dependent, with homogenised membrane (0 mV) differentially affecting affinity to functional assays, where membrane potentials are preserved.

Similarly, the presence of Cav auxiliary subunits endogenously expressed in native systems can also interfere with the ω-conotoxins affinities [3, 82, 86]. While in the binding assays auxiliary subunits might be disrupted with the membrane preparation, in the functional assays the complex is expected to be preserved (discussed in chapter 2 and in ref [3]). Auxiliary subunits are well known to shift the potency of the ω-conotoxins in different assays and conditions. Thus, differences in the influence of auxiliary subunits on the interaction of Cav2.2 with MoVIA and MoVIB could differentially influence the binding and functional affinity of MoVIB and [R13Y]-MoVIB.

109 Chapter 3

ω-Conotoxins affinities can differ in different species and preparations due to expression of different Cav2.2 splice isoforms (see chapter 2 and refs [87, 203, 225]. In chapter 2, we showed that the affinities of CVID and MVIIA were significantly different in rat and in the human SH- SY5Y cells. MoVIA and MoVIB affinities to displace 125I-GVIA from fish brain membranes were higher (approximately 10-fold for MoVIA and 100-fold for MoVIB) than that of the human SH-SY5Y membranes, while the analogue [R13Y]-MoVIB had similar affinities in both species. While the experiment conditions used for both membranes were the same, the reason for this shift in potency is unknown. These results suggest MoVIA and MoVIB interact

differently with the fish and human Cav2.2. The cloning and sequencing of fish Cav2.2 in future studies would be useful to confirm this hypothesis.

3.6 CONCLUSION

The results in this chapter indicate that -conotoxins MoVIA and MoVIB have a broad phylogenetic spectrum of activity, which may at least in part relate to the fact that these peptides appear in a vermivorous Conus sp., which evolved much earlier than piscivorous Conidae.

Thus, these toxins may have encountered ancestral forms of Cav channels [28, 273]. The discovery described in this chapter may be an example of convergent evolution [266, 267] of distantly related cone snail toxins that target a similar pharmacology (Cav2.2 and related channels) in different organisms. The "cross-phylum" pharmacological activity of MoVIA and

MoVIB may provide a useful pharmacological tool for examining how Cav2.2 channel outer vestibule structures have evolved. The results in this chapter may also be useful to guide future SAR studies and help to unfold structural features underlying the pharmacology of the ω-

conotoxin family at the Cav2.2 channels. MoVIB produced dose-dependent reduction in mechanical allodynia. However it unfortunately, also produced dose-related side-effects on a qualitative scale that were similar to that previously described for other ω-conotoxins [246, 247].

110 Chapter 4

Chapter 4

111

Chapter 4 Chapter 4 Discovery and Functional

Characterization of a Novel Cav2.2/Nav Channels Inhibitor Toxin, from a Tarantula Spider

4.1 FOREWORD

This chapter includes part of a review published in Toxins [1]. Most of the work presented in this Chapter is my own, except for work contributed by others, as indicated in the statement of contributions by others to the thesis, located at the beginning of this thesis. In collaboration with Prof. Paul Alewood laboratory, Dr Andreas Brust synthesised Cd1a. Dr Irina Vetter, Dr

Fernanda C Cardoso and myself from Prof. Richard Lewis group, have performed the Nav channel assays.

Chapter 4 Describes

 The discovery and pharmacological characterization of a novel spider venom toxin

with activity at Cav2.2 channels

 The selectivity profiling of the new toxin Cd1a at Cav and Nav channels

4.2 INTRODUCTION

Similarly to cone snails, spiders have evolved highly complex venoms rich in active peptide toxins to assist with their prey capture (for review see ref [1, 274]). Many of these peptides have activity at voltage-gated ion channels, including Cav2.2 (for review see Chapter 1 and ref [1, 274, 275]. While venoms from some spiders, such as Phoneutria nigriventer are dominated

by Nav inhibitors [276], in general, Cav inhibition dominates the pharmacology of spider venom

peptides (reviewed by ref [277]). However, activity of many spider toxins at Cav2.2 has not

been characterized extensively and many of these peptides preferentially target Cav channels

other than Cav2.2, such as Cav2.1, Cav2.3 or invertebrate Cav channels [158, 278-280]. In

contrast to cone snails, which have provided the most selective Cav2.2 blockers, spider toxins

have provided the most Cav2.1 and Cav2.3 selective inhibitors known [281, 282]. Given the similar overall structure shared by cone snail and spider venom peptides (for comparison see 112

Chapter 4 Figure 1.4 and Figure 1-4, Chapter 1), this divergent selectivity profile is surprising. As

illustrated by conopeptides, small differences in structure may result in profound effects on Cav selectivity [86].

While a range of studies in animals have suggested that spider venoms are sources of analgesic toxins [150], to date, relatively few Cav2.2 inhibitor toxins have been identified from

spiders; and even fewer have shown Cav2.2 subtype selectivity (see Table 1.5, chapter 1) [278,

279]. In contrast with conotoxins that have shown the most selective Cav2.2 inhibitors to date,

the majority of the spider peptides discovered display activity at Cav1, Cav2.1 and Cav2.3 channels [148-162]. Interestingly, some spider peptides, such as theraphotoxin-Hh1b–d even inhibit both Cav2.2 and Nav channels with similar potency (IC50 = 50 nM at N-type currents in

DRG and100 nM in TTX-s Nav currents [150]), and thus are designated ω-/µ-toxins based on their pharmacological profile. The toxin Hh1a or huwentoxin-X preferably inhibits vertebrate

Cav2.2 channels in rat DRG neurons and thus, this toxin is classified as ω-theraphotoxin [148].

Results from previous studies indicate that Cav channels inhibition by spider venoms may occur through two different mechanisms; a direct block of the Cav α subunit pore, or changing the voltage dependence and kinetics of gating. For example, ω-ctenitoxin-Pn4a and HWTX-X

decreased the N-type peak current from Cav2.2 channels with little effect on voltage- dependence of activation [159], and another unrelated spider toxin, ω-Agatoxin-Aa2a, displaced radiolabelled ω-conotoxin GVIA [162], suggesting that these peptides may indeed

act as pore blockers, rather than gating modifiers at Cav2.2. In contrast, ω-Aga-IVA a peptide

isolated from the venom of the funnel web spider Agelenopsis aperta inhibits Cav channels by altering the voltage dependence of gating [283].

In this chapter, I describe the isolation and characterization of Cd1a, a novel Cav2.2 inhibitor toxin from the venom of the spider Ceratogyrus darlingi (the African horned tarantula), using the high-throughput Cav2.2 screening assay established in chapter 2. Additionally, this chapter

describes the characterization of Cd1a on related Nav channel subtypes.

4.3 MATERIAL AND METHODS

4.3.1 Drugs and Chemicals

The drugs and chemicals were obtained from different sources, as detailed in appendix 1 and throughout the text. 113

Chapter 4

4.3.2 Cell Culture and FLIPR Ca2+ Imaging Assays

2+ The cell culture and the functional Cav2.2 assays using Ca imaging and SH-SY5Y cells were performed as described in chapter 2, section 2.4.5. The cell culture of the CHO cells was performed using DMEM (Dulbecco's Modified Eagle Medium, Invitrogen) with in addition, 10% FBS (Invitrogen).

4.3.3 Screening Bioactive Spider Venoms

A range of crude spider venoms (Source: 'from Dr. Volker Herzigs’ venom collection', University of Queensland, Australia) from adult female specimen were collected and freeze- dried. Lyophilized venom samples were redissolved in distilled water to 10 times the initial venom volume (1:10 dilution), centrifuged (14,000 rpm, 5 min) and the supernatant stored at – 2+ 20°C. Spiders were screened against Cav2.2 channels using the functional Ca imaging assays described in chapter 2, section 2.4.9, with slight modifications. Briefly, sixty spider venoms (estimated quantity: 4 µg/well) were pipetted into 96-well plates of the FLIPR Ca2+ imaging

assays for bioactivity testing at the human SH-SY5Y cells, endogenously expressing Cav2.2 channels. After each round of fractionation, ~ 1/10 of the fractions were aliquoted and freeze dried. Lyophilized fractions were diluted with PSS buffer (described in appendix 1) containing additional 10 µM nifedipine (to inhibit endogenous Cav1 channels), and incubated together 2+ with the Ca fluorescent dye Fluo 4 for 30 min. After the incubation period, a KCl/CaCl2 (90 2+ mM/5 mM) buffer solution was added to activate Cav2.2-mediated Ca responses in the cells (described in appendix 1).

4.3.4 HPLC Fractionation

Crude venom showing activity in the Cav2.2 assay was fractionated using a Vydac C18 reversed-phase analytical column (250 x 4.6 mm, 5 µm). Fractions were eluted at a flow of 1ml/min with a linear gradient of 0–80% solvent B over 70 min, using an Agilent solvent

delivery system (solvent A, 0.1% TFA (trifluoroacetic acid)/H2O; solvent B, 90% acetonitrile,

0.1% TFA). Peak fractions were collected manually and tested for Cav2.2 bioactivity using the FLIPR Ca2+ imaging assays. Active fractions were re-fractionated using an analytical Phenomenex Hydro-RP column (250 x 4.6 mm, 4 micron), with a gradient 0–30% solvent B for 60 min. This peak was re-tested for activity using the FLIPR and purity confirmed using a mixture of HPLC and MALDI-TOF MS (4700 Proteomics Analyzer, Applied Biosystems,

114

Chapter 4 Mulgrave, VIC) analyses. Approximately 1/3 of the peak containing sample was freeze dried and sent for outsourced Edman Degradation for sequencing.

4.3.5 N-terminal Sequencing Analysis

The aa sequencing analysis by Edman Degradation was performed by outsourced APAF (Australian Proteome Research Facility). Briefly, the dry peptide sample was dissolved in 25 mM ammonium bicarbonate, pH 8.0 and reduced with DTT at 56°C for 0.5 h, then alkylated using iodoacetamide at room temperature for 0.5 h. The sample mix was purified by RP-HPLC (reverse phase high-pressure liquid chromatography) using a Zorbax 300SB-C18 column (3 x 150 mm). A single major peak was identified at min 18.3. The peak fraction was collected, reduced in volume under vacuum and a total of 50 µL of sample was loaded onto a precycled Bioprene-treated disc for Edman sequencing. Automated Edman degradation was carried out using an Applied Biosystems 494 Procise Protein Sequencing System.

4.3.6 Chemical Synthesis of Spider Toxin

Chemical synthesis was performed using Boc chemistry methods, as previously described [284] and detailed in chapter 3, section 3.3.10.

4.3.7 Preparation of Membranes for the Radioligand Binding Assays

The method described by Wagner, et al., 1988 [200] was used with slight modifications, for adaptation to the SH-SY5Y cell membranes preparation, as described in chapter 2, section 2.4.7.

4.3.8 125I-GVIA Radioligand Binding Assays

[125I] Tyr22-GVIA radioligand binding assays were performed in triplicates using methods detailed in chapter 2, section 2.4.8.

4.3.9 Membrane Potential Nav Assays

The Nav channels membrane potential assays were performed as previously described [195].

Briefly, CHO cells heterologously expressing human clones of Nav1.1–Nav1.8 isoforms (Chantest, Ohio, USA) were loaded with the red membrane potential dye (Molecular Devices, 115

Chapter 4 Sunnyvale, CA, USA) reconstituted in PSS (appendix 1) at 37°C for 30 min. The cells were then transferred to the FLIPRTETRA fluorescent plate imaging reader and changes in fluorescence (excitation 510–545 nm; emission 565–625 nm) in response to addition of the antagonists (toxins or crude venoms) and agonist (veratridine 50 µM or deltamethrin 100 µM for Nav1.8) were measured every second for 300 s.

4.3.10 Statistical Analysis

The statistical analyses were performed as detailed in chapter 2, section 2.4.10, pg 48.

4.4 RESULTS

4.4.1 Screening and Assay-Guided Isolation

Spider venoms were screened for bioactivity at the human Cav2.2 channels, endogenously expressed in SH-SY5Y cells. From 60 crude spider venoms analysed, 50% inhibited at least 2+ 50% of the Cav2.2-mediated Ca responses (Figure 4-1), indicating spider venoms, like

conotoxins, are abundant sources of Cav2.2 inhibitors. The venom of the spider Ceratogyrus darlingi inhibited more than 50% of the responses and thus this venom was fractionated. Successive rounds of Ceratogyrus darlingi crude assay-guided RP-HPLC fractionation, resulted in the isolation of a novel hydrophilic Cav2.2 inhibitor peptide toxin, eluting at ~ 22% buffer B (Figure 4-2), which was named Cd1a. MALDI-TOF MS revealed the isolated peptide was free of contaminants and sample was dominated by a single mass (4028.21 Da, m/z, Figure 4-3).

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All 123456789101112

A

B

C

D

E

F

G

H

Figure 4-1. Cav2.2 Responses Are Inhibited by Ceratogyrus darlingi Venom. A layout of the FLIPRTetra 96 well-plate A.Crude venoms (4 µg/well estimated quantity) from 2+ 60 spider species were tested on Cav2.2-mediated Ca responses using the human SH-SY5Y cells. Inhibition was evident for crude C. darlingi venom (column 10, purple colour, rows C– D), an African tarantula spider. A range of other crude venoms were also active (≥ 50% inhibition) and are shown colored in the figure. PSS negative control (column 11 row A–H, light green colour) and KCl positive control (column 12, rows A–H, light blue colour), with in addition nifedipine (10 µM) to inhibit endogenous Cav1 channels.

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Chapter 4

2.0 3500 100 F/F) 1.5 KCl (+ control)  C. darlingi crude 1.0 PSS (- control) 3000 0.5 80 Fluorescence ( 0.0 2500 400 500 600 700 Time (sec) % SolventB 2000 60

1500 40 *

Absorbance at 214 nm 1000 214nm 280nm 20 500

0 0 0 10 20 30 40 50 60 Retention time (min)

Figure 4-2. Cd1a Isolation by Reverse Phase Liquid Chromatography. The chromatogram shows fractionation of the crude Ceratogyrus darlingi, which was an active

Cav2.2 inhibitor. Crude venom (1mg) was injected onto HPLC and run for 60 min using a

Reverse Phase-HPLC system, with a gradient of 0–80% sovent B. Activity at the human Cav2.2 Tetra channels was tested using the SH-SY5Y cell line and the FLIPR , as shown in Error!

Reference source not found.. A fraction eluting at ~ 28 min/22% solvent B inhibited Cav2.2- mediated Ca2+ responses (red asterisk over the peak). The isolation of the active fraction leaded to the discovery of a novel Cav2.2 inhibitor toxin, named Cd1a. Absorbance at 214 and 280 nM are represented in black and red coloured traces, respectively. Over the chromatogram on the right hand side a picture of the spider C. darlingi. The inset over the chromatogram on the left shows the well of the FLIPR in Figure 4-1 containing the active C. darlingi crude venom

(wells in column 10, rows C–D, purple colour, from Figure 5-1). Maximum Cav2.2-mediated Ca2+responses activated by KCl addition were used as positive control and are shown in the inset in light blue colour. PSS negative control is shown in green colour; and in purple, 2+ inhibition by the crude C. moncuri venom of the Cav2.2-mediated Ca responses activated by KCl.

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4.4.2 Mass Spectrometry and Synthesis

Automated N-terminal Edman degradation sequencing revealed an almost complete sequence. The peptide sequence was completed by MS/MS and found to belong to the family 1 of theraphoxin spiders (or tarantulas) [277]. This group of spider toxins belong to the inhibitor cystine knot motif (ICK) structural group (for review see ref. [148, 149, 285]. Cd1a is a 33-aa residue peptide, with the typical framework of four-loop toxins conforming to the canonical cysteine motif (–C1–C4–CC2–C4–C3–4–) and three disulphide bridges, found in all members of the ICK structural group (for review, see ref. [285]).

A B

C D

Figure 4-3. Cd1a Co-elutes With Synthetic Form. The Inset shows reconstructed spectra from LCMS of Ceratogyrus darlingi crude venom. A. Crude venom, B. Cd1a native form isolated from crude venom, C. Cd1a synthetic form and D. Overlapped synthetic and native MoVIB. Retention times (~ 38 min) and masses for Cd1a (4027.81 Da) overlapped, indicating the synthetic peptide was identical to the native form.

119

Chapter 4 Cd1a net charge calculation indicated the novel peptide toxin (aa sequence DCLGWFKSCDPKNDKCCKNYSCSRRDRWCKYDL-NH) is a basic peptide (calculated pI 8.37). The molecular masses measured by MALDI-TOF MS ([M+H]+ Cd1a, 4027.81 Da) agreed with those calculated from the sequence data, assuming the C-terminus was amidated (~ –1 Da difference with the molecular mass calculated with a carboxylic C-terminus. Predicted mass, 4027.81 Da; observed mass, 4028.21 Da). The toxin was synthesized to confirm identity and to allow further pharmacological characterization. The synthetic and native peptide were analysed by LC/MS and both forms co-eluted at min 28 and their extracted mass agreed (Figure 4-3 A–D), indicating the synthetic form was identical to the native peptide.

4.4.3 Cd1a did not displace 125I-GVIA

Cd1a did not displace 125I-GVIA (at up to [1 µM] (Fig 5-4). This result contrasts with the ω-conotoxins, which displace 125I-GVIA with picomolar concentrations (Table 1.4, chapter 1

and ref [3, 85, 86]), as well as the non-selective Cav2.2 inhibitor toxins from spiders, like ω- agatoxin-Aa3a (170 pM) (Table 1.5, chapter 1 and ref [155, 156]).

100

80

60

40 GVIA Cd1a 20 I-GVIA Binding (CPM%) I-GVIA Binding

125 0 -16 -14 -12 -10 -8 -6 Log Ligand [M]

Figure 4-4.125I-GVIA Displacement Assays. Cd1a did not displace 125I-GVIA from SH-SY5Y cell membranes (up to [1µ M]). This result

suggests Cd1a modulates Cav channels by a mechanism other than binding to Cav2.2 outer

vestibule. This is consistent with most spider toxins’ mechanism of inhibition at Cav/Nav channels, modifying gating properties instead of blocking the pore.

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4.4.4 Cd1a Inhibits Functional Cav2.2 Preferentially in SH-SY5Y Cells

Cd1a potency and selectivity was assessed at native Cav1.3, Cav2.2 and Cav3.1 channels

expressed in the human SH-SY5Y cells; using saturating concentrations of Cav2.2 inhibitor

CVID (3 µM), Cav1 inhibitor nifedipine (10 µM), or Cav3.1 inhibitor mibefradil (30 µM), as 2+ described in chapter 2, section 2.5.3. Cd1a inhibited functional Cav2.2-mediated Ca responses in SH-SY5Y cells at 3.45±0.04 µM (IC50 ± SEM; Figure 4-5). The Cav selectivity of Cd1a was assessed at native Cav1.3 and Cav3.1 channels expressed in the human SH-SY5Y cells (Chapter 2 and ref [3]. These responses were unaffected by Cd1a up to [30 µM] (Figure 4-5, Table 4.2),

indicating Cd1a was a modestly potent and selective Cav2.2 inhibitor.

100 100 80 80 60 60 Cd1a pIC50 = 5.275 40 40 1.3 Resp.(%) 2.2 Resp. (%) v v 20 20 Ca Ca 0 0 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 Log Cd1a [M] Log Cd1a [M]

2+ Figure 4-5. KCl Activated Ca Responses Mediated by Cav2.2 in SH-SY5Y Cells.

2+ Tetra A. Cav2.2-mediated Ca responses measured on the FLIPR . Toxin Cd1a fully inhibited

Cav2.2 in the human SH-SY5Y cells (Cd1a potency IC50 ± SEM [µM], 3.45 ± 0.05). B–C. Cd1a 2+ does not inhibit native Cav1 or Cav3-mediated Ca responses in SH-SY5Y Cells (at ≥ [30 µM]).

A blast sequence homology search indicated Cd1a shares 97% sequence identity with the spider toxins Cm1a (previously known as CcoTx1) and 94% with Cm1b (previously known as CcoTx2) [286] from the venom of the related theraphoxin C. marshallis [278], and both are

known Nav inhibitor toxins [287]. Cm1a–b and Cd1a differ only at position 21 (Cd1a has a serine while Cm1a-b have a threonine at this position) and 32, with an aspartic acid residue in Cm1a and Cd1a, and a tyrosine in Cm1b (Table 4.1 and Figure 4.7B).

121

Chapter 4 Table 4.1. Amino acid sequence of Cd1a, Cm1a and Cm1b.

µ/ω-theraphotoxin Cd1a DCLGWFKSCDPKNDKCCKNYSCSRRDRWCKYDL

β-theraphotoxin Cm1a DCLGWFKSCDPKNDKCCKNYTCSRRDRWCKYDL

β-theraphotoxin Cm1b DCLGWFKSCDPKNDKCCKNYTCSRRDRWCKYYL

4.4.5 Cd1a Inhibits Native and Recombinant Nav Channels

Due to the high homology of Cd1a with Nav channel inhibitor teraphotoxins from family 1 of spider toxins (ref [277] see Figure 4.7 B), the ability of Cd1a to inhibit veratridine-activated Nav responses was tested (Table 4.2). These assays revealed that Cd1a was in fact more potent at most Nav channel subtypes than Cav channels (IC50 ± SEM [µM]), including Nav1.1 at

0.71 ± 0.26; Nav1.2 at 0.60 ± 0.22; Nav1.3 at 0.71 ± 0.29; Nav1.4 at 4.16 ± 1.91; Nav1.5 at

1.10 ± 0.17; Nav1.6 at 0.83 ± 0.25; Nav1.7 at 0.12 ± 0.05; and Nav1.8 at 3.1 ± 2.4. Table 4.2

summarises published electrophysiology data for Cm1a–b [287] and functional FLIPR Cav/Nav assays and binding assays data, collected for Cd1a.

The pharmacological profile of Cm1a and Cm1b have been previously characterized by electrophysiological measurements on cloned Nav subtypes expressed in Xenopus laevis oocytes by Bosman, et al. [287], and their selectivity profile is shown in Table 4.2 for

comparison with Cd1a. Cd1a displays moderate activity against Nav1.1, Nav1.2, Nav1.4,

Nav1.5, Nav1.6 and Nav1.8, similar to both Cm1a and Cm1b. In contrast Cd1a potency at

Nav1.2 was 600-fold lower than that of Cm1a and Cm1b (Table 4.2, Figure 4.7). Interestingly,

Cd1a is at least 6-fold more potent at Nav1.7 than at any other Nav subtype or Cav2.2 channels.

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6 M) 

50 5 4 3 2 1

Cd1a (IC Inhibition 0 .1 .2 .3 .4 .5 .6 .7 .8 1 1 1 1 1 1 1 1 a v a v a v a v a v a v a v a v N N N N N N N N h h h h h h h h

Figure 4-5. Functional Selectivity of Cd1a at Nav Channels.

The potency and selectivity of Cd1a at Nav subtypes are represented in bar graphs. Cd1a inhibits a range of Nav channel subtypes with variable potency (IC50 mean ± SEM, [µM]);

Nav1.1: 0.71 ± 0.26; Nav1.2: 0.60 ± 0.22; Nav1.3: 0.71 ± 0.29; Nav1.4: 4.16 ± 1.91; Nav1.5:

1.10 ± 0.17; Nav1.6: 0.83 ± 0.25; Nav1.7: 0.12 ± 0.05, and Nav1.8: 3.1±2.4.

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Table 4.2. Promiscuous Pharmacology of Toxins Cd1a, Cm1a and Cm1b. Nav/Cav/Kv Selectivity β-TRTX-Cm1a (nM) β-TRTX-Cm1b (nM) µ/ω-Cd1a (nM)

Nav1.1/β1 523 407 710±0.26

Nav1.2/β1 3.0 8.0 600±0.22

Nav1.3/β1 Inactive at up to 2µM 88.0 710±0.29

Nav1.4/β1 888 400 4160±1.91

Nav1.5/β1 323 1634 1.100±0.17

Nav1.6/β1 ND ND 830±0.25

Nav1.7/β1 ND ND 120±0.05

Nav1.8/β1 55% inhibition (2µM) 40% inhibition (2 µM) 3.100±2.4

LVA Cav (Cav3) ND ND inactive Inactive at up to 100 nM in mice Inactive up to 100 nM in mice sensory sensory neurons. Inactive [up to 10 µM] HVA (Cav2.2) neurons. Inactive [up to 10 µM] on Cav 3.45±0.04 on Cav responses elicited by responses elicited by KCl in SH-SY5Y KCl in SH-SY5Y

Kv subtype ND ND ND Kv1.4/ Kv2.1/ Kv3.4/ Kv4.2

Kv1.3 20% block (2µM) oocytes 20% block (2µM) oocytes ND Note: Cm1a and Cm1b were tested at Xenopus oocytes. Data is from Bosman, et al., 2006: ref. [287]. Cd1a (this work) was tested in human cells lines expressing either Cav or Nav channels, as detailed in the methods section. ND: not determined.

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4.5 DISCUSSION AND CONCLUSION

In this Chapter we describe the assay-guided isolation of Cd1a, a toxin from the venom of

the African spider Ceratogyrus darlingi. Cd1a was discovered with a Cav2.2 assay using the human SH-SY5Y cell line. Cd1a was synthesized and the synthetic form adopted the same structure and had the same functional properties as the native peptide. Cd1a belongs to the four-

loop, ICK peptide group [287, 288]. SHSY5Y cells express Cav1.3, Cav2.2 and Cav3.1, endogenously (Chapter 1 and ref [3]. While the activity at Cav2.1 needs to be investigated in

another expression system, the potency of Cd1a at the human Cav2.2 channels was in the micromolar range and unlike -conotoxins this peptide did not displace 125I-GVIA from SH-

SY5Y cell membranes. In addition, Cd1a was inactive at other Cav subtypes (Cav1.3 and

Cav3.1) at concentrations up to 30 µM in the functional assays, indicating selectivity for Cav2.2 over these other Cav subtypes.

A blast homology search revealed that Cd1a amino acid sequence was highly homologous

to the sequences of two other spider toxins, which are Nav channel inhibitors, Cm1a and Cm1b, from the venom of the tarantula theraphoxin C. marshallis [287]. Thus, selectivity data previously published from Cm1a and Cm1b have been used in this chapter for comparison with data from Cd1a (Table 4.2). Cd1a also shares high sequence homology with other peptides previously isolated from tarantula venoms (Table 4.1 and Figure 4.7) that belong to family 1 (for review see ref [277, 285]). Toxins in this family are known as “pharmacologically promiscuous” as they can modulate Nav, Cav and Kv channels, mechano-sensitive cationic channels, and proton-gated ion channels [288-291].

While the effect of Cd1a at Kv, mechano-sensitive cationic channels and proton-gated ion channels was not determined, data from this Chapter reveal that Cd1a, in addition to being a

Cav2.2 inhibitor, is also a Nav channel inhibitor, like the related spider toxins huwentoxin IV and hainantoxin IV from the tarantulas Ornithoctonus huwena and Selescosmia hainana, respectively [292, 293]. Thus, Cd1a has been classified as a µ/-toxin, because of its pharmacological profile. Cm1a and Cm1b, in agreement with literature data [287], were

inactive on the Cav responses elicited by KCl in SH-SY5Y at concentrations as high as 10 µM.

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Figure 4-6. Cd1a Sequence Alignment. Cd1a aminoacid sequence (aa) is aligned with the aa sequences of other spider toxins from family 1, a family of promiscuous theraphoxins (or tarantulas). A. Cd1a belongs to the ICK structural group. The cysteine framework in red colour, connected by disulfide bridge (highlighted in blue colour). B. Cd1a Sequence alignment. The degree of aa conservation, quality of the data and consensus are shown under the alignment.

Cav channels comprise a common structural motif, consisting of four domains each with six transmembrane segments and a pore loop. Their pores are formed by the S5/S6 segments and the pore loop between these segments and are gated by bending of the S6 segments at a hinge

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Chapter 4 glycine or proline residue. The voltage sensor domain consists of the S1-S4 segments, with positively charged residues in the S4 segment serving as gating charges. For gating modifier toxins, voltage-sensing and voltage-dependent gating effects depend on the S1–S4 segments of the Cav channels [232, 294] but the exact binding epitope is known only for ω-Aga-IVA, and it is at the voltage sensor domain.

A range of tarantula toxins from family one, such as hainantoxins I, III, IV, V, and

huwentoxin IV have been classified as pore blockers (Figure 4), acting by occlusion Nav channels, in a manner similar to that of site 1 toxins [151]. Site 1 spider toxins block Nav currents entirely, without affecting either activation or inactivation currents [292, 293, 295,

296]. In contrast, Cm1a and Cm1b modulate Nav channels with similar properties to typical gating-modifier toxins, both by causing a depolarizing shift in gating kinetics and by blocking

the inward component of the Nav current (Table 4.2) [287] and shifting the current-voltage relationship. The high sequence homology of Cd1a with Cm1a and Cm1b suggests that Cd1a

is a gating modifier at Nav channels, similarly to other related spider toxins protoxin I, protoxin II, and jingzhaotoxin III [297-299].

To identify the mechanisms by which Cd1a binds and inhibits Cav2.2 channels we will need to perform further studies. Site direct mutagenesis may identify the exact amino acids that are

involved in the binding site of this toxin on Cav2.2 and increase our understanding of structure

activity relationship between these class of toxins and Cav2.2 channels. Understanding the mechanisms involving voltage and state dependency of inhibition of this toxin may allow the classification of Cd1a as a pore blocker or gating modifier toxin and thus will help the investigation of Cd1a therapeutic potential.

A range of spider toxins inhibit Cav activity by modifying their biophysical properties, either changing the kinetics or voltage dependence of activation or inactivation of the channel. Thus, these toxins are known as “gating modifiers. Consistent with being a gating modifier toxin, Cd1a did not share the same binding site with ω-conotoxins, which are believed to

mechanistically inhibit Cav2.2 channels by blocking and occluding the channels’ pore (for review see: [49]).

To confirm the hypothesis that Cd1a is a gating modifier toxin we will have to identify, in future studies, if Cd1a has the ability to change Cav2.2 channels kinetics. Electrophysiological experiment need to be performed varying voltage protocols and plotting current-voltage relationships in (eg. I-V curves to identify the voltage dependence of activation/inactivation). 127

Chapter 4 Activity of gating modifier toxins will be voltage-dependent and entail a modification of channel gating by interfering with the voltage dependence and kinetics of channel opening and closing. One of the characteristics of gating modifier toxins is despite complete inhibition of all inward currents, outward currents can still be observed in the presence of toxin [286, 307] [287, 300].

In FLIPR-based membrane potential assays, the potency of Cd1a at a range of Nav subtypes was similar to Cm1a and Cm1b (Nav1.1, Nav1.4, Nav1.5, and Nav1.8). However, compared to electrophysiological data reported by Bosmans et al [287] Cd1a was a moderate inhibitor at

Nav1.2, while Cm1a and Cm1b where highly potent at this Nav isoform (~ 200 times more potent than Cd1a) [287]. Bosmans et al reported that Cm1a, which differs from Cm1b by only

one aa residue at the C-terminal, does not inhibit Nav1.3 currents at concentrations up to 2 µM,

while Cm1b potently inhibits this Nav subtype (88 nM) [287]; and we found that Cd1a has only intermediate potency at Nav1.3 (~ 10-fold lower potency than Cm1b). While Cd1a had the

highest affinity for Nav1.7 (~ 120 nM), Cm1a and Cm1b have not been tested at this Nav subtype. It would be interesting for future studies to analyse the potency of both toxins Cm1a and Cm1b at Nav1.7 for comparison.

The pharmacological profile of Cd1a indicates that this peptide, when used at concentrations

near their IC50, could be used to selectively discriminate Nav1.7 currents in neurons in which

the other Nav subtypes is co-expressed. Based on data presented in this chapter, the presence of either an acidic group in Cm1a and Cd1a (N32) or an aromatic residue in Cm1b (Y32) appears to drive selectivity preferences of these peptides, most likely due to slight changes on

the surface charges [287]. Reports defining the mechanisms involving Cav2.2 inhibition by spider toxins have raised the possibility of different binding sites and at least two different modes of action [293, 299, 301]. For example, spider toxins ω-agatoxin-IVA, ω-grammotoxin-

SIA and the scorpion toxin kurtoxin act at Cav2.2 as gating modifiers [293, 299, 301]; ω- agatoxin-Aa2a displaced radiolabelled ω-conotoxin GVIA [162], suggesting that this peptide

may act as pore blocker, rather than gating modifiers at Cav2.2. ω-segestritoxin-Sf1a shares little structural homology identity with the ω-conotoxins, and is a large peptide stabilized by 4 disulfide bonds; however, it shares a common binding site with the ω-conotoxins, as ω- segestritoxin-Sf1a was able to displace radiolabelled MVIIA from rat brain synaptosomes [162].

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125 Cd1a was unable to displace I-GVIA from SH-SY5Y cells, but was active in the Cav2.2 functional assays, suggesting Cd1a, similarly to these other homologous toxins acts at Cav2.2 channels as a gating modifier. We have only used SH-SY5Y cells in the studies of this chapter and this cell line is known to express Cav1.3, Cav2.2, Cav3.1 subtypes, but not Cav2.1; and a range of auxiliary subunits (chapter 2 and ref [3]). Therefore, it wasn’t possible to investigate in our system the effect of auxiliary subunits at Cd1a inhibitory activity. Since little is known about the influence of Cav auxiliary subunits on the inhibition by spider toxins, given that the pharmacology of cone snail toxins is known to be affected by auxiliary subunits [82, 85, 86], and the large potential of spider venoms as new lead sources, future studies should be extended

to examine the influence of the Cav auxiliary subunit at spider toxins inhibitory activity using heterologous system.

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Chapter 5

Chapter 5

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Chapter 5 Chapter 5 General Discussion

Chronic or persistent pain affects more than 1.5 billion people in the world. For many patients, pain produces severe distress, which dominates and disrupts their quality of life [7, 34]. Besides the emotional and financial impact it has on the patients and their families, chronic pain also imposes a significant economic burden to the world’s health systems [7].

Chronic neuropathic pain is complex and represents a major therapeutic challenge, because currently available analgesics suffer from poor efficacy, produce side effects or have other

drawbacks. For example, clonidine an α2 adrenergic agonist, although effective against a range of pain states, causes severe side effects including, cardiovascular depression and sedation [137]. Local anaesthetics have shown efficacy against some types of pain [137], however, most local anaesthetics are not orally available and systemically administered local anaesthetics, such as lignocaine have a very narrow therapeutic window due to dose-limiting side effects. Ketamine, an NMDA receptor antagonist produces other side effects, including primarily sedation, dysphoria and hallucinations; and thus this drug is not routinely used [137].

When the side effects from systemically administered analgesics are intolerable or when pain relief is insufficient, the second line of option includes interventional approaches, such as nerve block, surgeries, spinal or intrathecal injection of drugs such as morphine, hydromorphone, fentanyl, clonidine and a range of local anaesthetics, given alone or in combination [40]. However, serious adverse events can restrict the use of systemic analgesics and its long-term usage is often associated with additional side effects and the development of tolerance. Opioids, in addition to dependence and tolerance, can cause dose-related respiratory depression, euphoria and sedation [302].

To address these shortcomings, new treatment approaches to pain and new therapeutic

targets are required. In recent years, Cav2.2 has emerged as one such analgesic target. For

example, gabapentin which inhibits Cav2.2 indirectly, has produced efficacy in patients with hypersensitivity and chronic pain; and have shown tolerability in a range of in vivo studies [303, 304], which have made this drug a useful drug candidate for the treatment of chronic pain. However, a number of cases of delirium tremens have been reported upon gabapentin withdrawal (for review see: [39]).

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Cav2.2 channels play an essential role in neurotransmission and nociception. Cav2.2 inhibition at the spinal cord level produces analgesia in animal models of pain [46, 86] and in humans [69]. Indirect inhibitors such as gabapentin and morphine, and the direct inhibitors ziconotide and other ω-conotoxins, have validated Cav2.2 as an analgesic target [44]. Ziconotide has shown some advantage over other currently used analgesic drugs; for example ziconotide is advantageous over opioids in terms of habituation and tolerance [13, 44]. However dose-related side effect problems and difficulties with delivery, makes ziconotide lack the “ideal candidate” status. However ziconotide can be safely used if intrathecally titrated to prevent side effects. This can be an alternative to patients with chronic pain that no more respond to opioid treatment [44, 237]. Importantly, ziconotide acts synergistically with morphine [13, 69, 235], producing efficacy in neuropathic pain and reducing dose-related side effects of both drugs.

Venomous animals, such as snakes and scorpions, cone snails and spiders, are abundant sources

of remarkably potent and selective Cav2.2 inhibitors. There are probably 100 different venom components per species of cone snails, leading to an estimate of 50,000 different pharmacologically active components present in venoms of all living cone snails. Spiders are the most successful venomous animals, with an estimated 100,000 extant species. The vast 22 majority of spiders employ a lethal cocktail to rapidly paralyse their prey, some species produce venoms containing >1000 unique peptides. The development of miniature high throughput screening assays described

in this thesis allowed the accelerated identification and characterization of novel Cav2.2 inhibitor toxins from large venom libraries and the identification of new analgesic leads.

Cav2.2 inhibitors still remain under-exploited as therapeutical leads and Cav2.2 still remains under-exploited as an analgesic target despite large available natural libraries from animal venoms, which have evolved over millions of years and produced some of the most subtype- selective Cav2.2 inhibitors known [1, 80].

To accelerate the discovery of Cav2.2 inhibitors, this thesis has established sensitive and

robust HTS assays using a human system expressing Cav2.2 channels endogenously, the SH-

SY5Y neuroblastoma cells. The expression profile of the Cav subtypes was characterised, which helped interpretation of the pharmacological data from this cell line. Importantly, the

studies in this thesis have shown that the expression of different Cavα splice variants, in conjunction with auxiliary subunits in a native context, modulates the pharmacology of ω- conotoxins, a result that may have implications for strategies used in the identification of therapeutic leads at Cav2.2 channels. 132

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The characterization of Cav subtypes expressed in these cells, additionally allowed assays’

adaption, for detection of other Cav subtype inhibitors (see chapter 2), and for Cav channel

selectivity studies. Importantly, the Cav2.2 assays established in this thesis have proved useful for the discovery and pharmacological characterization of novel potent and selective Cav2.2 inhibitors; and are expected to be useful to identify modulators of other human Cav channel- related diseases.

Consistent with animal venoms being abundant sources of active Cav2.2 inhibitor toxins, the Cav2.2 HTS assays established in this thesis has identified a range of active venoms from spiders and cone snails. Assay-guided fractionation lead to the isolation of the two first toxins, MoVIA and MoVIB, discovered from a vermivorous cone snail, C. moncuri. The sequencing of the new ω-conotoxins MoVIA and MoVIB revealed a sequence with a range of unique features, including an R13 instead of the Y13 most commonly found in fish hunting snail toxins, which is essential to their activity [80]. We replaced R13 for Y13 in MoVIB ([R13Y]- MoVIB) expecting to see an increase in the affinity of the analogue. However, surprisingly, the Y13 version had similar potency to the wild type MoVIB in binding assays; and in functional assays, the potency of the analogue was instead reduced, compared to the wild type. Since the [R13Y]-MoVIB had a similar structural conformation to that of the wild type MoVIB it appears that R13 can replace Y13, at least in MoVIB. While R13 in MoVIB overlays the Y13 in the other ω-conotoxins, MoVIA and MoVIB might have a different mode of interaction

with Cav2.2 compared to ω-conotoxins from fish hunting snails. It will be interesting for future studies to assess the replacement of the Y13 in other ω-conotoxins for a R13, to confirm our hypothesis of possible ionic interactions between R13 in these toxins and the binding site at

Cav2.2 channels.

MoVIA and MoVIB fully displaced 125I-GVIA binding from SH-SY5Y membranes.

Interestingly, MoVIA and MoVIB had the highest affinity for fish Cav2.2, compared with the conotoxins from fish hunting cone snails. Nevertheless, MoVIA and MoVIB potently inhibited functional Cav2.2 responses in SH-SY5Y human cells and in rat DRG neurons; therefore, showing that these toxins have a broad phylogenetic spectrum of activity. The "cross-phylum" pharmacological activity of MoVIA and MoVIB may provide a useful pharmacological tool

for examining how Cav2.2 channel outer vestibule structures have evolved in a range of animal species. The results described in chapter 3 may also be useful to guide future SAR studies and help to unravel structural features underlying the pharmacology of the ω-conotoxin family at

the Cav2.2 channels. In addition the high affinity and selectivity profile of ω-conotoxins 133

Chapter 5 provide structural insights for use as a guide on the development of small organic molecules peptide mimetic.

“Further studies will need to be performed in order to draw definitive conclusions regarding 2+ the mechanism of action of MoVIA and MoVIA. Ca influx through Cav2.2 channels activates K+ channels responsible for the AP (action potential) after-depolarization and it is conceivable that toxins may alter spike frequency at concentrations below levels that inhibit release from terminals. Thus, despite the sequence and structural similarities between MoVIA and MoVIB and the classical ω-conotoxins (which are known to inhibit Ca2+ influx through Cav channels and consequently neurotransmitter release), it is conceivable the mechanism of action of MoVIA and MoVIA may be different from those ω-conotoxins. Electrophysiological techniques such as current clamp on the action potential may be used in future studies to indicate if MoVIA and MoVIB can potentially alter spike frequency at concentrations lower than that required to inhibit release from nerve terminals.

Consistent with Cav2.2 inhibition by ω-conotoxins being a powerful analgesic-strategy, intrathecal MoVIB produced potent and long-lasting, dose-dependent reduction in mechanical allodynia in a partial nerve ligation model in rats. However it unfortunately also produced dose- related side-effects on a qualitative scale that were similar to that previously described for other ω-conotoxins (49, 50). Despite the small therapeutic/safety window of MoVIB in the model studied in this thesis, MoVIB was highly effective in treating neuropathic pain. Further studies are needed to investigate the potential of MoVIA and MoVIB as analgesics leads in different pain models and using different routes of administration. Recovery from block may influence how effectively ω-conotoxins reverse different painful conditions in vivo, and could indicate whether administration of these peptides can be controlled to avoid unwanted side effects [86].

MoVIB pharmacokinetics has not been investigated to date. However due to MoVIB amino- acid sequence and structure similarities with ziconotide and other members of the ω-conotoxin family comparison with ziconotide may explain these effects. Ziconotide produces delayed onset and off set of analgesia and adverse events. This delay occurs probably as result from the fact that ziconotide is a large and highly hydrophilic molecule compared with non-peptidergic drugs, meaning that it moves only slowly from the cerebrospinal fluid to its target site in the dorsal horn of the spinal cord [305]. Thus onset of pain relief after intrathecal ziconotide can be delayed 2–4 h, maximum effects might develop after 8–12 h, and analgesic effects can last

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Chapter 5 up to 48 h after discontinuation [45]. Ziconotide should be titrated on an individual basis to a desired level of response to ensure efficacy and safety [45].

The lower doses of MoVIB administrated might have been too low to reach sites in the spinal cord and show a desirable effect with regarding nociception, but enough to reach synapses that control normal function. Factors such as the intrinsic reversibility of MoVIB, the presence of auxiliary subunits, which also regulate functions such as reversibility, and the

expression of splice variants of the Cav2.2 would affect how the peptide relates to observed side effects, which are probably related to accumulation in some of the CNS areas where Cav2.2 controls transmitter release. Additionally, side-effects may develop at a high interindividual and intraindividual variability. While in some animals we tested MoVIB produced only mild side effects, ataxia was more pronounced in other animals.

Different routes have however been studied for ziconotide and also produce adverse effects. If given intravenous (IV) or subcutaneous, ziconotide has shown efficacy diminishing inflammation in rat models [272, 306]. However, early studies have shown that ziconotide may not be sufficiently delivered to the CNS to be effective, via this route. Also toxicity may be increase with systemic exposure (ziconotide seems to block sympathetic efferent activity). Epidural injections have shown minimal efficacy, presumably due to a median CSF (central spinal fluid) bioavailability [272].

Alternatively mutational studies could be performed to try to produce analogues that show better recovery from block than native conotoxins. Recovery from block may influence how effectively ω-conotoxins reverse different painful conditions in vivo, and could indicate whether administration of these peptides can be controlled to avoid unwanted side effects [86].Thus, despite the small therapeutic/safety window of MoVIB in the model studied in this thesis, MoVIB was highly effective to treat neuropathic pain; and further studies are needed to investigate the potential of MoVIA and MoVIB as analgesics leads in different pain models and using different routes of administration.

The results in this thesis provide hope that a ω-conotoxin with improved characteristics can be found from cone snail venoms. The use of HTS assays have accelerated the primarily identification of new analgesic leads and secondarily, accelerated the pharmacological characterization of these leads. With the large natural scaffold library available from animal venoms, the results in this thesis prove a concept, and support a hope that in near future a better analgesic from animal venom will reach the market. 135

Chapter 5 Although our data suggests that the FLIPR assays used may identify state-dependent and

state-independent Cav2.2 inhibitors (state dependant blockers such as mibefradil and pore blockers such as CVID were active in our assays), it is not known the proportion of channels that exist in the different states in the SH-SY5Y cells. Therefore, our FLIPR assays have limitations in terms of characterising state-dependant inhibitors, as we cannot control membrane potential with these assays. It would be interesting if future studies can adapt the

FLIPR assays to identify state dependant inhibitors, which may be able to inhibit Cav channels in diseases states, while sparing function in normal physiological states. That has been suggested to possibly avoid or minimize the side effects caused by currently used drugs [197].

2+) The mobility of extracellular calcium (Ca via Cav channels in neurons is involved in triggering multiple physiological cell functions but also the abnormal, pathophysiological responses that develop as a consequence of different types of injury. In conditions of chronic 2+ neuropathic pain, Cav channels are involved in providing the Ca signal, which is essential for the sustained neuronal firing and neurotransmitter release characteristic of these syndromes. The voltage dependence and kinetics of N-type Ca2+ currents vary considerably in different

neurons and ionic conditions used. At physiologic or resting membrane potential, Cav channels are normally closed. They are activated at depolarized membrane potentials (i.e., opened) and this is the source of the "voltage-dependency”. Nowycky et al, 1985 was the first to describe the N-type Ca2+ currents from chick DRG neurons, which activation varied between –30 to – 10 mV (HP= –100 to –80 mV), reaching a peak at +20 mV. The averaged current recorded decays almost completely by the end of 130 ms test pulse. A steady holding potential of –20 mV also produced complete inactivation [56].

It is believed that the therapeutic utility of currently used ion channel drugs reside in the fact that they recognize specific ion channel conformations that are present in the disease state, but absent when the same channel resides in a normal environment. Cav2.2 channels are highly

expressed in pain pathways. The ability to identify compounds selective for Cav2.2 versus other

Cav subtypes, but also drugs that demonstrate effective blockade during the pathophysiological states of neuropathic pain without compromising channel activity associated with sustaining

normal housekeeping cellular function, seems to be imperative for the success of a Cav2.2 inhibitor as a drug (for review see: [190]). An interesting approach to obtain this target profile is to identify compounds that are more potent in blocking Cav2.2 during higher frequencies of firing as compared to the slower more physiologically-relevant frequencies. This may be achieved by identifying compounds with enhanced potency for the inactivated state of the 136

Chapter 5

channels [307]. Thus, it has been proposed that a state-dependent Cav2.2 inhibitor that preferentially binds to channels in open or inactivated states may provide efficacy with an improved therapeutic window over ziconotide [15, 183, 185, 190, 307, 308].

SH-SY5Y cells have a higher (more depolarised) resting potential than neurons (~ –30 mV). This means that channels are in both the resting and inactivated states, as opposed to just the resting state seen at –80 mV), allowing this assay to identify potentially use-dependent inhibitors that preferentially act on the inactivated states.

Approaches to modify the FLIPR assays to help identify state dependant effects of drugs have been previously described [183, 185, 307, 308]. For example, stable cell lines expressing

functional Cav2.2 channels were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, was controlled by external potassium concentration. Following cell incubation of the drugs with varying concentrations of K+, a high potassium trigger solution can be added to elicit calcium influx through available, unblocked channels. State dependent inhibitors that preferentially bind to channels in the open or inactivated state may be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. With

this approach, although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity [183].

Over 41,000 described species of spiders have evolved from a common ancestor that lived at least 350 million years ago. Since that time, spider venoms have been evolving as predatory tools in the context of diversifying tactics of prey capture [285]. Spider venoms are extraordinarily refined cocktails comprised of hundreds of toxins endowed with exquisite selectivity and affinity [277, 285]. Overall, the biochemical diversity of venom toxins from spiders may be up to forty million components [285], and thus represents a truly phenomenal potential for discovery. However, to date venoms of only a few spider family trees have been analysed and less than 1500 toxins described [278]. The HTS assays using the human SH-

SY5Y cell line identified a range of spider venom Cav2.2-inhibitors, with about 50% of the

spider venoms screened showing activity at Cav2.2. This data confirmed the potential of spider venoms as abundant sources of analgesic leads, similarly to cone snails.

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Chapter 5 Assay-guided isolation of the venom of C. darlingi, a tarantula spider, leaded to the

discovery of a novel Cav2.2 inhibitor toxin, Cd1a, which was sequenced and synthesized.

Synthetic Cd1a inhibited Cav2.2 with low micromolar potency. Similarly to other close-related spider toxins, Cd1a exhibited promiscuous pharmacological activity. In addition to preferably

inhibiting Cav2.2 channels in SH-SY5Y cells, Cd1a inhibited a range of Nav channel subtypes and thus have been classified as µ/ω-toxin. Interestingly, Cd1a did not displace 125I-GVIA,

indicating this toxin acts at Cav2.2 through a different mechanism from that of cone snail ω-

conotoxins. While ω-conotoxins are thought to block Cav2.2 via occluding the Cavα subunit pore (for review see: [80]), the mechanisms of Cav2.2 inhibition by spider venom toxins seems to vary, as both pore blockers and gating modifier toxins have been isolated from different spider toxin families (for review see chapter 1 and ref [1]).

The results in this thesis suggest Cd1a most likely inhibits Cav2.2 via modifying gating properties. To confirm this hypothesis we will have to identify, in future studies, if Cd1a has the ability to change Cav2.2 channels kinetics. One of the characteristics of gating modifier toxins is despite complete inhibition of all inward currents, outward currents can still be observed in the presence of toxin [287, 300]. Electrophysiological experiment need to be performed varying voltage protocols and plotting current-voltage relationships in (eg. I-V curves to identify the voltage dependence of activation/inactivation). Activity of gating modifier toxins will be voltage- dependent and entail a modification of channel gating by interfering with the voltage dependence and kinetics of channel opening and closing. Importantly The HTS assays established in this

thesis can easily detect active Cav2.2 channel modulators independently of the mechanism of action. The studies in this thesis prove ω-conotoxins are potent analgesic toxins which may have improved characteristics to that of current drugs and suggest novel ω-conotoxins might still reach the clinic in the future.

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Chapter 6

Chapter 6

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Chapter 6 Chapter 6 References

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1.1. List of Chemicals and Buffers

Name Source

DMEM Invitrogen, California, USA

RNase-Free DNase Set Qiagen, USA

RPMI 1640 Invitrogen, USA

SH-SY5Y cells Victor Diaz, Germany

CHO cells Chantest, USA

HEK cells ATCC, USA

Calcium 4 assay kit Molecular Devices, USA

Fluo-4 AM Invitrogen, USA

Synthetic Conotoxins Alewood group, UQ, AU

Iodine-125 (125I) PerkinElmer, USA

ω-Conotoxin [125I]Tyr22 – GVIA PerkinElmer, USA

Trizol Reagent Invitrogen, Carlsbad, USA

Omniscript Reverse Transcription Kit Qiagen, Germantown, USA

Taq Polymerase Kit New England Biolabs, USA

DNA molecular weight makers New England Biolabs, USA

SYBR Safe DNA gel stain Invitrogen, Carlsbad, USA

6x Loading dye New England Biolabs, USA

Wizard SV Gel and PCR clean-up system Promega, USA

FBS Invitrogen, USA

2 mM GlutaMAX Invitrogen, USA 157

Appendix 1 Trypsin/EDTA Invitrogen, USA

Cell lysis buffer Roche Diagnostics, AU

Agarose gel Sigma-Aldrich, USA

Trypsin/EDTA Invitrogen, USA

Bicinchoninic acid (BCA) assay reagent Pierce Rockford, USA

RNAlater Ambion, Invitrogen, USA

α-cyano-4-hydroxycinnamic acid (CHCA) Sigma Aldrich, USA

FirstChoice RLM-RACE kit Ambion, Invitrogen, USA

Agatoxin-IVA Abcam, AU

Mibefradil Tocris biosciences

Nifedipine Sigma Aldrich, USA

Isoflurane Abbot, USA

QIAquick Gel Extraction kit Qiagen, USA

FastStart Taq DNA polymerase Roche Applied Science

Red membrane potential dye Molecular Devices, USA

Veratridine Sigma Aldrich, USA

Deltamethrin Sigma Aldrich, USA

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Appendix 1 1.2. Composition of Solvents, Mediums and Buffers

Analytical RP-HPLC Solvents

1. Analytical RP-HPLC solvents used in chapter 3. Solvent A: 90% water, 0.01% formic acid; Solvent B: 90% acetonitrile, 10% water, 0.09% formic acid. 2. Analytical RP-HPLC solvents used in chapter 2: Solvent A: 0.1% TFA (trifluoroacetic acid)/H2O; Solvent B: 90% acetonitrile, 0.1% TFA.

3. External electrophysiology recording solution (mM): 150 TEA-Cl, 2 BaCl2, 10 D- Glucose and 10 HEPES, adjusted to pH 7.4 with TEA-OH (Electrophysiology, Chapter 3).

4. Intracellular electrophysiology solution (mM): 140 CsCl, 1 MgCl2, 5 BAPTA and 10 HEPES adjusted to pH 7.2 with CsOH (Electrophysiology, Chapter 3

Mediums

1. Dulbecco’s Modified Eagle Medium (DMEM). Obtained from Invitrogen:

30 mg/L glycine, 84 mg/L L-arginine, 63 mg/L L-cystine , 584 mg/L Lglutamine, 42 mg/L L-Histidine, 105 mg/L L-isoleucine, 105 mg/L L-leucine, 146 mg/L L-lysine HCl, 30 mg/L L- methionine, 66 mg/L L-phenylalanine, 42 mg/L L-serine, 95 mg/L L-threonine. 16 mg/L L- tryptophan, 104 mg/L Ltyrosine disodium salt dehydrate, 94 mg/L L-valine, 4 mg/L choline chloride, 4 mg/L D-Ca2+ pantothenate, 4 mg/L folic acid, 7.2 mg/L i-inositol, 4 mg/L niacinamide, 4 mg/L pyridoxine HCl, 0.4 mg/L Riboflavin, 4 mg/L thiamine, 200 mg/L Ca2+ chloride, 0.1 mg/L ferric nitrate, 97.67 mg/L magnesium sulphate, 400 mg/L potassium chloride, 3700 mg/L sodium bicarbonate, 6400 mg/L sodium chloride, 125 mg/L sodium phosphate, 4500 mg/L D-glucose, 15 mg/L phenol red, 110 mg/L sodium pyruvate.

2. Roswell Park Memorial Institute Medium (RPMI). Obtained from Invitrogen:

Glycine 10 mg/mL, L-Arginine 200 mg/mL, L-Asparagine 50 mg/mL, LAspartic acid 20 mg/mL, L-Cystine 2HCl 65 mg/mL, L-Glutamic Acid 20 mg/mL, L-Glutamine 300 mg/mL, L-Histidine 15 mg/mL, L-Hydroxyproline 20 mg/mL, L-Isoleucine 50 mg/mL, L-Leucine 50 mg/mL, 0.382 L-Lysine hydrochloride 40 mg/mL, L-Methionine 15 mg/mL, L-Phenylalanin 15 mg/mL, L-Proline 20 mg/mL, L-Serine 30 mg/mL, L-Threonine 20 mg/mL, LTryptophan 5 5 mg/mL, L-Tyrosine disodium salt dihydrate 29 mg/mL, LValine 20 mg/mL, Biotin 0.2

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Appendix 1 mg/mL, Choline chloride 3 mg/mL, D-Calcium pantothenate 0.25 mg/mL, Folic Acid 1 mg/mL, Niacinamide 1 mg/mL, Para-aminobenzoic acid 1 mg/mL, Pyridoxine hydrochloride 1 mg/mL, Riboflavin 0.2 mg/mL, Thiamine hydrochloride 1 mg/mL, Vitamin B12 0.005 mg/mL, i-Inositol 35 mg/mL, Calcium nitrate 100 mg/mL, Magnesium Sulfate 48.84 mg/mL, Potassium Chloride 400 mg/mL, Sodium Bicarbonate 2000 mg/mL, Sodium Chloride 6000 mg/mL, Sodium Phosphate dibasic anhydrous 800 mg/mL, D-Glucose 2000 mg/mL, Glutathione 1 mg/mL, Phenol Red 5 mg/mL.

Buffers

3. Binding Assay Buffer- pH: 7.4

 20 mM HEPES

 75 mM NaCl

 0.2 mM EDTA

 0.2 mM EGTA

 0.1–0.2% BSA

4. Binding Assay- Washing Buffer

1. 20 mM HEPES

2. 125 mM NaCl

5. Physiological Salt Solution (PSS), pH: 7.4

 5.9 mM KCl

 1.5 mM MgCl2

 1.2 mM NaH2PO4

 5.0 mM NaHCO3

 140 mM NaCl

 11.5 mM Glucose

 5.0 mM CaCl2 160

Appendix 1

 10 mM HEPES

Containing in addition 0.1 % BSA and 10 µM nifedipine (Cav2.2 assays), 3 µM CVID (Cav1 assays), or 30 µM mibefradil (Cav3 assays).

6. Hanks Balanced Salt Solution (HBSS), pH: 7.4. Obtained from Multicell, Worthington

Biochemical, NJ, USA

 140 mg/L CalCl2

 100 mg/L MgCl2

 100 mg/L MgSO4-7H2O

 400 mg/L KCl

 60 mg/L KH2PO4

 350 mg/L NaHCO3

 8000 mg/L NaCl

 48 mg/L NaHPO4

 1000 mg/L D-glucose

7. Cell Lysis Buffer. Obtained from Roche Diagnostics, AU

 0.15 mM NaCl

 5 mM EDTA

 10 mM Tris

 1 % Triton X100

 1 protease inhibitor tablet/10 mL

8. TBE buffer (10X), pH 8.0

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 890 mM Tris

 890 mM Boric acid

 20 mM EDTA

9. Gel loading dye buffer 6x (New England Biolabs), pH 8.0

 2.5% Ficoll®-400

 11mM EDTA

 3.3mM Tris-HCl

 0.017% SDS

 0.015% bromophenol blue

10. Wizard SV Gel and PCR Clean-Up System 50 preps (Promega)

 20 ml Membrane Binding Solution

 15 ml Membrane Wash Solution (concentrated)

 3.75 ml Nuclease-Free Water

 50 Wizard SV Mini-columns

 50 Collection Tubes (2 ml)

11. Dulbecco’s Phosphate Buffered Saline (DPBS). Obtained from Invitrogen

 KCl

 KH2PO4

 NaCl

 Na2HPO4-7H2O

12. Phosphate-Buffered Saline (PBS), pH: 7.4

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 137 mM NaCl

 2.7 mM KCl

 10 mM NaH2PO4

 1.8 mM KH2PO4

13. KCl Activation Buffer for the Ca2+ Imaging Assays

 PSS buffer

 KCl 90 mM

 CaCl2 5 mM

 10 µM nifedipine for Cav2.2 assays

 µM CVID for Cav1 assays

 or 30 µM mibefradil for Cav3.1 assays

14. Trypsin-EDTA. Obtained from Invitrogen

 8000 mg/L NaCl

 400 mg/L KCl

 90 mg/L NaH2PO4-7H20

 60 mg/L KH2PO4

 350 mg/L NaHCO3

 1000 mg/L D-Glucose

 10 mg/L Phenol Red

 380 mg/L Na-EDTA

 2500 mg/L Trypsin

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