Venoms-based discovery of novel modulators of human neuronal α7 and

α3* nicotinic acetylcholine receptors

Rosa Esther Prahl

MSc. Cell Biology

A thesis submitted for the degree of Master of Philosophy at

The University of Queensland in 2016

Institute for Molecular Bioscience

 

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ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated channels

that contain combinations of α and β subunits (heteromeric) or only five α subunits

(homomeric). To date, 12 subunits have been cloned. Although different types of

neuronal nAChRs are expected to occur in vivo, most structure-activity relationship

studies have been carried out for just a few neuronal subtypes. Clinical and basic

research has shown that cholinergic receptors play a role in several disorders of

the nervous system such as chronic pain, Alzheimer’s disease and addiction to

nicotine, alcohol and drugs. Unfortunately, the lack of selective modulators for

each subtype of nAChR makes their pharmacological characterization difficult,

which has slowed the development of therapeutic nAChR modulators with high

selectivity and absence of off-target side effects.

Animal venoms have proven to be an excellent natural source of bioactive

molecules with activity against ion channels. Venoms from different snakes and in

particular molluscs of the Conus genus have been an important source of

modulators of cholinergic receptors. However, spider and scorpion venoms have

been studied less extensively and there are only two examples of cholinergic

receptor modulators reported from arachnid venom. Thus, the primary goal of this

thesis was to identify novel modulators of the human ganglionic α3* nAChR

(where the asterisk indicates that β subunits form part of the heteropentameric

receptor) and the homomeric neuronal α7 nAChR using spider and scorpion

venoms.

“Hit” venoms from a selection of “modern” and “primitive” spiders were identified

via high-throughput screens against the α3* and α7 nAChRs endogenously

expressed in SH-SY5Y cells. A total of 71 spider venoms were screened, with

representatives from nine different taxonomic families. The resulting data showed

that 91% of the venoms presented some activity against the nAChR subtypes

tested and most of these were non-selective blockers of the α3* and α7 subtypes.

Toxins directed against vertebrate nAChRs likely evolved through frequent

interactions between spiders and small vertebrate predators. Our data revealed

that nAChR agonists are likely to be present in venom of the “primitive” spiders

 

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and therefore were probably basally recruited in spider venom. In summary, the

screening of spider venoms against vertebrate cholinergic receptors provided

information about the origin and evolution of toxins against vertebrate receptors.

The search for nAChR modulators was extended to scorpion venoms that are

recognized as sources of toxins that affect primarily the properties of voltage-gated

sodium and potassium channels. In this work, venoms from Androctonus australis,

Androctonus crassicauda, Leirurus quinquestriatus and Grosphus grandidieri

(Buthidae) and Heterometus spinnifer (Scorpionidae) were screened against the

α3* and α7 nAChRs using SH-SY5Y cells. The venom of Heterometrus spinnifer

was found to contain an agonistic compound. Activity guided-fractionation allowed

isolation of the “hit” toxin, and it was identified by high-resolution mass

spectrometry as senecioylcholine. This non-peptide toxin has not previously been

identified in arachnids but it was previously isolated from some marine gastropods

(Muricidae). It shows high toxicity against vertebrates. Thus, this non-peptide toxin

is likely to function in defence against vertebrate predators.

 

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DECLARATION BY 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 policy and procedures of The University of

Queensland, the thesis be made available for research and study in accordance

with the Copyright Act 1968 unless a period of embargo has been approved by the

Dean of the Graduate School.

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.

 

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PUBLICATIONS DURING CANDIDATURE

No publications.

PUBLICATIONS INCLUDED IN THIS THESIS

No publications.

 

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CONTRIBUTION BY OTHERS TO THE THESIS

Prof. Richard Lewis and Dr Irina Vetter (Institute for molecular Bioscience)

supported during the screening of “hit” toxins of the 71 venoms with their expertise

in FLIPR assays.

Chapter 1 Dr Volker Herzig (Institute for Molecular Bioscience) performed HPLC fractionation

of crude venom from Macrothele gigas.

Dr Irina Vetter (Institute for Molecular Bioscience) performed one FLIPR assay that

was mentioned in section 2.3.4.

Chapter 2 HPLC fractionation of crude venom from Heterometrus spinnifer was performed by

Dr Niraj Bende (Institute for Molecular Bioscience).

Dr Angela Salim identified the “hit” toxin from Heterometrus spinnifer as

senecioylcholine using high-resolution electrospray ionization mass spectrometry.

She also performed as well as the co-elution of the native compound with its

synthetic form, and two of its synthetic isomers (tigloylcholine and

angeloylcholine).

Pratik Neupane carried out the chemical synthesis of senecioylcholine,

tigloylcholine and angeloylcholine.

Angela and Pratik were involved in the writing of the protocol for identification and

chemical synthesis of senecioylcholine. Angela and Pratik are members of the

group of Prof. Robert Capon at The Institute for Molecular Bioscience.

 

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STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF

ANOTHER DEGREE

None.

 

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ACKNOWLEDGEMENTS

I thank Prof. Glenn King, Prof. Richard Lewis, Prof. Robert Capon, Dr Amanda

Carozzi, Dr Irina Vetter, Dr Angela Salim, Pratik Neupane and Sassan Ranhama

for their time, efforts and suggestions during the course of my thesis.

This work was funding and supported by the following institutions: Postgraduate

studies of The University of Queensland (UQ), the UQ Institute for Molecular

Bioscience, and a program grant from the Australian National Health & Medical

Research Council.

I dedicate this thesis to the Holy Father, my husband Torben and my son Clouseau.

 

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KEYWORDS

spider venom, scorpion venom, ligand gated calcium channel, α7 nicotinic

acetylcholine receptors, α3* nicotinic acetylcholine receptors, FLIPR, high

throughput screening, choline ester, senecioylcholine

AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS

(ANZSRC)

ANZSRC code: 060101, Analytical Biochemistry, 80%

ANZSRC code: 060199, Biochemistry and Cell Biology not elsewhere classified,

20%

FIELDS OF RESEARCH (FOR) CLASSIFICATION

FoR code: 0601, Biochemistry and Cell Biology, 80%

FoR code: 0699, Other Biological Sciences, 20%

 

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TABLE OF CONTENTS

Abstract  ........................................................................................................................  2  

Declaration  by  author  ...................................................................................................  4  

Publications  during  candidature  ....................................................................................  5  

Publications  included  in  this  thesis  ................................................................................  5  

Contribution  by  others  to  the  thesis  ..............................................................................  6  

Statement  of  parts  of  the  thesis  submitted  to  qualify  for  the  award  of  another  degree  .  7  

Acknowledgements  .......................................................................................................  8  

Keywords  ......................................................................................................................  9  

Australian  and  New  Zealand  Standard  Research  Classifications  (ANZSRC)  .....................  9  

Fields  of  Research  (FoR)  Classification  ...........................................................................  9  

Table  of  Contents  .........................................................................................................  10  

List  of  figures  and  Tables  ..............................................................................................  12  List of abbreviations  ................................................................................................................................................  14  

CHAPTER  1  —  Introduction  ...........................................................................................  16  1.1   Nicotinic acetylcholine receptors  ...........................................................................................................  17  1.1.1   Homopentameric/heteropentameric architecture of neuronal nAChRs  ....................  17  1.1.2   The use of toxins to elucidate the molecular architecture of nAChRs  .......................  19  1.1.3   Location and physiological function of nAChRs in vertebrates and invertebrates   21  1.1.4   Role of nAChRs in human disease  ................................................................................................  24  

1.2   Modulators of nAChRs  ...............................................................................................................................  26  1.2.1   Modulators of nAChRs as drugs and insecticides  .................................................................  26  1.2.2   Novel subtype-selective modulators of vertebrate nAChRs are required to develop accuracy pharmacological tools and potential therapeutic leads  ..............................  28  

1.3   Venom-derived modulators of nAChRs  .............................................................................................  32  1.3.1   Important role played by subtype-selective α-conotoxins in study of vertebrate nAChRs  ..........................................................................................................................................................................  32  1.3.2   nAChR modulators from other venomous animals  ...............................................................  34  1.3.3   Rationale for examining spider venoms as a source of novel nAChR modulators   34  

1.4   Significance and aims of the current work  .......................................................................................  35  1.4.1   Significance  ..................................................................................................................................................  35  1.4.2   Aims  ..................................................................................................................................................................  36  

CHAPTER  2  —  nAChR  modulators  from  spider  venoms  ..................................................  37  2.1   Introduction  .......................................................................................................................................................  37  2.2   Methods and Materials  ...............................................................................................................................  39  2.2.1   Tissue culture reagents  .........................................................................................................................  39  2.2.2   Spider venoms  ...........................................................................................................................................  40  2.2.3   Cell lines and culture  ..............................................................................................................................  40  2.2.4   Preparation of the Calcium 4-dye  ...................................................................................................  41  2.2.5   High-throughput FLIPR calcium assay  ........................................................................................  42  2.2.6   Activation of α3* nAChR by nicotine  .............................................................................................  43  2.2.7   Activation of α7 nAChR by choline in the presence of the positive allosteric modulator PNU-120596  ........................................................................................................................................  45  

 

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2.2.8   Identification of blockers and agonists for α3* and α7 nAChR using the FLIPRTETRA system  ...................................................................................................................................................  46  2.2.9   Data analysis  ...............................................................................................................................................  49  2.2.10   Statistical Analyses  ...............................................................................................................................  49  

2.3   Results  ................................................................................................................................................................  50  2.3.1   Screening of 71 spider venoms using the high-throughput FLIPRTETRA system  ..  50  2.3.2   Screening of venoms from “modern” spiders (Infraorder Araneomorphae)  ...........  50  2.3.3   Screening of venoms from “primitive” spiders (Infraorder Mygalomorphae)  .........  55  2.3.4   Identification of α7/α3* nAChR agonists in the venom of theraphosid spiders  ....  55  2.3.5   Venoms that selectively inhibit vertebrate α3* nAChRs  ....................................................  58  2.3.6   Venoms that non-selectively block both α3* and α7 nAChRs  .......................................  61  2.3.7   Theraphosid venoms with no apparent effect on nAChRs  ...............................................  63  2.3.8   Effect of non-theraphosid mygalomorph venom on vertebrate nAChRs  .................  64  2.3.9   Taxonomic distribution of nAChR modulators within Araneae  .......................................  64  

2.4   Discussion  .........................................................................................................................................................  66  CHAPTER  3  —  nAChR  modulators  from  scorpion  venom  ................................................  75  3.1   Introduction  .......................................................................................................................................................  75  3.1.1   Scorpion toxins and their pharmacological action  .................................................................  75  

3.2   Methods  ..............................................................................................................................................................  77  3.2.1   Figure 3.1 shows an example of the protocol as applied to scorpion venoms  .....  77  3.2.2   Scorpion venoms  ......................................................................................................................................  80  3.2.3   Venom fractionation  ................................................................................................................................  80  3.2.4   Discovery of a choline ester from the venom of H. spinnifer  ..........................................  80  3.2.5   Determination of mass and purity  ...................................................................................................  81  3.2.6   Identification of active compound using HRMS and LCMS  .............................................  81  3.2.7   Chemical synthesis of choline esters  ............................................................................................  82  

3.3   Results  ................................................................................................................................................................  84  3.3.1   High-throughput FLIPR assay  ...........................................................................................................  84  3.3.2   Isolation of the active compound from H. spinnifer venom  ..............................................  87  3.3.3   Identification of the active compound by high-resolution mass spectrometry  ......  90  

3.4   Discussion  .........................................................................................................................................................  92  CHAPTER  4  —  Conclusions  ............................................................................................  94  

Summary  and  future  directions  ....................................................................................  95  

References  ...................................................................................................................  97  

Appendix  I  ..................................................................................................................  125  

 

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LIST OF FIGURES AND TABLES FIGURE 1.1. GENERAL ARCHITECTURE OF PENTAMERIC ACETYLCHOLINE RECEPTORS.  .....................................  18  FIGURE 1.2 α-CONOTOXINS AS RESEARCH TOOLS TO STUDY THE STRCUTURE AND FUNCTION OF NACHRS.  ...............................................................................................................................................................................................................  21  FIGURE 1.3. STRUCTURES OF TOXINS AND TOXIN DERIVATIVES THAT TARGET NACHRS.  ..................................  31  TABLE 1.1 AMINO ACID SEQUENCES OF SELECTED α-CONOTOXINS. SI CONOTOXIN FROM CONUS STRIATUS THAT TARGETS THE NEUROMUSCULAR SUBTYPE CHOLINERGIC RECEPTOR; MII CONOTOXIN FROM CONUS MAGUS THAT TARGETS WITH HETEROMERIC NACHRS WITH HIGHER AFFINITY THAN THE HOMOMERIC

SUBTYPE α7 NACHR; AUIA CONOTOXIN ISOLATED FROM CONUS AULICUS, WHICH TARGETS THE

NEURONAL HETEROMERIC SUBTYPE α3β4. THE ASTERISKS (*) REPRESENT C-TERMINAL AMIDATION. TABLE ADAPTED FROM (LEWIS, 2012; MOLGÓ, 2013). LOOP 1 AND LOOP2 ARE THE TWO INTERCYSTINE LOOPS.  33  α-CONOTOXIN  ...................................................................................................................................................................................  33  FIGURE 2.1. BASIC TWO-ADDITION FLIPR PROTOCOL TO MEASURE CALCIUM RESPONSES EVOKED BY

ACTIVATION OF α7 OR α3* NACHRS.  .....................................................................................................................................  47  FIGURE 2.2. ‘TWO-ADDITION’ FLIPR PROTOCOL USED TO IDENTIFY NACHR AGONISTS.  ...................................  48  FIGURE 2.3. SCREENING OF VENOMS FROM “MODERN” SPIDERS AGAINST HUMAN α3* AND α7 NACHRS USING A HIGH-THROUGHPUT FLIPR ASSAY.  .........................................................................................................................  52  TABLE 2.1. TAXONOMIC DISTRIBUTION OF NACHR MODULATORS FROM ARANEOMORPH SPIDER VENOMS. TWO SPECIES SPARASSIDAE SP. WERE ASSAYED; THE NUMBERS 1 AND 2 INDICATE THEY WERE COLLECTED IN TWO DIFFERENT AREAS IN PNG. VENOMS WITH HIGH BLOCKING EFFECT ARE INDICATED WITH TWO

SYMBOLS DENOTED AS √√, WHILE THOSE WITH PARTIAL EFFECT ARE INDICATED WITH ONE √. THE GREEN AREA INDICATES THAT OTHER EFFECTS ON THE VENOM IS NOT OBSERVED.  .............................................................  54  FIGURE 2.4. AGONISM OF HUMAN NACHRS BY THERAPHOSID VENOMS.  ..................................................................  57  TABLE 2.2. APPARENT AGONISTIC EFFECT CAUSED BY VENOMS FROM FOUR THERAPHOSID SPIDERS. AFTER VENOM ADDITION, THE FLUORESCENT CALCIUM RESPONSES EVOKED BY NACHRS IN THE ABSENCE OR PRESENCE OF PNU WERE CONVERTER INTO AREA UNDER CURVE USING SCREENWORKS SOFTWARE, VERSION 3.1. THE VALUES ARE AVERAGES OF THREE INDEPENDENT EXPERIMENTS WITH N = 3. THE MEAN

OF THE CONTROL NICOTINE RESPONSE WAS 509 ± 50.40 WHILE THE CONTROL CALCIUM RESPONSE

ELICITED BY CHOLINE WAS 1088 ± 38.31  ..............................................................................................................................  58  TABLE 2.3. MYGALOMORPH SPIDERS WITH VENOMS THAT ARE LIKELY TO CONTAIN AGONISTS OF α7 NACHRS. N.D INDICATES THE EFFECT WAS NOT DETERMINED AND A FURTHER ANALYSIS IS REQUIRED TO

CONFIRM THAT THE VENOM EFFECT IS VIA ACTIVATION OF α7 NACHRS IN THE PRESENCE OF PNU.  ............  58  FIGURE 2.5. THERAPHOSID SPIDER VENOMS THAT PREFERENTIALLY BLOCK α3 NACHRS.  ..............................  59  TABLE 2.4. THERAPHOSID SPIDERS WITH VENOMS THAT SELECTIVELY BLOCK α3* NACHRS.  .........................  59  SPECIES WITH CHARACTERISTIC COLORS ARE INDICATED. (M) AND (F) REFER TO GENDER. THE NUMBERS BESIDE THE NAME OF EACH THERAPHOSID SPIDER INDICATES THAT THEY ARE INDIVIDUALS FROM THE SAME SPECIES, ALTHOUGH WERE COLLECTED FROM DISTINCT LOCALITIES.  .........................................................................  59  FIGURE 2.6. MANY THERAPHOSID VENOMS NON-SELECTIVELY BLOCK BOTH α3* AND α7 NACHRS.  ...........  61  TABLE 2.5. THERAPHOSID SPIDERS WITH VENOMS THAT NON-SELECTIVELY BLOCK BOTH α3* AND α7 NACHRS. (M) AND (F) REFER TO GENDER. THE NUMBERS BESIDE THE NAME OF EACH THERAPHOSID SPIDER INDICATES THAT THEY ARE INDIVIDUALS FROM THE SAME SPECIES, ALTHOUGH WERE COLLECTED FROM DISTINCT LOCALITIES.  ........................................................................................................................................................  62  FIGURE 2.7. FOUR TARANTULA VENOMS WITHOUT POTENT MODULATORS OF α3* OR α7 NACHRS.  ............  63  TABLE 2.6. VENOMS FROM AMERICAN THERAPHOSIDS THAT LACK OF MODULATORS OF HUMAN α3* AND α7 NACHRS.  ............................................................................................................................................................................................  64  FIGURE 2.8. SUMMARISE OF THE HIGH-THROUGHPUT SCREENING OF SPIDER VENOMS.  ....................................  65  FIGURE 3.1. A. CRASSICAUDA VENOM CAN INDUCE CALCIUM RESPONSES ELICITED VIA ACTIVATION OF NAV CHANNELS.  .........................................................................................................................................................................................  79  FIGURE 3.2. FLIPR SCREENING OF SCORPION VENOMS AGAINST α7 AND α3* NACHRS.  ................................  85  FIGURE 3.3. NON-SELECTIVE ACTIVATION OF α7 AND α3* NACHRS EXPRESSED IN SH-SY5Y BY CRUDE VENOM FROM H. SPINNIFER.  .......................................................................................................................................................  86  FIGURE 3.4. HPLC FRACTIONATION OF CRUDE VENOM FROM H. SPINNIFER. CHROMATOGRAM RESULTING FROM FRACTIONATION OF CRUDE VENOM VIA C18 RP-HPLC.  .....................................................................................  87  FIGURE 3.5. ACTIVITY OF F10 FROM FRACTIONATION OF CRUDE VENOM FROM H. SPINNIFER.  .......................  88  

 

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FIGURE 3.6. PURIFICATION OF THE “HIT’ TOXIN BY RP-HPLC.  .....................................................................................  89  FIGURE 3.7. AGONISTIC ACTIVITY OF THE “HIT” TOXIN.  .....................................................................................................  89  FIGURE 3.8. CO-ELUTION OF BIOACTIVE COMPOUND FROM H. SPINNIFER VENOM AND SYNTHETIC CHOLINE ESTERS.  ..............................................................................................................................................................................................  91  TABLE I. LIST OF SPIDERS WHOSE VENOMS WERE USED IN THIS WORK  ..................................................................  125  TABLE II. LIST OF SCORPIONS WHOSE VENOMS WERE USED IN THIS WORK  ...........................................................  128  

 

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List of abbreviations ACh Acetylcholine

AChBP acetylcholine binding protein

AuIB α-conotoxin AuIB

AFU arbitrary fluorescent units

BSA bovine serum albumin

Ca2+ calcium ion

Cav2.3 P/Q-type calcium channel

CHCA α-cyano-4-hydroxycinnamic acid

CNS central nervous system

Da Dalton

DRG dorsal root ganglion

EDTA ethylenediaminetetraacetic acid

ESIMS electrospray ionization-mass spectrometry

FBS foetal bovine serum

FITC fluorescein isothiocyanate

FLIPR fluorescent imaging plate reader

GABA

GABAARs

γ-aminobutyric acid

γ-aminobutyric acid type-A receptors

HEPES 4-(2-hydroexyethyl)-1-piperazineethane sulfonic acid

HILIC hydrophilic interaction liquid chromatography

HPLC high-performance liquid chromatographic

HRMS high- resolution mass spectrometry

kDa Kilodalton

LsIA α-conotoxin LsIA

MALDI-TOF matrix assisted laser desorption/ ionization time-of-

flight

MS mass spectrometry

NMR nuclear magnetic resonance

NSAID non-steroidal anti-inflammatory drugs

nAChRs nicotinic acetylcholine receptors

PNS peripheral nervous system

 

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PNU-120596 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-

3-yl) urea

RLU relative light units

PSS physiological salt solution

RPMI Rosewell Park Memorial Institute

RP-HPLC reversed phase high-performance liquid

chromatography

SAR structure-activity relationship

TFA trifluoroacetic acid

TRITC Tetramethylrhodamine

TTX Tetrodotoxin

 

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CHAPTER 1 — INTRODUCTION Recent studies indicate that imbalance of the cholinergic system in human brain

via nicotinic acetylcholine receptors (nAChRs) are linked to various neurological

disorders (AhnAllen, 2012; Chu, 2005; Nides, 2006; Tregellas, 2011; Yu, 1996)

while mutations in the nAChR can lead to frontal lobe epilepsy (for a review see

(Steilein, 2001)). These findings have prompted research into the development of

drugs that target neuronal cholinergic receptors. Nevertheless, most of the

chemical components under trial lack selectivity, or hardly improve the medical

condition (for reviews, see (Arneric, 2007; Jensen, 2005; Pammolli, 2011)). The

discovery of new therapeutic agents is currently a challenge because of the

complex arrangement of subunits of neuronal cholinergic receptors expressed in

human brain (Gotti, 2007) and the fact that their roles under physiological and

pathological conditions are not fully understood (for a review, see (Tuesta, 2011)).

To facilitate studies of the pharmacology of nAChRs, a search for new nAChR

modulators in spider and scorpion venoms was carried out using a high-

throughput FLIPR assay to rapidly identify “hit” venoms. The probability of finding

native cholinergic modulators was expected to be high because arachnid venoms

are estimated to contain about 1 million different components, and even though

less than 0.01% of these molecules have been studied (for reviews, see

(Escoubas, 2006b; King, 2014; King and Coaker, 2014; King and Hardy, 2013),

many of them are peptidic toxins that act on diverse types of ionic channels

(Meves, 1984; Quintero-Hernandez, 2013; Shahbazzadeh, 2007; Xie, 2012).

This chapter provides a review of what is known about the molecular structure of

nAChRs and their physiological functions in vertebrates and invertebrates.

Furthermore, I review the use of animal toxins as research tools to characterize

nAChRs, as well as their roles in the development of therapeutic drugs and

bioinsecticides.

 

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1.1 NICOTINIC ACETYLCHOLINE RECEPTORS

1.1.1 Homopentameric/heteropentameric architecture of neuronal nAChRs nAChRs are members of the pentameric Cys-loop ligand-gated ion channel

superfamily that are activated by the neurotransmitter acetylcholine (ACh) (for a

review, see (Unwin, 2005)). Our current knowledge of the molecular structure of

neuronal nAChRs began with determination of the N-terminal sequence of the

nAChR isolated from the electric organ of the marbled ray Torpedo marmorata,

followed by the cloning of its subunits. These subunits were subsequently shown

to be closely related to cDNAs cloned from mammals that encoded 17 structurally

homologous subunits, denoted α1–α10, β1–β4, γ, δ and ε. Each subunit

comprises four α-helical transmembrane regions (M1–M4), of which M1-M3 are

conserved and M4 has a variable extracellular C-terminal sequence; a conserved

large N-terminal extracellular domain (ECD) of approximately 200 amino acid

residues; a short intracellular C-terminal tail (see Figure 1.1A); and a variable

cytoplasmic domain between the transmembrane regions M3 and M4 (Unwin,

2005). In addition, all subunits contain in the first extracellular domain a cysteine-

loop (Cys–loop) that consists of a pair of cysteines, and in the case of mammals,

the Cys-Cys pair is separated by 13 residues. This structural template describes

the molecular backbone of different types of nAChRs.

 

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Figure 1.1. General architecture of pentameric acetylcholine receptors. (A) Ribbon representation of the three-dimensional structure of the pentameric nAChR from Torpedo marmorata determined using cryo-electron microscopy (Unwin, 2005). The three domains are indicated: extracellular domain (ECD), transmembrane domain and intracellular domain (ICD). The illustration was modified from the original in the review by Molgó et al. (Molgó, 2013). (B) Schematic representation of the arrangement of a heteromeric nAChR. The subtype α3β4 (left) contains two α3 and three β4 subunits. The heteromeric subtype has two binding sites for ACh (black circles) between α3 and β4 subunits. Homomeric subtypes like α7 (right) contains five identical subunits, and they have five bindings sites for ACh (black circles). The illustration was adapted from a review by Changeux (Changeux, 2010).

According to their localization and subunit composition, nAChRs are classified into

two types, the so-called muscle-type nAChRs and neuronal nAChRs. Muscle-type

nAChRs contain five classes of subunits denoted α1, β1, δ, and either γ or ε, which

are arranged in a circular order of barrel-like staves around a central channel in

the muscle sarcolemma, and with a stoichiometry of 2:1:1:1, which corresponds to

two α1 subunits plus β1, δ, and γ/ε subunits (Miyazawa, 1999; Unwin, 1993).

α7

Heteromeric α3β4

β4

β4

β4

α3

Homomeric α7

α7

α7

α7

α7

α3

A B

 

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In contrast to the muscle-type nAChRs, the neuronal-type receptors are more

diverse. Neuronal nAChRs are encoded by nine α (α2–α7, α9, α8, α10) and three

β (β2–β4) subunit genes. In the case of sensory and neuronal mammalian tissues,

eleven subunits have been cloned, which are α2–α7, α9, α10, and β2–β4, but a

homologue of the avian α8 subunit has not been described (reviewed in

(Albuquerque, 2009; Nicke, 2004)). Neuronal nAChRs also contain five subunits

that form a functional receptor via combinations of different α and β subunits

(heteropentamers), such as α3β2 and α4β2. The ACh binding site is located at the

interface between an α subunit and an adjacent β subunit (see Figure 1.1B). Also,

a third subunit can be included in the receptor (α4)2(β2)2α5. In addition, the

pentamers can consist of only α subunits, such as homopentamers of α7, α9 or

α10 (Cooper, 1991); in these subtypes, the ACh binding site is located between α

subunits (see Figure 1B).

The molecular architecture of heteropentamers and homopentamers is similar

(see Figure 1.1B). Nevertheless, each subtype differs in function (Elgoyhen, 2001;

Zhou, 2003). If novel and selective modulators for heteropentameric and

homopentameric nAChRs were available some relevant questions in terms of the

functional characterization of neuronal nAChRs could be answered. For example,

the α6 and β3 subunits are co-expressed in brain areas such as the substantia

nigra and the ventral tegmental area (Quik and McIntosh, 2006) which leads to two

questions: firstly, why are these subunits co-expressed only in these areas of the

brain, and secondly, is co-expression of these subunits related to the

symptomatology of Parkinson’s disease or addiction to nicotine.

1.1.2 The use of toxins to elucidate the molecular architecture of nAChRs

Toxins derived from animal venoms have been a valuable source of

pharmacological tools for studying structure-activity relationships of nAChRs. For

example, the muscle-type nAChR was characterized for the first time by Changeux

and colleagues in 1970 thanks to the use of α-bungarotoxin, which was purified

from the venom of an elapid snake (Bungarus multicinctus), on membrane

preparations derived from the electric organ of the eel Electrophorus electricus

 

20

(Changeux, 1970). In addition, the proposed binding site for ACh in nicotinic

receptors, which lies in a cleft at the interface between the subunits, was

determined from x-ray crystallographic structures of the acetylcholine binding

protein (AChBP), a homolog of nAChRs, in complex with a variety of ligands (for a

review, see (Tsetlin and Hucho, 2009)). AChBP is a soluble protein that shares

sequence similarity to the ligand-binding site of α1 or α7 nAChRs. Following its

discovery, several crystal structures have been determined of the complex formed

AChBP and various α-conotoxins (see Figure 1.2A,B). For instance, co-

crystallization of the complexes formed between AChBP and the α-conotoxins IMI

and PnIA revealed that these peptide toxins bind at a similar spatial position in the

ACh binding domain despite having divergent primary structures (see Figure

1.2C). Moreover, it was shown that hydrophobic interactions between the ACh

ligand- binding domain, and a conserved proline residue in both conotoxins could

lead to a differential selectivity to nAChRs (for reviews, see (Lebbe, 2014b; Lewis,

2012)).

A large number of α-conotoxins have been used to characterise many of the

neuronal subtypes of nAChRs (for a review, see (Lebbe, 2014b)) and structural

studies of the α-conotoxins : nAChR interaction have been invaluable (Bourne,

2005; Lewis, 2012). Nowadays, we can predict what amino acids residues can be

modified to improve α-conotoxin affinity and selectivity. For example, it was shown

that the selectivity of α-conotoxin, Lo1a from Conus longuriunis (see Figure 1.1D)

could be switched from the neuronal α7 subtype to adult muscle-subtype nAChR

by deleting an Asp residue from its C-terminus (Lebbe, 2014a). An α-conotoxin

that can differentially target the muscle subtype over the neuronal subtype could

be used to engineered probes for imaging studies of nAChRs in the peripheral

nervous system. Because α marine cone snails represent the largest source of

tools for studying vertebrate nAChRs, most of the modulators discovered to date

are antagonists. In contrast, very few nAChRs agonists have been reported.

Therefore, further research is needed to obtain new, subtype-selective nAChRs

modulators that might be useful therapeutics and/or useful tools for addressing

fundamental questions about nAChRs.

 

21

Figure 1.2 α-Conotoxins as research tools to study the strcuture and function of nAChRs. (A) Top view of the X-ray structure of the complex between Aplysia californica AChBP (AcAChBP) and α-conotoxin PnIA (for a review see (Armishaw, 2010)). Each of the five subunits is shown in a different color (orange, green, blue, red and cyan). (B) A single subunit of AcAChBP interacting with an α-conotoxin , with the C-loop is an open position (cyan). The arrow indicates the difference in conformation between the C-loop in the open (cyan) and closed (red) conformations (for more details see review (Armishaw, 2010)). (C) Co-crystal structures between AcAChBP (transparent image, only two subunits shown for clarity) and α-conotoxin ImI. Residues Arg11 and Arg7 (labelled) that are the primary residues on ImI that interact with AcAChBP. Figure taken from Dutertre et al. (Dutertre, 2007). (D) NMR solution structure of α-conotoxin Lo1A. Disulfide bonds are shown in yellow. Image from Lebbe et al. (Lebbe, 2014a).

1.1.3 Location and physiological function of nAChRs in vertebrates and invertebrates In vertebrates, nAChRs are expressed in most cells of the central and peripheral

nervous system (Albuquerque, 2009; Cooper, 1991), and in non-neuronal cells

(Macklin, 1998; Plummer, 2005). It seems that the role of ACh and nAChRs in the

CNS is mainly modulatory because the cholinergic signal can be excitatory or

C D

 

22

inhibitory of synaptic pathways in different cerebral areas (for a review, see

(Picciotto, 2012)). The different roles of neuronal nAChRs are made possible by

different combinations of α and β (Gotti, 2007). To date, the function of each

nAChR subtype in each place of expression is still not fully understood.

Human imaging studies in vivo have allowed mapping of the distribution of

nAChRs in the human brain (Kimes, 2006). Cholinergic projections are widely

distributed in the brain. For example, neuronal nAChRs have been reported to be

expressed in the white matter (Ding, 2004) and are likely to be involved in the

regulation of neuronal excitability in the thalamocortical area (Bucher and

Goaillard, 2011). Another cerebral region is the ventral tegmental area where the

release of glutamate and dopamine are related to the reward responses generated

by nicotine administration (Picciotto and Kenny, 2013). It contains GABAergic,

glutamatergic, and dopaminergic neurons; and their activities are regulated by a

combination of α4β2* nAChRs expressed mainly in GABAergic neurons and α7

nAChRs expressed in terminals of the glutamatergic neurons (Mansvelder, 2002).

Neuronal nAChRs are not restricted to the postsynaptic membranes; they can also

be expressed along the neuronal body, including axons and presynaptic terminals,

and different subtypes of nAChRs can be co-expressed in a neuron (for a review,

see (Picciotto, 2012)). Other cholinergic projections come from the basal forebrain

complex to the cerebral cortex. Their terminals contain β2* nAChRs; and are

distributed across all the cerebral cortex. Activation of the receptor lead to release

of glutamate into the prefrontal cortex, and this mediates neuronal processes such

as memory, attention and cue detection (Parikh, 2010).

To date, it is known that in humans the distribution of nAChRs is not limited to the

nervous system. Cholinergic receptors are expressed in human aortic endothelial

cells (α3, α5, α7, α10, β2-4) (Wada, 2007), vascular smooth muscle cells (α2-5,

α7, α10) and immune cells, such as human platelets (α7) (Schedel, 2010); human

mononuclear leukocytes (α7, β2) (Albaugh, 2004), human macrophages (α1, α7,

α10), B-lymphocytes (α3-5, α7), T-lymphocytes (α3, α4, α7, β2, β4), (for a review,

see (Bauwens, 2015)). The function of these receptors is linked to homeostasis of

the inflammatory response by the cholinergic anti-inflammatory pathway. For

 

23

instance, when terminals of afferent fibres located in the vagus are stimulated by

pro-inflammatory stimuli, action potentials are generated, and this causes efferent

fibres to activate α7 expressed on macrophages by acetylcholine (for a review see

(Egea, 2015)). In the end, α7 control the production of pro-inflammatory cytokines

such as TNF-α and IL-6 (Zhang, 2015a) without affecting the levels of anti-

inflammatory cytokines by inhibiting NF-kB nuclear translocation and activating

several pathways including JAK2/STA3 (Li, 2015). However, the cholinergic anti-

inflammatory pathway is not fully understood (for a review, see (Egea, 2015)).

In invertebrates, nAChRs have been found in the central nervous system, where

they mediate the excitatory synapsis of ACh. Studies of insect brain nAChRs have

been limited because there has only been limited success with heterologous

expression of cloned insect nAChRs (Landsdell, 2012). The main problem is that

cholinergic currents are evoked at high concentrations of ACh and the inhibitory

currents are usually too small to be accurately detected with electrophysiology

experiments using Xenopus oocytes, although co-expression of insect nAChRs

with subunits of vertebrate nAChRs has partially facilitated the expression of insect

nAChRs (Landsdell, 2012). There are still important nAChRs from insect species

such as honeybee Apis mellifera that are important to study to design novel and

safe insecticides. Today, it is known insect nAChRs have similar molecular

structure to vertebrate nAChRs. For instance, in the genome of Drosophila

melanogaster, 10 nAChR subunit genes of nAChRs have been identified: seven

encoded the α subtype (Dα1-7), and three corresponded to β subtype (Dβ1-3)

(Jones, 2007). The sequences of Dα5, Dα6 and Dα7 subunits are similar to each

other, and to α7 subunit from vertebrate nAChRs (Grauso, 2002; Jones and

Satelle, 2009). The Dα6 subunit is the most similar to the vertebrate α7 nAChR

(Sattelle, 2005). This similarity may allow insect brains to serve as a model system

for studying brain insect as an alternative animal model to study the role of

nAChRs in different pathologies. For example, more than 40% of bee brain

consists of Kenyon cells that are the major cells of the cerebral structure, known

as mushroom bodies or corpora pedunculata. This structure processes multiple

sensory information derived from the antennal lobe (Akalal, 2006; McGuire, 2001).

It was shown that neonicotinoids cause up-regulation of nAChRs (Moffat, 2015);

 

24

and because nAChRs can cause mitochondrial depolarization it was suggested

the effect of these insecticides may be mediated in part by up-regulation of

nAChRs in Kenyon cells leading to mitochondrial dysfunction, memory deficits,

and poor navigation (Fischer, 2014; Williamson, 2013).

To summarize, nAChRs engaged in different physiological functions that are

guided by the variability of their subunits (McGehee and Role, 1995). Many efforts

have been made to determine the role of various nAChR subtypes in the nervous

system but most studies have focussed on the homomeric α7 and heteromeric α4

nAChRs (Koga, 2014; Schedel, 2010; Zhou, 2003). This is reflected in the

increasing number of drugs that target α7 and α4β2, which are under development

for treatment of several neurological conditions. However, the lack of specific

modulators for other nAChR subtypes makes it challenging to elucidate the role of

each subtype under both physiological and pathological conditions. Thus, there is

still a significant need to discover or develop highly selective modulators of

neuronal nAChRs.

1.1.4 Role of nAChRs in human disease

The role of ACh is as important as their molecular target, nAChRs. It is

synthesised by the enzyme choline acetyltransferase that transfers an acetyl

group from acetyl-CoA to choline (Korey, 1951). Its precursor, choline, is

endogenously produced, although it is also absorbed in the small intestine to

reach the levels required to accomplish several functions (Zeisel and da Costa,

2009). For example, choline is a precursor for the biosynthesis of constituents of

plasma membrane such as phosphatidylcholine and sphingomyelin (Zeisel, 1992;

Zeisel and da Costa, 2009). Furthermore, choline is a methyl group donor; and

methylation is needed to regulate of expression of genes and mediate biosynthetic

reactions (Zeisel and da Costa, 2009). A deficiency in choline has been related to

liver damage (Buchman, 1995) and impairment of cognitive functions in older

people (Fioravanti and Yanagi, 2005). Indeed, choline supplementation enhances

cognitive functions in patients with schizophrenia (Knott, 2015). De-regulation of

cholinergic function is linked to the function and expression of nAChRs. For

 

25

instance, chronic administration of nicotine can induce an increase in the number

of neurons containing α4β2 nAChRs in the glutamatergic subthalamic nucleus, a

cerebral area related to movement control (Xiao, 2015), or down-regulate the

function of other subtype nAChRs. Below is discussed different pre-clinical and

clinical evidence of the consequences of smoking, and links between de-regulation

of nAChRs and major symptoms in neurodegenerative diseases.

Post-mortem studies in the brain of people who suffer from Alzheimer’s and

Parkinson’s disease (Rinne, 1991), epilepsy and schizophrenia showed that the

density of α4β2 nAChRs is extremely low. The degeneration of cholinergic circuits

was confirmed in human brains after the development of nicotine radioligands for

imaging studies in vivo. For example, in vivo studies of brains of people suffering

from Alzheimer’s disease showed a decrease of α4β2 nAChRs (Kendziorra,

2010), and the density of nAChRs in arteries appeared to be affected as well

(Bauwens, 2015).

In patients suffering from Alzheimer’s disease and Parkinson’s disease, there is a

notable loss of cholinergic projections into the cerebral cortex, and this has been

considered as part of the reason for the impaired cognition observed in these

patients, although the loss of the cortical cholinergic activity in people with

Parkinson’s disease is notable (for a review see (Müller and Bohnen, 2013)). In

addition, depressive symptoms in patients with Parkinson’s disease is strongly

correlated to loss of α4β2 nAChRs in the neuronal frontal corticomesostriatal

circuitry, while those patients with mild cognitive impairment were associated with

low levels of α4β2 in the neuronal frontal corticobasal ganglia-limbic-cerebellar

circuitry that includes some areas of the former circuitry (Meyer, 2009).

Furthermore, α4β2* nAChRs are widely distributed in the brain and are not

restricted to cholinergic or dopaminergic neurons. Thus, down-regulation of this

heteromeric subtype may lead to dysfunction of other neuronal circuitries (Emre,

2003). Cholinergic deregulation in the hippocampus may be associated with

cognitive problems because this cerebral region is related to functions of learning

and memory (Kutlu and Gould, 2015) and this correlation between dysfunction of

 

26

neuronal circuitry and cognitive functions is observed in various neuronal disorders

(Drevets, 2008).

nAChRs have also been identified in immune cells, and up-regulation of its activity

in this cells has been linked to cardiovascular diseases such as atherosclerosis in

smokers (Aicher, 2003; Egleton, 2009; Wada, 2007). Recent evidence suggests

that the mechanism underlying this process could be activation of α7 nAChRs that

trigger transcription of cell-adhesion proteins on endothelial cells (Alamanda,

2012). These proteins, such as E-selectin, favour the attachment of monocytes,

and it might lead to abnormal formation of fibrous plaques in vascular smooth

muscle cells (Alamanda, 2012). Notably, the duration and amount of nicotine

administered via smoking are dependent variables, and when these reach critical

values it may facilitate the activation of numerous nAChR subtypes (Koga, 2014;

Wada, 2007) that are linked to signalling cascades (Wada, 2007) and cause

abnormal proliferation of neuronal and non-neuronal cells (He, 2014; Wada, 2007).

1.2 MODULATORS OF NACHRS

1.2.1 Modulators of nAChRs as drugs and insecticides

Several clinical trials are being carried out to evaluate potential drugs that target

nAChRs. For instance, some nicotine analogues are been used to facilitate

quitting smoking (Rollema, 2007). Varenicline (trade name Chantix®) is currently

being used for treatment of nicotine addiction and is a partial agonist of the α4β2

and α3* receptors. However, varenicline also activates the α7 nAChR (Mihalak,

2006), and it has been suggested that its mechanism of action involves is by

modulation of several nAChRs (Bordia, 2012; Lotfipour, 2012) that contributes to

decreasing the levels of dopamine that were previously increased by nicotine in

the limbic system (Corrigall, 1992, 1994), thus, preventing the reward response

induced by smoking (Mihalak, 2006; Rollema, 2007). Imaging studies in vivo of

smokers under treatment with varenicline showed that α4β2* nAChRs are

saturated after administration of the drug at low or high doses, and withdrawal

effects were not detected. Unfortunately, varenicline can also cause adverse

effects in smokers under treatment; nausea is very common and is one of the

 

27

primary reasons for withdrawal for treatment (Jorenby, 2006; Lam and Patel,

2007). The side-effect is likely to be mediated by activation of ganglionic nAChRs.

This is likely to be a common problem when using therapeutic agents that target

multiple subtypes of nAChRs distributed across the peripheral nervous system.

As mentioned previously, nAChRs are also important insecticide targets, and the

so-called neonicotinoid insecticides that target insect nAChRs are one of the most

successful classes of chemical insecticides. Nicotinoid insecticides are derived

from nicotine, the major alkaloid derived from leaves and stems of the tobacco

plant Nicotiana tabacum; nicotine is a secondary metabolite that is used by the

plant to defend against herbivorous insects (Isman, 2006). Nicotine was used to

control crop pests as far back as the 1700s (Siegwart, 2015) but its use was

limited because of toxic effects on mammals and only moderate potency against

insects. The neonicotinoids are chemically similar to nicotine but they have low

toxicity to mammals as they were engineered to bind selectively to insect nAChRs

(Brown, 2006). However, like the majority of chemical insecticides, most

neonicotinoids are broadly active against a wide range of insect pests, including

beneficial insects such bees (Déglise, 2002; Faucon, 2005). Honeybees are

efficient pollinators that mediate plant reproduction, and therefore it would be

highly desired to engineer insecticides that preferentially target nAChRs in insect

pests but not beneficial insects such as pollinators or predators of the targeted

pest.

Structure-activity relationship studies are helping to guide the development of

more selective neonicotinoid insecticides. Crystal strictures of the complex formed

between neonicotinoids and AChBP have been used to identify rational methods

of improving the potency and/or selectivity of particular neonicotinoids (Matsuda,

2009). For instance, Matsuda et al. (Matsuda, 2009) compared the complex

formed between imidacloprid, the most successful neonicotinoid, and α2β1

nAChRs from the honeybee Apis mellifera and the green peach aphid Myzus

persicae (Matsuda, 2009). They found a remarkable structural difference between

the binding sites for imidacloprid in the two nAChRs, with a hidden and broader

binding groove in the aphid nAChR compared with the bee receptor. The authors

 

28

suggested that linking imidacloprid to a molecular fragment that fits the groove of

the aphid nAChR could improve the selectivity for insect pests over bees.

To summarise, neonicotinoids are generally safe for humans but they can be toxic

for small vertebrates such as birds and beneficial insects such as honeybees.

Thus, further research is needed to develop more selective neonicotinoids with

reduced ecological impact.

1.2.2 Novel subtype-selective modulators of vertebrate nAChRs are required to develop accuracy pharmacological tools and potential therapeutic leads

The selectivity of some toxins for nAChRs has facilitated the development of

fluorescent binding probes and radioligands for use in biological sciences

(Hannan, 2015; Schmaljohann, 2006) and nuclear medicine (Schmaljohann, 2005,

2006), respectively. It has helped to determine the distribution of nAChRs in the

brain of rodents (Perry, 2002; Rueter, 2006) and humans (Ding, 2004; Kimes,

2006), including in people suffering from Alzheimer’s and Parkinson’s disease

(Schmaljohann, 2005, 2006). For example, α-bungarotoxin a 74-residue peptide

isolated from venom of Bungarus multicinctus, was used to affinity purify the

nAChR from the electric organ of T. marmorata (Changeux, 1970). α-Bungarotoxin

has also been useful for visualizing the distribution of nAChRs in the brain and the

in the electric organ from Torpedo californica, and for identifying clones expressing

epitopes from nAChR in Escherichia coli (Gershoni, 1987). Recently, it was shown

that Alexa Fluor 555-labelled α-bungarotoxin labels GABAA-Rs expressed in HEK-

293 cells (Hannan, 2015), and this binding is inhibited by bicuculline, a competitive

antagonist at GABAA-Rs, and d-tubocurarine, a non-selective nAChRs antagonist

and a competitive antagonist at GABAA-Rs (Caputi, 2003) (see Figure 1.2A-C).

This finding is not surprising because type-A GABAergic receptors are members of

the pentameric Cys-loop ligand-gated ion channel family, like nAChRs (Corringer,

2012). The selectivity of α-bungarotoxin has been used to discover new functional

subunits of nAChRs, such as the α7 subunit of nAChRs. The vertebrate neuronal

subunit α7 was cloned for the first time in 1990 by Couturier et al. (Couturier,

 

29

1990). They found the subunit formed an oligomeric channel in the membrane of

Xenopus laevis oocytes and it was activated by nicotine and choline, and

desensitised rapidly. This was a receptor similar to the heteropentameric nAChR

reported previously because α-bungarotoxin blocked its activation and was able to

bind the subunit α7. Furthermore, iodinated α-bungarotoxin has been used in

competition studies to evaluate the specificity of radioligands towards α7 nAChRs.

Nicotine radioligands are being used for imaging studies using positron emission

tomography (PET) (for a review, see (Bauwens, 2015)). Note that radioligands are

typically used for imaging studies at sub-therapeutic doses, and consequent safety

rarely poses an issue. The first nicotine-based radioligand developed to label

nAChR was [11C]-nicotine (Halldin, 1992) (see Figure 1.3E). It was synthesised at

high purity but had low specificity when tested on human brain in vivo (Nybäck,

1994). Other radioligands that are chemically related to nicotine are still under

development with some promising results, such as [18F]-ZW-104 and [18F]-AZAN-α

(for a review, see (Bauwens, 2015)) (see Figures 1.3F-G). The first probe is a

selective ligand for the β2 subunit of nAChR and has been tested pre-clinically.

[18F]-AZAN-α has been assayed in human studies with promising results (for a

review, see (Bauwens, 2015)). It reached the brain in less than 20 min after

injection, and selectively bound the α4β2 subtype (Wong, 2013). In this study the

specificity for α4β2 was evaluated through administration of varenicline, a

selective partial agonist of α4β2 that is used to aid smoking cessation (Rollema,

2007). The specificity of the radiotracer for the receptor was confirmed after the

drug blocked totally the uptake of [18F]-AZAN-α. Another radio-chemical tracer is

[18F]-flubatine, which is an analogue of epibatidine, an alkaloid isolated from the

skin of the frog Epipodobates tricolor (Fisher, 1994) (see Figures 1.3D,H).

A-85380 is another example of a radiotracer (Kimes, 2006; Sullivan, 1996) that

has high affinity for α4β2 nAChRs (for a review, see (Lotfipour, 2011)). It allowed

mapping of the distribution of nAChRs in the brain of smokers and non smokers

(Kimes, 2006). Furthermore, it has been used to confirm that expression of α4β2

in the brain of patients with Alzheimer’s disease and dementia is significantly

decreased, and is correlated with the degree of cognitive impairment (Colloby,

 

30

2010).

The development of agents to be used for human brain imaging in vivo has been

extended to design of tracers selective for the α7 subtype. Some promising results

has been obtained even where the homomeric α7 in the human brain is found in

low concentrations compared to α4β2. For example, [18F]-NS-10743 and [18F]-

ASEM (see Figure 1.3I-J) showed high affinity toward α7 and the distribution of

these radioligands were found in brain areas where α7 is densely expressed, such

as frontal cortex and hippocampus. To prove the specificity of the agents, the

specific ligand SSR180771, which is a selective and partial agonist at human and

rodent subtype α7, was used in competition studies. The tracers showed their

specificity for α7 when these were displaced by SSR180771.

As discussed above, part of the problem with nAChR modulators as drug leads,

insecticides, and pharmacological tools is their lack of selectivity. This has made it

difficult to dissect the role of nAChRs in different physiological processes (García,

2015) as well as their role in pathophysiological processes such as cognitive

dysfunction, depression, chronic anxiety, analgesia, inflammation and

neurodegenerative diseases. Thus, there is still a great need to develop novel

nAChR modulators that are both potent and selective. As discussed below, animal

venoms have provided some of the most potent and selective nAChR modulators

described to date, and represent a potential source of new nAChR modulators.

 

31

Figure 1.3. Structures of toxins and toxin derivatives that target nAChRs. (A) α-bungarotoxin. Image adapted from https://en.wikipedia.org/wiki/Alpha-Bungarotoxin. (B) Bicuculline. Image from http://halofanon.wikia.com/wiki/Bicuculline. (C) Tubocurarine chloride. Image from https://en.wikipedia.org/wiki/Tubocurarine_chloride. (D) Epibatidine. (E) 11C-nicotine. (F) [18F]-ZW-184. Image from http://www.iphc.cnrs.fr/dirac/productReadHit.php?id=923. (G) [18F]-AZAN-α. Image adapted from (Bauwens, 2015). (H) [18F]-Flubatine. Image adapted from (Bauwens, 2015). (I) [18F]-NS-10743. Image adapted from (Bauwens, 2015). (J) [18F]-ASEM. Image from Horti et al. (Horti, 2014). The pyridine and the pyrrolidine rings are shown in orange and green, respectively. The chemical structure of nicotine is similar to that from epibatidine such as pyridine rings and nitrogen atoms (in orange color). The tracers are shown in blue.

E) F)

A) B) C)

α-bungarotoxin Bicuculline Tubocurarine chloride

[18F]-NS-10743

D)

I) J)

Epibatidine

[18F]-AZEM

N

N Cl

N H3[11C]

N

11C-nicotine [18F]-ZW-104 [18F]-AZAN-α [18F]-Flubatine

N

O

18F G) H)

N

N

N

18F

N N

18F

N N

O 18F N

N N

18F O O

S

 

32

1.3 VENOM-DERIVED MODULATORS OF NACHRS 1.3.1 Important role played by subtype-selective α-conotoxins in study of

vertebrate nAChRs Venoms from marine cone snails (genus Conus) have been extensively studied,

although scarcely 0.1% of the predicted total number of venom compounds have

been characterized so far (Lebbe, 2014b). Conus venoms are replete with

disulfide-bridged peptides, named conopeptides or conotoxins that target a wide

range of voltage and ligand-gated ion channels, including nAChRs (Lebbe, 2014b;

Lewis, 2012; McIntosh, 1999). Indeed, the venoms of marine cone snails are the

largest natural source of potent and selective nAChR blockers (for reviews, see

(Lewis, 2012; McIntosh, 1999)). There are at least seven families α-conotoxins

that bind specific muscle and neuronal nAChR subtypes with high selectivity

(Wang, 2015). α-Conotoxins are the smallest conopeptides (generally 12–20

amino acid residues) that competitively binds to the ACh binding site of nAChRs.

Their general framework is denoted CC-Xm-C-Xn-C, where C represents a

cysteine residue and X corresponds to the two inter-cysteine loops that contain a

variable region of amino acid residues. The number of amino acid residues is

indicated as m/n and α-Conotoxins with a 3/5 loop size combination are usually

purified from the venom of Conus species that feed on fish and are typically active

on mammalian neuromuscular nAChRs, while other conopeptides with 4/3, 4/4,

4/5, 4/6 and 4/7 loop combinations preferentially act on mammalian neuronal

nAChRs (for reviews, see (Lebbe, 2014b; Lewis, 2012; Terlau and Olivera, 2004)).

See table 1.1 below.

 

33

Table 1.1 Amino acid sequences of selected α-conotoxins. SI conotoxin from Conus striatus that targets the neuromuscular subtype cholinergic receptor; MII conotoxin from Conus magus that targets with heteromeric nAChRs with higher affinity than the homomeric subtype α7 nAChR; AuIA conotoxin isolated from Conus aulicus, which targets the neuronal heteromeric subtype α3β4. The asterisks (*) represent C-terminal amidation. Table adapted from (Lewis, 2012; Molgó, 2013). Loop 1 and Loop2 are the two intercystine loops. α-conotoxin Amino acid sequence Selectivity

C Loop 1 C Loop 2 C*

SI (3/5 loop) I CC NPA C GPKYS C* Muscle type α12βγδ

MII (4/7 loop)

G CC SNP V C HLEHSNL C* Neuronal subtype α6α3β2β3>α3β2>α7

AuIA (4/7 loop)

G CC SYP P C FATNSDY C* Neuronal subtype α3β4

Several studies of the interaction between α-conotoxins and AChBP have

contributed to our understanding of how α-conotoxins can be selective for a

particular nAChR (Dutertre, 2007). Basically, the different electrostatic interactions

and hydrogen bonds between aromatic residues of α-conotoxins and amino acid

residues outside of the ACh binding pocket are found in the conotoxins that block

nAChRs while those conotoxins with fewer interaction are more likely to be

agonists (Dutertre, 2004). These studies have also helped develop a structural

model of the pharmacophore of α-conotoxins. It is delimitated by the two loops

between the cysteine residues. One is conserved and determines the binding to

the nAChR and the second loop consists of a variable sequence that determines

their selectivity for a particular subunit of the nAChR (for reviews, see (Lebbe,

2014b; Lewis, 2012; Terlau and Olivera, 2004)).

 

34

1.3.2 nAChR modulators from other venomous animals Snake venom is another large source of proteins and peptides that target nAChRs

(Brahma, 2015; Utkin, 2015). The most abundant proteins described so far in

elapid, colubrid and psammophid snakes are the three-finger toxins (3FTx)

(Sunagar, 2013) that contain between 60 to 74 amino acid residues with molecular

masses ranging between 6000 and 8000 Da (Manjunatha and Robin, 2010; Roly,

2014). Most 3FTxs target nAChRs. These toxins are divided into three types

differentiated by the number of cysteines and the inter-cysteine spacing. Type I

and type III α-neurotoxins with eight cysteine residues target neuromuscular

nAChRs. Type II α-neurotoxins contain an extra pair of cysteine residues which

form an additional disulfide bond that stabilises loop 2 (Fry, 2003; Sunagar, 2013).

This additional disulfide bond facilitates interaction with neuronal α7 and α9-10

nAChRs, while maintaining affinity for the neuromuscular α1 nAChR (Fry, 2003).

Another interesting group of 3FTxs are the so-called “non-conventional toxins” that

have an additional disulfide bond that is not in the central loop II but instead in the

N-terminal loop I (for a review see (Tsetlin, 2015)). Examples of non-conventional

toxins that block nAChRs are candoxin from Bungarus candidus and the weak

toxin (WTX) from Naja Kaouthia.

1.3.3 Rationale for examining spider venoms as a source of novel nAChR modulators Spiders are very known to produce venoms that contain diverse chemical

structures such as peptides up to 10 kDa, proteins, amino acids, and

acylpolyamines (for a review, see (Estrada, 2007)). This complex and

heterogeneous mixture of compounds allows spiders to prey on, and defend

themselves against a wide range of prey and predators. The different repertoire of

acylpolyamines appears to have evolved to allow predation of different species of

invertebrates and small vertebrates.

Acylpolyamines can selectively block ionotropic glutamate receptors from insects

and vertebrates including mammals (for a review see (Olsen, 2011)). Because

glutamate is the main neurotransmitter at the neuromuscular junctions in insects,

 

35

block of glutamatergic receptors by acylpolyamines induces paralysis (Piek, 1971;

Piek and Njio, 1975). However, acylpolyamines toxins are also reported to

antagonise of nAChRs (Willians, 1997). The basic backbone of acylpolyamines is

an aromatic acyl group and the polyamine chain that form the essential part of the

molecule (Estrada, 2007). These two features are generally present in

acylpolyamines derived from funnel-web, trap door and tarantula spiders

(Strømgaard, 2005). The length of the polyamine chain and the degree of

hydrophobicity determines the affinity for nAChRs.

Recently, a toxin denoted VdTX-1 was purified from the venom of the Brazilian

theraphosid spider Vitalius dubius (Rocha-E-Silva, 2013). This component has a

molecular weight of 728 Da and it non-competitively blocks nAChRs. Surprisingly,

this toxin was photosensitive, which might be why this toxin, and perhaps related

toxins in other spider venoms, was not identified in previous studies. In any case,

there is clearly some evidence for the presence of small-molecule nAChR

modulators in spider venoms, suggesting that these venoms should be explored

more systematically as a potential source of novel modulators.

1.4 SIGNIFICANCE AND AIMS OF THE CURRENT WORK 1.4.1 Significance nAChRs are considered a key molecular target for treatment of several

neurological and vascular disorders (Bauwens, 2015; van Enkhuizen, 2015). Key

knowledge about the pharmacology and distribution of nAChRs in the vertebrate

nervous system has been achieved thanks to the use of animal toxins as research

tools. However, we are still a long away from fully understanding of role of different

subtypes of nAChRs in neurons and non-neuronal cells due largely to the lack of

selective pharmacological modulators. Although animal venoms have proved to be

an excellent source of nAChR modulators, most work has been carried out using

venoms from snakes and cone snails, while spiders and scorpions remain largely

unstudied as a potential source of nAChR modulators. Since spiders and

scorpions use their venom to either hunt, or defend against, vertebrates, their

venoms represent a potential source of compounds that can modulate the activity

 

36

of vertebrate nAChRs. Thus, in this thesis, I aimed to explore arachnid venoms for

the presence of compounds that modulate the activity of vertebrate α3* and the

neuronal α7 nAChRs.

1.4.2 Aims Aim 1: Determine the taxonomic penetrance of nAChR modulators in venoms

from the two major infraorders of spiders: the “primitive” spiders (Mygalomorphae)

and “modern” spiders (Araneomorphae)

Aim 2: Isolate and pharmacologically characterize a non-peptide nAChR

modulator identified in venom of the scorpion “Heterometrus spinnifer”.

 

37

CHAPTER 2 — NACHR MODULATORS FROM SPIDER VENOMS 2.1 INTRODUCTION

Venomous animals use their venom to hunt prey and/or protect themselves

against predators (Billen, 2008; Olsen, 2011). Most animal venoms are a complex

mixture of salts, small organic compounds, peptides and proteins (Escoubas,

2006b; Zhang, 2015b), with activity against a wide range of receptors (Inserra,

2013; Wang, 2015) and ion channels (Billen, 2008; DeBin, 1993; Nirthanan, 2002).

Consequently, over the past 20 years, a number of venom molecules have been

purified which have proved useful clinically, to agronomy, or as research tools

(Harrison, 2014; King and Coaker, 2014; King and Hardy, 2013; Lewis, 2012;

Nicke, 2004; Windley, 2012b).

One potentially rich source of compounds active against ligand-gated channels,

which remains largely unexplored, is spider venoms (Nørager, 2014). It is

predicted that all venoms from the estimated 100,000 extant species of spiders

contains approximately 20 million bioactive molecules and scarcely 0.008% have

been characterized so far (for reviews, see (Estrada, 2007; King and Coaker,

2014; King and Hardy, 2013; Windley, 2012b)). This has been due in part to the

small amount of venom that can be obtained from arachnids, making traditional

biochemical characterization challenging. However, the development of high-

throughput functional assays has made it more feasible to functionally characterize

venom from small venomous animals (for a review, see (Vetter, 2011)).

There are two reasons why spider venoms were considered to be a likely possible

source of novel molecules that target human neuronal nAChRs. Firstly, each

venom is estimated to contain several hundred to more than a thousand molecules

in the desired molecular weight range of between 0.1 and 10 kDa (for reviews, see

(Escoubas, 2000, 2006a, b; Estrada, 2007; King and Hardy, 2013)), with most of

those already isolated having been shown to predominantly act on voltage- and

ligand-gated ion channels (for reviews, see (Escoubas, 2000, 2006a, b; King and

Hardy, 2013)). Secondly, even though spider toxins are designed to target mainly

insect receptors (because insects are the main prey of spiders) (Corzo, 2002;

 

38

Vassilevski, 2008), the similarity of invertebrate receptors to their counterparts in

vertebrates (Lansdell, 2012) suggests that modulators of human nAChR receptors

might be found in spider venoms.

Some authors have divided spider toxins into two major groups: acylpolyamines

and cysteine-rich peptide toxins (Liang, 2008; Vassilevski, 2009). The

acylpolyamines were first isolated from theraphosid spiders (for reviews, see

(Estrada, 2007; Strømgaard, 2001) and shown to cause fast paralysis in insects

(Escoubas, 2000) but they are found in many spider venoms (Moore, 2009;

Strømgaard and Mellor, 2004) They are known to block both ionotropic

glutamatergic receptors (Nørager, 2014; Strømgaard and Mellor, 2004) and

nAChR receptors in vertebrates and invertebrates (Mellor and Usherwood, 2004;

Strømgaard, 2001). Their potential therapeutic and insecticidal use has led

researchers to synthesise acylpolyamines (Strømgaard and Mellor, 2004) and

study their structure-activity relationships in detail in order to improve their affinity

for either glutamatergic (Kromann, 2002) or cholinergic receptors (Olsen, 2006).

Recently, a small molecule of 728 Da was isolated from the venom of the

theraphosid spider Vitalius dubius that blocked cholinergic receptors in vertebrate

nerve-muscle preparations (Rocha-E-Silva, 2013). The structure of the molecule

was not determined but its sensitivity to light and its small size suggests that it

likely to be a polyamine.

In addition to polyamines, other small molecules can be found in spider venoms,

such as amino acids (GABA, glutamate, acetylcholine), adenosine triphosphate

(ATP) and adenosine monophosphate (ADP) (for reviews, see (Estrada, 2007;

King and Hardy, 2013; Olsen, 2006, 2011). These molecules are endogenous

modulators of ion channels and receptors both in invertebrates and vertebrates but

their lack of selectivity makes these molecules unsuitable as drug leads (for a

review, see (King and Hardy, 2013)).

Most spider venoms are dominated by high concentrations of small cysteine-rich

peptides (King and Hardy, 2013). Most of these peptide toxins conform to a

structural scaffold known as an inhibitor cysteine knot (Nicke) or "knottin" fold (for

a review, see (King and Hardy, 2013)). Numerous spider-venom ICK peptides that

 

39

target ion channels and receptors have been identified in the venom of both

mygalomorph (Ayroza, 2012; Corzo, 2008) and araneomorph (Trachsel, 2012;

Vassilevski, 2013) spiders. In contrast, there is a small group of spiders whose

venoms contain cytolytic peptides that consist of linear amphipathic domains (for

reviews, see (King and Hardy, 2013; Windley, 2012a). These peptides, which are

devoid of disulphide bridges, have been identified in venoms of araneomorph

spiders from the families Ctenidae, Lycosidae and Oxyopidae (for reviews, see

(King and Hardy, 2013; Kuhn-Nentwig, 2003).

Apart from the discovery of huwentoxin-I (HWTX-I), which blocks vertebrate

cholinergic transmission, from venom of the theraphosid Haplopelma schmidti

(formerly Selenocosmia huwen Selenocosmia (now Ornithoctonus huwena) (Zhou,

1997), there is no evidence of spider-venom peptides with effects on cholinergic

receptors. In this work, a systematic approach was undertaken to screen 71 spider

venoms, extracted from representative species of both “primitive” and “modern”

spiders, for modulators of vertebrate neuronal nAChRs, using a high-throughput

FLIPRTETRA screening approach. It was hoped the data derived from the screening

of spider venoms would identify lead peptide toxins that could be further

characterised as potential therapeutic agents. However, no “hit” peptide toxin

could be identified. Nevertheless, cholinergic modulatory activity was identified in

venoms from a number of species and this data was used to map the taxonomic

distribution of modulators that target human neuronal nAChRs.

2.2 METHODS AND MATERIALS

This chapter describes the strategy followed to identify modulators of nAChRs in

crude spider venoms using a high-throughput FLIPR assay. The protocol used for

the high-throughput FLIPR assay was based on that described by Vetter and

Lewis (Vetter, 2012; Vetter and Lewis, 2010).

2.2.1 Tissue culture reagents

RPMI (Roswell Park Memorial Institute) medium, (+)-L glutamine; DPBS

(Dulbecco’s Phosphate Buffered Saline) without CaCl2 and MgCl2; 0.25 %

 

40

Trypsin/EDTA were from Gibco Life Technologies (Mulgrave, Vic, Australia).

Fetal Bovine Serum (FBS) were from Scientific Life (Clayton, Vic, Australia). PNU-

120596 (1-(5-chloro-2,4 -dimethoxyphenyl)-3-(5-methylisoxazol-3-yl) urea) was

from Sigma-Aldrich. FLIPRTETRA pipet tips, and Calcium-4 no-wash kit were from

Molecular Devices (Sunnyvale, CA, USA).

384-Well cell/imaging microplates (flat-well, clear-bottom black polystyrene) and

384-Well reagent microplates (clear-well, round-bottom polypropylene) were from

Corning® Incorporation (Corning, Lowell, MA, USA). Nicotine and choline were

from Sigma-Aldrich (Castle Hill, New South Wales, Australia), and were prepared

as a stock solution of 100 mM in MilliQ water. The composition of the Physiological

Salt Solution (PSS) used in all the assays was 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, and pH adjusted at 7.4 using NaOH (Vetter 2012).

2.2.2 Spider venoms

Crude venoms were extracted from spiders by Dr Volker Herzig (Institute for

Molecular Bioscience, The University of Queensland) via electrical stimulation of

the chelicerae. Venoms were then lyophilized and stored at –20°C. Before starting

fractionation of venoms using reverse-phase high-performance liquid

chromatography (RP-HPLC), crude venom (1 mg) was dissolved in 5% Buffer B

(90% CH3CN, 0.05% TFA in H2O).

Crude venom from 71 spiders was used in this research (see Appendix I for the list

of spiders whose venom was analysed for nAChR activity using the FLIPRTETRA

system). For the FLIPR screens, four different concentrations of spider venom

were prepared using MilliQ water as diluent. All venoms and toxins were dissolved

in MilliQ water before running the FLIPR calcium assay.

2.2.3 Cell lines and culture

In this project SH-SY5Y cells were used for all FLIPR screens because they are

 

41

reported to endogenously express the neuronal α7 nAChR and the “ganglionic”

nAChRs, named α3β2 and α3β4 (Dajas-Bailador, 2002). To facilitate the

description of these heteromeric receptors, an asterisk (*) is included to denote the

possibility of various β subunits in combinations with α3. The calcium responses

evoked by these subtypes after stimulation with choline and PNU-120596, and

nicotine, respectively, were characterized previously by Vetter and Lewis (Vetter

and Lewis, 2010).

SH-SY5Y human neuroblastoma cells (a kind gift from Victor Diaz, Max Planck

Institute for Experimental Medicine, Göttingen, Germany) were maintained at 37°C

with 5% (v/v) CO2 in RPMI medium which contained 2 mM L-glutamine and 15%

(v/v) FBS. Cells were passaged every 3–5 days using 0.25% trypsin/EDTA at a

dilution of 1:7. The SH-SY5Y cells were passaged a maximum of 10–20 times in

antibiotic-free medium, prior to use for assay. The SH-SY5Y cells used in this

project did not show irregular expression of α3* and α7 nAChRs as the control

calcium responses, which were obtained in each high-throughput screening, were

comparable to the control responses obtained by Vetter and Lewis (Vetter and

Lewis, 2010) during the characterization of α7 and α3* nAChRs expressed in SH-

SY5Y cells.

Two days prior to assay, a confluent culture of SH-SY5Y cells was collected by

trypsinization and resuspended in RPMI with 15% (v/v) FBS, then 20,000 SH-

SY5Y cells per well were seed in uncoated black–walled 384-well imaging plates

and maintained at 37°C and 5% CO2 for 48 h before running the experiment

(Vetter and Lewis, 2010). This time is the optimum to obtain a homogenous

monolayer of cells, thus avoiding problems associated with cellular differentiation

and changes in the levels of endogenous expression of receptors and ion

channels (Vetter, 2012).

2.2.4 Preparation of the Calcium 4-dye

A stock solution of calcium 4-dye was prepared according to the instructions of

Molecular Devices. Briefly, 10 ml of PSS is added to the component A to make a

 

42

10 x concentrated stock solution, then the solution is protected from light with

aluminium foil and stored in aliquots of 100 µL at –20°C. Aliquots can be reused,

at least once, if refrozen at –20°C immediately post-experiment.

2.2.5 High-throughput FLIPR calcium assay

The FLIPR-based calcium-influx assay has been shown to be a good high-

throughput approach to identify and characterize toxins derived from animal

venoms (Inserra, 2013; Jin, 2014; Wang, 2015). In this research project, a FLIPR-

based calcium assay was used to expedite the discovery of bioactive molecules

from complex mixtures of spider venoms.

71 spider venoms were screened using the fluorescent calcium FLIPR assay, and

either agonistic or antagonistic function at α7 and α3* nAChRs was measured

according to the methodology described by Vetter and Lewis (Vetter and Lewis,

2010). Briefly, 20,000 SH-SY5Y cells per well were seeded on uncoated black–

walled-384-well walled imaging plates and maintained at 37°C and 5% CO2 for 48

h before running the experiment. Once a 95% confluent monolayer was obtained,

the RPMI medium was removed from the cell plate by flicking it, the cells were

immediately loaded with Calcium 4-no-wash-dye (Molecular Devices), prepared as

a 1 x solution in PSS as indicated above, for 30 min at 37°C and 5% CO2. A

washing step after medium removal and after dye loading was not required as a

no-wash calcium dye was used and all the control calcium responses elicited by

cholinergic receptors stimulated by nicotine and choline were comparable to the

calcium responses obtained in previous work (Vetter and Lewis, 2010).

It is noteworthy that the 30 min of incubation time is optimal for dye-uptake, and

de-esterification of the acetoxymethyl ester (AM) by endogenous esterases

(Vetter, 2012; Vetter and Lewis, 2010). The cell preparation was always protected

from the light during the incubation to prevent photobleaching of the fluorophore.

After dye loading, the cell plate was transferred to the FLIPRTETRA fluorescence

plate reader and a protocol test signal was executed to obtain an appropriate level

 

43

of fluorescent baseline that is approximately 1,000 arbitrary fluorescence units with

a standard deviation less than 5%. In general, the excitation intensity was adjusted

in 80% and camera gain around 150. All fluorescent calcium responses were

recorded using a ‘two-addition’ protocol and a cooled CCD camera with excitation

wavelength of 470–495 nm and an emission wavelength of 515–575 nm for over a

period of 10 min.

After baseline fluorescence readings were recorded every second for 5 s, variable

amounts of crude venom (2 µg; 1 µg; 0.2 µg; 0.02 µg), venom fractions (1% of

venom fraction in relation to 1 mg of crude venom fractionated by RP-HPLC was

tested) or pure toxin (1% of pure toxin in relation to 1 mg of crude venom dissolved

in MilliQ water was tested) were added to the cell plate (addition 1) and the

calcium response was measured every second for 300 s. At the end of this time,

cells were subsequently stimulated with nicotine (30 µM) or choline (30 µM) +

PNU-120596 (10 µM) (addition 2), and readings continued every second for an

additional 300 s.

From the stock solution of nicotine (100 mM) and choline (100 mM), a working

solution for each agonist was prepared at 120 µM in MilliQ water. The final

concentration of nicotine or choline in each well of the plate that contains the SH-

SY5Y cells was 30 µM.

2.2.6 Activation of α3* nAChR by nicotine

The activation of the α3* nAChR by nicotine (30 µM) was measured using the

‘two-addition’ protocol mentioned above. This protocol is described in Section

2.2.8 and a schematic of the protocol is shown in Figure 2.1A.

The reagent plate with venoms or venom fractions takes a long time to prepare

when testing 71 venoms at different concentrations. To avoid loss of incubation

time once cells were loaded with calcium 4-dye, the reagent plate with venoms or

venom fractions was prepared before loading the cells.

 

44

After loading the SH-SY5Y cells, a fluorescent baseline was recorded for 5 s then

PSS was added to the cells (addition 1), followed by readings each second for

300s. During this recording time, calcium responses are not expected to be

different from the baseline. Finally, nicotine was added (addition 2) to yield a final

concentration in the assay of 30 µM, and the fluorescence was measured for 300

s. At the end of this time there is a transient increase in the fluorescent calcium

signal that corresponds to activation of α3* by the agonist nicotine.

To confirm that the observed calcium responses were elicited by activation of

nAChRs, d-tubocurarine, a non-selective blocker of nAChRs (Wenningmann and

Dilger, 2001), was added to the SH-SY5Y cells before stimulation of α3* by

nicotine. A stock solution of d-tubocurarine was prepared at 100 mM in MilliQ

water and its final concentration in the assay was 30 µM. All FLIPR experiments

included a positive control (calcium response elicited by activation of α3* by

nicotine) and a negative control that corresponds to inhibition of these responses

by d-tubocurarine.

Figure 2.1B shows the ‘two-addition’ protocol that was employed to detect

blockers of the calcium responses elicited by α3* nAChRs. After recording the

fluorescent baseline for 5 s, d-tubocurarine was added to the cells (addition 1),

followed readings each second for 300 s. No calcium responses are expected to

occur during this time. Then, a second fluorescent baseline was read for 5 s,

followed stimulation of α3* by nicotine (addition 2), and the fluorescent calcium

responses were measured for another 300 s. As expected, no increase in the

fluorescent calcium response was detected after nicotine addition. Figure 2.1D

shows a typical recording of the inhibition of nicotine-induced calcium responses

by d-tubocurarine.

The calcium responses caused by stimulation of α3* with nicotine were generally

smaller than those induced by choline. Furthermore, the kinetics of activation and

inactivation were slow and they can be detected on the FLIPR assay without

incubation of the SH-SY5Y cells with PNU-120596 (Figure 2.1C).

 

45

2.2.7 Activation of α7 nAChR by choline in the presence of the positive

allosteric modulator PNU-120596

The α7 nAChRs have fast kinetics of deactivation and long-lasting desensitization

that makes it difficult to measure its activity (Lopez, 1998). An experimental

strategy to detect its activity in SH-SY5Y cells was the addition of PNU-120596 (10

µM) in the Calcium 4-no-wash-dye solution, followed by incubation in SH-SY5Y

cells for 30 min at 37°C and 5% CO2 (Vetter and Lewis, 2010). The stock solution

of PNU-120596 was prepared at 100 mM in MilliQ water and 5% (v/v) DMSO, and

stored in aliquots of 20 µL at –20°C. Aliquots can be reused if refrozen at –20°C

immediately post-experiment.

The activation of the α7 nAChRs by choline (30 µM final concentration) was

measured basically using the same protocol as that for the study of the α3*

subtype. Briefly, after reading the fluorescent baseline for 5 s, PSS was added

(addition 1) to the cells and fluorescence measurements were made for a further

300s. Then, the agonist choline was added (addition 2) to stimulate the α7

nAChRs, followed by fluorescence measurements for another 300 s. As expected,

the activation of fluorescence nAChRs by choline elicited an increase in the

fluorescent calcium response that corresponds to an increase in the concentration

of intracellular calcium.

A typical trace of increase of intracellular calcium after stimulation of α7 nAChR by

choline is shown in Figure 2.1E. The positive allosteric modulator PNU-120596 (10

µM) was included in assays to delay the deactivation kinetics of α7 nAChR and

allow recording of the fluorescent Ca2+ responses (Vetter and Lewis, 2010).The

calcium responses are mostly caused by activation of α7 when PNU and choline

were used, although subtypes α3β2/α3β4/α5 are also activated and they were part

of the calcium response (Vetter and Lewis, 2010).

The same ‘two-addition’ protocol was used to confirm whether the calcium

responses were elicited by α7 nAChR. Briefly, d-tubocurarine (30 µM final

concentration) was added to the cells (addition 1) and the fluorescent calcium

 

46

response was read for 300 s, then choline was added (addition 2) to stimulate the

receptor, and a final reading of the fluorescence was carried out for another 300 s.

As expected, d-tubocurarine blocked the calcium response elicited by activation of

α7 nAChR (Figure 2.1F). The following Section 2.2.8 provides an explanation of

how the FLIPR assays were used to identify nAChR blockers and agonists in

arachnid venoms.

2.2.8 Identification of blockers and agonists for α3* and α7 nAChR using

the FLIPRTETRA system A ‘two-addition’ protocol was used to detect the presence of α3* and α7 blockers

in venoms or venom fractions. Crude venoms, venom fractions, or pure toxin were

added first to the cells, and then the fluorescence was measured for 300 s. Those

venoms or venom fractions that contain blockers inhibited the calcium responses

elicited by either α3* or α7 when they were stimulated by nicotine or choline

(addition 2), respectively.

On the other hand, an agonist compound contained in the venoms or venom

fractions caused an increase in the fluorescent calcium response before

stimulation of the receptors by agonists (addition 2). Inhibition by d-tubocurarine

(30 µM) was used to confirm that the observed calcium response was caused by

activation of the receptor rather than activation of voltage-gated calcium channels

(Sousa, 2013) or other endogenously expressed GPCRs or ion channels coupled

to an increase in intracellular Ca2+.

 

47

Figure 2.1. Basic two-addition FLIPR protocol to measure calcium responses evoked by activation of α7 or α3* nAChRs. (A) Schematic of the protocol to measure calcium responses caused by activation of the heteromeric α3* nAChR: SH-SY5Y cells were plated onto a 384-well plate and loaded with calcium 4-dye after discarding the RPMI medium. The method is based on two additions, and control experiments were always included in every assay. PSS or test compound was added first to the SH-SY5Y cells and the fluorescence measured for 300 s, then the α3* nAChR or α7 nAChR were stimulated by nicotine or choline (addition 2), respectively, and the resulting calcium responses were measured for another 300 s. (B) Schematic of protocol used to confirm that the calcium responses were elicited by activation of nAChRs. If the calcium responses observed in (A) were elicited by activation of α3* or α7 nAChRs they should be inhibited when d-tubocurarine (addition 1) was added before agonist addition, nicotine or choline, respectively. (C) Calcium responses recorded by the FLIPRTETRA system when α3* nAChRs were stimulated by nicotine (addition 2). (D) The calcium response was elicited by the activation of α3* because the addition of d-tubocurarine (addition 1) inhibited the calcium response elicited by the receptor when it was stimulated by nicotine (addition 2). (E) A typical calcium response elicited by activation of α7 nAChR using choline as agonist (addition 2). (F) The calcium response elicited by activation of subtype α7 nAChR using choline was inhibited when d-tubocurarine was added before the addition of choline (addition 2). The traces of control calcium responses are an average of one experiment with n = 4.

Ca2+dye+

SH(Sy5Y+cells+

addition+1+ addition+2+

PSS+ agonist+

A)#

Ca2++dye+

SH(Sy5Y+cells+

addition+1+ addition+2+

d(tubocurarine+ agonist+

B)#

0"

1"

2"

3"

4"

1" 151" 301" 451" 601"Maxim

un(calcium

(respon

se(

(RLU

)(

5me((seconds)(

addi5on(1((PSS)(

addi5on(2((Nico5ne)(

C)(

0"

1"

2"

3"

4"

1" 151" 301" 451" 601"Maxim

un(calcium

(respon

se(

(RLU

)(5me((seconds)(

addi5on(1((d=tubocurarine)(

addi5on(2((Nico5ne)(

D)(

0"

2"

4"

6"

8"

1" 151" 301" 451" 601"Maxim

un(calcium

(respon

se(

(RLU

)(

5me((seconds)(

F)(

addi5on(1((d=tubocurarine)(

addi5on(2((Choline)(

0"

2"

4"

6"

8"

1" 151" 301" 451" 601"Maxim

un(calcium

(respon

se(

(RLU

)(

5me((seconds)(

addi5on(2((Choline)(

E)(

addi5on(1((PSS)(

 

48

Figure 2.2. ‘Two-addition’ FLIPR protocol used to identify nAChR agonists. (A) Venom was added (addition 1), then fluorescent responses were measured for 300 s. After the cells were stimulated by nicotine or choline (addition 2), fluorescence measurements were continued for another 300 s. If any venom compound contains a blocker, the fluorescent calcium response induced by nicotine or choline (addition 2) will be inhibited. (B) If any venom compound contains an agonist there will be an increase in the fluorescence after addition 1. d-tubocurarine (30 µM) is added during the loading process, then venom is added (addition 1), if the following response is inhibited then the response was caused by activation of nAChRs. Finally, addition 2 occurs, followed a second reading for 300 s. (C) 1 µg of crude venom from H. hainanum was added (addition 1) to the SH-SY5Y cells, then the calcium fluorescent response was measured for 300 s. After choline was added (addition 2), the fluorescent signal was measured for other 300 s. (D) To confirm that the increase in fluorescence after venom addition was caused by cholinergic activity, d-tubocurarine (30 µM) was included in the Calcium 4-dye solution, and added to the cells. Then, the crude venom (H. hainanum) was added (addition 1) and the fluorescent response was measured. The response was inhibited, and after choline addition (addition 2) the response remained inhibited. The venom was assayed in the presence of PNU-120596 (10 µM). The traces shown in panels (C) and (D) are averages with n = 3.

0

1

2

3

1 151 301 451 601

Max

imun

calc

ium

resp

onse

(RLU

)

time (seconds)

crude venom H. hainanum 1 µg addition 1

0

1

2

3

1 151 301 451 601

Max

imun

calc

ium

resp

onse

(RLU

)

time (seconds)

crude venom H. hainanum 1 µg addition 1 choline 30 µM

addition 2 choline 30 µM addition 2

C) D)

Ca2+dye(

SH+Sy5Y(cells(

addition(1( addition(2(

Venom/(fraction(venom( agonist(

Ca2+(dye((+blocker(

addition(1( addition(2(

agonist(Venom/(fraction(venom(

A)( B)(

 

49

2.2.9 Data analysis

The 71 spider venoms were tested in triplicate in at least three independent

experiments. All data was analysed as followed: Raw fluorescence values

obtained after venom addition were subtracted from the baseline fluorescence

values measured during the reads between 0 and 5 s after starting the assay. In

addition, the raw data readings obtained after stimulation of the receptors by

nicotine or choline were subtracted from the baseline raw fluorescent values

registered between 300 and 305 s. Then, the resulting raw fluorescent reads were

converted to maximum calcium responses by calculating in relative light units the

maximum values reached over the baseline reads using the ScreenWorks,

software, version 3.1. Once the data was corrected using the baseline fluorescent

reads, it was exported and analysed using the software Prism, version 5.0.

Data are expressed as mean ± standard error of the mean (SEM) from n = 3

independent experiments or as the mean ± SEM of the values obtained in the

three independent experiments with a total n = 9.

FLIPR data were normalized to the maximum fluorescence calcium response

evoked by activation of α3* nAChR with nicotine (30 µM) or by activation of α7

nAChR with choline (30 µM) and indicated as FMax (%)

The fluorescent traces from the FLIPR data are reported as calcium response over

baseline that is corrected previously by subtracting the average of the

fluorescence elicited by the negative control (only components of buffer PPS)

using ScreenWorks software. This eliminates the artefacts caused by buffer

addition.

2.2.10 Statistical Analyses

Comparative analysis of the mean of calcium responses induced by crude venoms

of each species on α3* and α7 nAChRs versus control responses was carried out

 

50

through a standard one-way ANOVA and non-parametric statistical analysis, using

the analysis tool of GraphPad Prism®, version 5.0.

2.3 RESULTS

A high-throughput FLIPR screen was carried out to examine the effect of 12

araneomorph and 59 mygalomorph venoms (see Appendix I, Table 1-2 for a list of

species) on the calcium responses evoked by activation of α3* and α7 nAChRs

endogenously expressed in SH-SY5Y cells. Following the initial screen, at least

two additional screens were performed to enhance the statistical significance of

the data, although the data for each species was highly reproducible, indicating

that the venom composition of each species is constant across different pools of

venom. To facilitate analysis of the 71 spider venoms, the resulting data from

araneomorph spiders is presented first in accordance with an Araneae

phylogenetic tree. The second group corresponds to mygalomorph spiders, and

the data was compared in accordance with the geographical origin (state and

country) of each species. Then, common and different elements between both

suborders were described and later discussed (see discussion section) to

determine possible mechanisms of evolution of modulators for vertebrate neuronal

cholinergic receptors.

2.3.1 Screening of 71 spider venoms using the high-throughput FLIPRTETRA system

In all cases, unless specified, 2 µg of crude venom from each spider was tested

against α3* and α7 nAChRs endogenously expressed in human SH-SY5Y cells.

For the screening of these 71 spider venoms, a two-addition protocol was used

(for further details see the methods section).

2.3.2 Screening of venoms from “modern” spiders (Infraorder Araneomorphae)

A phylogenetic tree of the infraorder Araneomorphae is shown in Figure 2.3, each

superfamily is indicated on the main branches of the tree, and the name of each

 

51

representative spider considered in this study is indicated beside the superfamily

name.

To describe the results obtained with araneomorph spider venoms, the spiders

were group in three groups, and are described in order of evolutionary

appearance. The first group corresponds to the superfamily Austrochiloidea, and

the one species from this taxon is Hickmania troglodytes, found in Tasmania. The

spider makes and hangs beneath extensive sheet webs, and occasionally wraps

prey after biting. The crude venom of H. troglodytes at 2 µg did not modify the

calcium responses evoked in SH-SY5Y cells by the agonists nicotine and choline

(Figure 2.3A).

According to the tree, the superfamily Araneoidea appeared in the Cretaceous 145

million years ago after the family Austrochiloidea. Crude venoms from four

exemplars of the superfamily Araneoidea were used in this work: Cyrtophora

moluccensis, Eriophora transmarina, Nephila pilipes and Nephila plumipes. This is

a large taxon that consists of approximately 29% of the spiders described so far

(Griswold, 2005). Venom from E. transmarina and the two Nephila species

blocked both the α3* and α7 nAChRs with high potency whereas C. moluccensis

specifically blocked only the heteromeric α3* nAChRs (Figure 2.3A). This venom

had no effect on the choline response (Figure 2.3B), indicating that blockers are

likely to target mainly the α3*. In contrast, N. pilipes and N. plumipes venom had

equally potent inhibitory effects on α3* and α7 nAChRs, see Figures 2.3A and

2.3B, respectively.

 

52

Figure 2.3. Screening of venoms from “modern” spiders against human α3* and α7 nAChRs using a high-throughput FLIPR assay. The left panel shows a phylogenetic tree of the infraorder Araneomorphae. The name of the spider species whose venoms were used in this study is denoted next to the branch of each family. The tree was adapted from (Coddington and Levi, 1991); ma = millions of years. On the right panel is shown: A) 2 µg of crude venoms of the twelve araneomorph spiders were assayed in the absence of PNU-120596 (10 µM). B) 2 µg of venoms from the same araneomorph spiders were assayed in the presence of PNU-120596 (10 µM). Venom from H. troglodytes (Family Austrochiloidea) had no effect on α3* and α7 nAChRs in the absence and presence of PNU-120596, respectively. Venoms from E. transmarina, N. pilipes, and N. plumites (Superfamily araneoidea) had a potent blocking effect on α3* and α7 subtypes with the exception of venom from C. moluccensis that only had potent blocking effect on α3* nAChRs in the absence of PNU-120596. As seen on the phylogenetic tree (left panel), spiders from the RTA clade evolved after the araneoid spiders; and venoms from Sparassidae sp. (1) and (2), and Lycosidae sp. showed a partial blocking effect on α3* and α7 subtypes without and with PNU-120596, respectively, while venoms from H. jugulans, and Ancylometes sp. had no effect on nAChRs, although venom from V. sylvestriformis only had a partial blocking effect on α3* nAChRs in the absence of PNU-120596. Venom from M. australianus blocked completely α3* and α7 nAChRs in the absence and presence of PNU-120596, respectively. The data on panel A was normalized in relation to the maximum calcium response elicited with nicotine addition (30 µM) (positive control) in the absence of PNU-120596 (10 µM) and expressed in percentage while data from panel B was normalised in relation to the maximum

Austrochiloidea.

Araneoidea.

RTA.clade.

Dionychans.

Lycosidae.

ENTELEGYNAE(

ARAN

EOMORPHAE((

(“modern”(SPIDERS)(

Pisauridae.

Agelenidae.

Ctenidae.

Sparassidae.

Therididae.

Nephilidae.

H.#Jugulans#

Sparassidae#sp.1#Sparassidae#sp.2#

Lycosidae#sp.##

Ancylometes#sp.#V.#sylvestriformis#

M.#australianus#

H.#troglodytes#

C.#Moluccensis##E.#transmarina#N.#Pilipes#.N.#plumites#

ma.

200.

145.

65.

2.

Jurassic( Cretaceous(

d-tuboc

urarin

e (30

µM)

H. trog

lodyte

s

C. molu

ccensis

E. tran

smari

na

N. pilip

es

N. plumite

s

Sparas

sidae

sp. 1

Sparas

sidae

sp. 2

Lycosid

ae sp.

H. jugu

lans

Ancylom

etes s

p.

V. sylve

strifo

rmis

M. austr

alian

us

0

50

100

150

2 µg crude venom / araneomorph spider

Nor

mal

ized

calc

ium

resp

onse

(%)

PNU-120596 (10 µM)

********

****

*** ****

*

****

No effect on nAChRs

Partial effect on nAChRs

RTA CladeB)

d-tuboc

urarin

e (30

µM)

H. trog

lodyte

s

C. molu

ccensis

E. tran

smari

na

N. pilip

es

N. plumite

s

Sparas

sidae

sp. 1

Sparas

sidae

sp. 2

Lycosid

ae sp

.

H. jugu

lans

Ancylom

etes s

p.

V. sylve

strifo

rmis

M. austr

alian

us

0

50

100

150

2 µg crude venom / araneomorph spider

Nor

mal

ized

cal

cium

res

pons

e (%

)

***************

**

****

*

**

Partial effect on nAChRs

**

No effect on nAChRs

RTA CladeA)

 

53

calcium response elicited with choline addition (30 µM) (positive control) in the presence of PNU-120596. The negative control corresponds to the inhibition of the calcium response of nAChRs by d-tubocurarine addition at 30 µM in the absence or presence of PNU-120596 (10 µM). The values are averages of three independent experiments with n = 3. Dotted lines represent the control response in the absence of venoms. Using the unpaired t-test, the means between the effect of each venom and the control response without venom were compared. The number of (*) indicates the degree of significance at P < 0.05 probability level. Those values that lack of significance do not have the symbol (*). The third group of spiders used herein belong to the Retrolateral Tibial Apophysis

(RTA) clade, and venoms from six exemplars of this group were assayed. Table

2.1 list these species and their geographic distribution. Most species are from

Australasia, although the two ctenid species (Ancylometes sp. and Viridasius

sylvestriformis) are from Central and South America. Of the six venoms tested, the

two sparassid venoms (Sparassidae sp. 1 and Sparassidae sp. 2), and the one

lycosid venom (Lycosidae sp.) showed to have a partial blocking effect on α3* and

α7, while one sparassid venom (H. jugulans), and the two ctenid venoms had no

effect on vertebrate α3* and α7 nAChRs, although venom from V. sylvestriformis

had a partial blocking effect on α3*. In striking contrast, venom from the giant

water spider Megadolomedes australianus (family Pisauridae) blocked both

nAChR subtypes with high potency.

 

54

Table 2.1. Taxonomic distribution of nAChR modulators from araneomorph spider venoms. Two species Sparassidae sp. were assayed; the numbers 1 and 2 indicate they were collected in two different areas in PNG. Venoms with high blocking effect are indicated with two symbols denoted as √√, while those with partial effect are indicated with one √. The green area indicates that other effects on the venom is not observed.

Spider Effect on human neuronal α3* and α7 nAChRs Block of α3*

& α7 Block of α3*

No effect

Location

Infraorder Araneomorphae Family Austrochiloidea Hickmania troglodytes

√ Tasmania, Australia

Family Araneoidea Cyrtophora moluccensis

√√ Gardens across South East and coast from Queensland, Northern and western Australia

Eriophora transmarina

√√ Gardens across eastern and southern Australia

Nephila pilipes √√ Gardens Northern Australia, PNG, China, Japan

Nephila plumites √√ Sydney region RTA Clade Family Sparassidae Heteropoda jugulans

√ Queensland, Australia

Sparassidae sp. 1

√ Papua New Guinea

Sparassidae sp. 2

√ Papua New Guinea

Family Ctenidae Ancylometes sp. √ Central and South

America Viridasius sylvestriformis

√ Amazon, South America

Family Lycosidae Lycosidae sp. √ Western Australia Family Pisauridae Megadolomedes australianus

√√ South Eastern Australia

 

55

2.3.3 Screening of venoms from “primitive” spiders (Infraorder Mygalomorphae)

Mygalomorphs are the oldest group of spiders. They are known as “primitive”

spiders because they retain some ancestral morphological features, such as

downward pointing chelicera (Coddington and Levi, 1991). In this study, venoms

were derived from 59 mygalomorph spiders. The infraorder Mygalomorphae is

comprised of 15 taxonomic families (Bond, 2012), of which two were considered

here, namely Hexathelidae (1 species) and Theraphosidae (58 species).

Four mygalomorph venoms were found to contain nAChR agonists (Section 2.9.4),

24 venoms selectively blocked α3* nAChRs (Section 2.9.5), 26 venoms inhibited

both α3* and α7 nAChRs (Section 2.9.6), and four mygalomorph venoms were

found to have no effect on α3* or α7 nAChRs (Section 2.9.7). Each of these four

groups is discussed in the following sections.

2.3.4 Identification of α7/α3* nAChR agonists in the venom of theraphosid

spiders

The two-addition protocol was used to identify potential agonists of vertebrate

nAChRs. Addition of venom (1 or 2 µg per well) from Haplopelma hainanum,

Pamphobeteus sp., Sericopelma rubronites and Xenesthis sp. caused an

increased calcium response in the FLIPR assay both in the absence and presence

of PNU (Table 2.2). Based in a previous FLIPR assay performed by Dr Vetter

(data not showed), the fluorescent responses evoked by 1 µg of venom from H.

hainanum, Pamphobeteus sp., Sericopelma rubronites and in the absence of PNU

were not abolished with addition of d-tubocurarine, indicating that these venoms

lack of agonists of α3* nAChRs. Interestingly, the same venoms at 1 µg were

potentiated in the presence of PNU, although were no assayed in the presence of

d-tubocurarine (data not showed). Based on these previous results, I evaluated if

the calcium responses evoked for the same venoms at 1 µg and in the presence

of PNU were related to activation of the α7 nAChRs (Table 2.3). The venom

responses from H. hainanum, Pamphobeteus sp., S. rubronites, and Xenesthis sp.

 

56

were assayed with the previous addition of d-tubocurarine (Figure 2.4A-D). The

traces derived from the FLIPRTETRA system are presented to show the strategy

used to identify agonists in these venoms. After crude venom addition (addition 1)

at 1 µg, there was an increase of the calcium response that was extinguished at

the end of the 300 s (Figure 2.4B-D) although the venom from H. hainanum

caused an increase of the fluorescence that was sustained during this period of

time (0–300 s) (Figure 2.4A). Then, after choline addition (addition 2), the calcium

response was not different from that evoked by stimulation of the α7 nAChRs by

choline (positive control) (Figure 2.4B-D). Interestingly, venom from H. hainanum

caused complete inhibition of the calcium response evoked by choline stimulation

of the α7 nAChR (see Figure 2.4A). A further analysis was not carried out to

determine if the differences of calcium responses in the presence of PNU and

addition of d-tubocurarine were statistically significant due to the limitation of

amount of these venoms, although the venom from H. hainanum is likely to have

an agonist compound.

 

57

Figure 2.4. Agonism of human nAChRs by theraphosid venoms. All traces are averages of the maximum calcium responses over baseline (with n = 3) elicited by crude venom from (A) Haplopelma hainanum, (B) Pamphobeteus sp., (C) Sericopelma rubronites, and (D) Xenesthis sp.. All responses elicited between 5 s and 300 s are caused by venom addition. The responses after choline addition were measured between 300 s and 600 s. All crude venoms were tested at 1 µg/well in the presence of PNU-120596 (10 µM). Dotted lines of each graphic correspond to averages of calcium responses over baseline (with n=3) elicited by the venoms in the presence of d-tubocurarine (30 µM) and PNU-120596 (10 µM).

0

1

2

3

1 151 301 451 601

Max

imun

cal

cium

res

pons

e (R

LU)

time (seconds)

crude venom Xenesthis sp. 1 µg + PNU-120596 (10 µM)

crude venom Xenesthis sp. 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

time (seconds)

0

1

2

3

1 151 301 451 601

Max

imun

calc

ium

resp

onse

(RLU

)

time (seconds)

crude venom S. rubronitens 1 µg + PNU-120596 (10 µM) crude venom S. rubronitens 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

0

1

2

3

4

1 151 301 451 601Max

imun

calc

ium

resp

onse

(R

LU)

time (seconds)

crude venom Pamphobeteus sp . 1 µg + PNU-120596 (10 µM) crude venom Pamphobeteus sp. 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

0

1

2

3

1 151 301 451 601

Max

imun

cal

cium

resp

onse

(R

LU)

time (seconds)

crude venom H. hainanum 1 µg + PNU-120596 (10 µM) crude venom H. hainanum 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM) A)#

C)# D)#

B)#

 

58

Table 2.2. Apparent agonistic effect caused by venoms from four theraphosid spiders. After venom addition, the fluorescent calcium responses evoked by nAChRs in the absence or presence of PNU were converter into area under curve using ScreenWorks software, version 3.1. The values are averages of three independent experiments with n = 3. The mean of the control nicotine response was 509 ± 50.40 while the control calcium response elicited by choline was 1088 ± 38.31 Spider Calcium response

over baseline after venom addition at 2 µg

Calcium response over baseline after venom addition at 2 µg when PNU-120596 (10 µM) was included in the assay

Haplopelma hainanum

345.6 ± 136 822.2 ± 127

Pamphobeteus sp. 542.2 ± 164.2 517 ± 179 Sericopelma rubronites

241.1 ± 101.5 696 ± 137

Xenesthis sp. 325.5 ± 151.4 678.8 ± 80 Table 2.3. Mygalomorph spiders with venoms that are likely to contain agonists of α7 nAChRs. N.D indicates the effect was not determined and a further analysis is required to confirm that the venom effect is via activation of α7 nAChRs in the presence of PNU.

Location and affect on human α7 nAChRs Spider Agonistic effect Location Haplopelma hainanum √ Southern China and

Vietnam Pamphobeteus (platyomma) sp. (F)

N.D Ecuador

Sericopelma rubronites (F) N.D Panama Xenesthis sp. (F) “white colour”

N.D Colombia

2.3.5 Venoms that selectively inhibit vertebrate α3* nAChRs

24 theraphosid venoms were found to selectively block human α3* nAChRs

(Figure 2.5A). The 24 species are listed in Table 2.4 along with their geographic

distribution. These venoms did not have a substantial effect on calcium responses

in the FLIPR assay when PNU-120596 was included (Figure 2.5B).

 

59

Figure 2.5. Theraphosid spider venoms that preferentially block α3 nAChRs. First screening of venom from “primitive” spiders against human α3* and α7 nAChRs was carried out by using a high-throughout calcium assay. The calcium responses elicited by crude venom was calculated as the area under fluorescence curve normalized to the maximum α3* response evoked by nicotine (A) or stimulation of α7 by choline (B). Each value corresponds to averages of normalized calcium responses over baseline of at least three independent experiments and n = 3. All venoms were tested at 2 µg/well, in the absence (A) and presence (B) of PNU-120596. For a species identification see Table 2.4. Dotted lines represent the control response in the absence of venoms. On graphic A and B, the control responses corresponds to stimulation of α3* with nicotine (30 µM), and α7 choline (30 µM), respectively. Using one-way ANOVA, the means between each venom and the control responses (nicotine response were compared). The number of (*) indicates the degree of significance at P < 0.05 probability level. Those values that lack of significance do not have the symbol (*).

Table 2.4. Theraphosid spiders with venoms that selectively block α3* nAChRs. Species with characteristic colors are indicated. (M) and (F) refer to gender. The numbers beside the name of each theraphosid spider indicates that they are individuals from the same species, although were collected from distinct localities. Number Theraphosid Geographic distribution

1-A Avicularia metallica (F)

Guyana (coast), Suriname (coast)

2-B Avicularia ulrichea (F) All Brazil

3-C Avicularia sp. 1 (M) “purple” Peru-Amazonas

4-D Coremiocnemis tropix Australia

d-tu

bocu

rarin

e1-

A2-

B3-

C4-

D5-

E6-

F7-

G8-

H 9-I

10-J

11-K

12-L

13-M

14-N

15-O

16-P

17-Q

18-R

19-S

20-T

21-U

22-V

23-W

24-X

0

50

100

150

Nor

mal

ized

cal

cium

resp

onse

(%)A)

**** ****

****

**** *******

********

**

****

****

**********

*******

*****

******

****

****

***

****

****

d-tu

bocu

rarin

e1-

A2-

B3-

C4-

D5-

E6-

F7-

G8-

H 9-I

10-J

11-K

12-L

13-M

14-N

15-O

16-P

17-Q

18-R

19-S

20-T

21-U

22-V

23-W

24-X

0

50

100

150

Nor

mal

ized

cal

cium

resp

onse

(%) PNU-120596 (10 µM)

****

****

*******

B)

 

60

5-E Chilobrachys sp. 1 Malaysia-Penang island

6-F Grammostola grossa 8 (F)

Pampas from Argentina and Paraguay. Brazil south and north of Paraguay

7-G Grammostola iheringi (M) Brazilian south, Entre Rios

8-H Euathlus sp. 1 (F) “green” Chile

9-1 Euathlus sp. 2 (F) “red” Chile

10-J Euathlus vulpinus (F) “crème” Chile highland

11-K Orphnaecus philippinus

Philippines

12-L Orphnaecus sp. Philippines-Paqnai-island

13-M Paraphysa scrofa (F) Chile

14-N Pelinobius muticus West Papua New-Guinea (PNG)

15-O Poecilotheria striatus Sri Lanka, India

16-P Pterinuchilus murinus

Central, Eastern, Southern Africa

17-Q Selenocosmia crassipes

Australia-East Coast Queensland

18-R Selenocosmia effera East PNG

19-S Selenocosmia sp. 1 PNG

20-T Selenocosmia sp. 2 PNG

21-U Selenotholus foelschei Australia

22-V Thrixopelma cyaneolum (F) Peru

23-W Thrixopelma sp. (F) Peru – Cajamarca

24-X Thrixopelma lagunas sp. (F)

Peru – Lagunas

 

61

2.3.6 Venoms that non-selectively block both α3* and α7 nAChRs

26 theraphosid venoms were found to non-selectively block both α3* (Figure 2.6A)

and α7 nAChRs (Figure 2.6B). The 26 species from which these venoms were

obtained are listed in Table 2.5 along with their geographic distribution.

Figure 2.6. Many theraphosid venoms non-selectively block both α3* and α7 nAChRs. Normalised calcium responses obtained from FLIPR screen of venoms in the absence (A) and presence (B) of PNU-120596. Each value corresponds to averages of normalized calcium responses over baseline of at least three independent experiments and n = 3. For a list of species see Table 2.5. Dotted lines represent the control response in the absence of venoms. On graphic A and B, the control responses corresponds to stimulation of α3* with nicotine (30 µM), and α7 choline (30 µM), respectively. Using one-way ANOVA, the means between each venom and the control responses (nicotine response were compared). The number of (*) indicates the degree of significance at P < 0.05 probability level. Those values that lack of significance do not have the symbol (*).

d-tub

ocura

rine1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021 22 23 24 25 26

0

50

100

150

Nor

mal

ized

cal

cium

resp

onse

(%)

***

A)

********* ********* ************************

********* ************

***

************

d-tub

ocura

rine1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

0

50

100

150

Nor

mal

ized

cal

cium

resp

onse

(%)

PNU-120596 (10 µM)

* ***********

*

***

**** ********

B)

****

****

****

*

**

***********

********

******** ********

****

****

 

62

Table 2.5. Theraphosid spiders with venoms that non-selectively block both α3* and α7 nAChRs. (M) and (F) refer to gender. The numbers beside the name of each theraphosid spider indicates that they are individuals from the same species, although were collected from distinct localities.

Number Theraphosid species Location

1 Acanthoscurria cf insubtilis (F) Peru, Bolivia

2 Ceratogyrus bechunanicus

Namibia, Zimbabwe, Mozambique, South Africa

3 Ceratogyrus ezendami Mozambique

4 Ceratogyrus marshalli Zimbabwe, Mozambique

5 Chilobrachys sp. 2 North India-Meghalaya (State)

6 Eupalaestrus campestratus (F)

Argentina, Brazil, Paraguay

7 Ephebopus uatuman (F) Argentina, Brazil, Paraguay

8 Grammostola porteri (F) All Chilean coast

9 Grammostola rosea (M) Bolivia, Argentina, and all Chilean coast

10 Grammostola sp. 1 (F) (Chilean North) 11 Grammostola sp. 2 (F) Concepcion, Chile 12 Grammostola sp.3 (F) Maule River, Chile 13 Holothele sp.1 (F) Carabobo, Colombia 14 Holothele sp. 2 (F) Peru 15 Idiothele nigrofulva South Africa 16 Lasiodorides striatus (F) Brazil 17 Lasiodora striatipes (F) Brazil

18 Nhandu chromatus 5 (F) Savannahs from Brazil and Paraguay

19 Nhandu tripepii (M) State Para, Brazil 20 Nhandu vulpinus Brazilian South

21 Phormictopus cancerides (F)

Dominican Republic, Cuba, Haiti

22 Pamphobeteus cf petersi Ecuador, Peru

23 Psalmopoeus cambridgei (F) Trinidad and Tobago

24 Psalmopoeus langenbucheri (F) Venezuela

25 Psalmopoeus pulcher Panama

 

63

(F) 26 Theraphosinae sp. (F) Chile

2.3.7 Theraphosid venoms with no apparent effect on nAChRs Figure 2.7 summarizes FLIPR data obtained with venom from four American

theraphosids (Aphonopelma crinitum, Avicularia sp., Homoeomma sp. and

Pseudhapalopus sp.; Table 2.6). These venoms had only a minor effect on α3*

nAChRs (Figure 2.7A) and no effect on α7 nAChRs (Figure 2.7B).

Figure 2.7. Four tarantula venoms without potent modulators of α3* or α7 nAChRs. Normalized FLIPR responses resulting from a screen of crude venom against human α3* (A) and α7 nAChRs (B). All venoms were tested at 2 µg/well. Each value corresponds to averages of normalized calcium responses over baseline of at least three independent experiments and with n = 3. For legend see table 2.5. Dotted lines represent the control response in the absence of venoms. On graphic A and B, the control responses corresponds to stimulation of α3* with nicotine (30 µM), and α7 choline (30 µM), respectively. Using one-way ANOVA, the means between each venom and the control responses (nicotine response were compared. The number of (*) indicates the degree of significance at P < 0.05 probability level. Those values that lack of significance do not have the symbol (*).

d-tub

ocura

rine (

30 µM)

Aphon

opelm

a crin

itum

Avicula

ria sp

. 2

Homoe

omma s

p.

Pseuda

phalo

pus s

p.

0

50

100

150

Nor

mal

ized

resp

onse

(%)

*** **** ****

*

****

A)

d-tub

ocura

rine (

30 µM)

Aphon

opelm

a crin

itum

Avicula

ria sp

. 2

Homoe

omma s

p.

Pseudh

apalo

pus s

p.

0

50

100

150

Nor

mal

ized

resp

onse

(%)

****

B) PNU-120596 10 µM

 

64

Table 2.6. Venoms from American theraphosids that lack of modulators of human α3* and α7 nAChRs.

Theraphosid spider Geographic location Aphonopelma crinitum

(F) Northern Mexico

Avicularia sp. (F) 2 Bolivia-Rio Madre Homoeomma sp. (blue) Peru

Pseudhapalopus sp. (F)

South America

2.3.8 Effect of non-theraphosid mygalomorph venom on vertebrate nAChRs

Unfortunately, I only had access to one non-theraphosid mygalomorph venom for

this study, namely Macrothele gigas from the family Hexathelidae (see condensed

phylogenetic tree in Figure 2.8). M. gigas venom blocked both the α3* and α7

nAChR subtypes. At 2 µg, M. gigas venom reduced the nicotine-evoked calcium

response in the FLIPR assay by 28 ± 22% and the response evoked by choline in

the presence of PNU-120596 by 46 ± 27%.

2.3.9 Taxonomic distribution of nAChR modulators within Araneae

Figure 2.8 summarizes the data obtained from the high-throughput FLIPR-based

screen of spider venoms against human nAChRs. Figure 2.8A shows the

distribution of blockers, agonists and inactive venoms within the infra-orders

Mygalomorphae and Araneomorphae, while Figure 2.8B shows the distribution of

nAChR modulators mapped onto a condensed phylogenetic tree of spiders (order

Araneae).

 

65

Figure 2.8. Summarise of the high-throughput screening of spider venoms. (A) Pie charts summarising the different types of nAChR modulators found in the venoms of mygalomorph spiders and araneomorph spiders. (B) Distribution of nAChRs modulators found in spider venoms mapped onto a condensed phylogenetic tree. Circles to the right of the tree denoting biological activities are sized according to the dominance of that pharmacology in the venom. NS denotes "not sampled".

PALAEOZOIC MESOZOIC CAINOZOICCARBONIFEROUS PERMIAN TRIASSIC JURASSIC CRETACEOUS PALEOGENE NGENE

299 251 200 145 65 23 2

ATYPIDAEHEXATHELIDAE

THERAPHOSIDINA

RASTELLOIDINA

LAGONOMEGOPIDAE

MESOTHELAE

ARANEAE

OPISTHO-THELAE

NOT VENOMOUS

ERESOIDAE

DEINOPIDAE

ULOBORIDAE

ARANEOIDAE

AUSTROCHILDAE

ARCHAEIDAE & HUTTONIDAE

HAPLOGYNAE

PALEOCRIBELLATE

RTA CLADE

ENTELE-GYNAE

NEO-CRIBELLATE

DIVIDEDCRIBELLUM

CLADE

MYGLAMORPHAE(primitive spiders)

ARANEOMORPHAE(modern spiders)

NOT VENOMOUS

MESOTHELAE

359

NS

NS

NS

NSNS

NS

NSNSNS

NS

No effect on human _3* or _7 nAChRs

Non-selective inhibitor of human _3* and _7 nAChRs

Selective inhibitor of human _3* nAChRs

Non-selective agonist of human _3* and _7 nAChRs

KEY

no effect 7%

Non-selective

block of α3* and α7 46%

Selective block of α3*

41%

Agonists 6%

Mygalomorphae

no effect 34%

Potent non-selective

block of α3* and α7 34%

No substancial

non-selective

block of α3* and α7 24%

Selective block of α3*

8%

Agonists 0%

Araneomorphae

A) B)

 

66

2.4 DISCUSSION

Today, it is recognized that an imbalance of the cholinergic pathways in humans

may lead to several pathologies (Picciotto, 2012). Therefore, nAChRs are now

molecular targets; and several studies have been carrying out to understand their

mechanisms of regulation, but few advances have been made (Bencherif, 2014;

Pammolli, 2011; Russo, 2014). In the field of agronomy, invertebrate nAChRs are

also targets of neonicotinoids that are used to control insect pests (Fischer, 2014;

Matsuda, 2009); and these are one of the most successful classes of chemical

insecticides ever developed, although it is no selective for insect pests, it has been

found to be lethal to other insects of importance in agriculture such as pollinators

(Moffat, 2015). Part of the problem is that many studies of cholinergic receptors

are limited by the number and type of selective modulators for nAChRs (Bauwens,

2015; Gotti, 2007).

Since spiders are the most successful insect predators on the planet (for a review

see (King and Hardy, 2013)), it would be surprising if they had not developed

nAChR modulators as part of their venom arsenal. To date, however, there are

very few reports of this pharmacological activity in spider venoms. We therefore

decided to screen spider venoms for nAChR modulators. Since cloned insect

nAChRs were not available, we performed screens against two subtypes of

vertebrate nAChRs (α3* and α7) based on the hypothesis that nAChR modulators

might also be useful for deterring vertebrate predators.

Several high-throughput FLIPR screenings were carried out to identify “hit”

venoms that were to contain promising lead compounds to further use as research

tools, or lead drugs. However, the data indicated that most of the spider venoms

contained predominantly blockers highly potent for subtype α3* nAChRs, and

blockers for α7 nAChRs; and a non-selective modulator was not considered to

isolate because adverse effects of some agents under current study are

associated with low selectivity for cholinergic receptors (Kalayci, 2013; Koga,

2014; Mihalak, 2006). Even when a toxin lead, which was expected not being

acylpolyamine type but selective for either α3* or α7 nAChRs, was not identified,

 

67

the resulting data was suitable to further analyse the distribution of modulators

across the phylogenetic tree of spiders.

Because spiders are venomous animals with more than 45,000 extant species

spread across 114 taxonomic families (see http://www.wsc.nmbe.ch/statistics/), it

was impossible in this study to sample broadly across this immense phylogenetic

tree, so we instead chose 71 species spread across the major suborder

Opisthothelae, which is comprised of the infraorders Araneomorphae ("modern"

spiders") and Mygalomorphae ("primitive spiders"). Note that Mesothelae, the

other taxonomic suborder with Araneae, comprises only ~100 extant species that

are thought to be non-venomous (for a review, see (Xu, 2015)).

With the caveat that it is difficult to make firm conclusions in the absence of deeper

taxonomic sampling across the phylogenetic tree, two trends emerge from the

data summary shown in Figure 2.8; and the discussion is based on the trends:

1) Although nAChR modulators can be found in distantly related species across

the phylogenetic tree, they were absent from 3 of the 6 venoms that we sampled

from the RTA clade as well as the single member of the family Austrochilidae that

we sampled. In addition, the 3 left venoms from the RTA clade had a partial

blocking effect on nAChRs. This indicates that modulation of vertebrate nAChRs

was either a basal pharmacology in spider venoms that was secondarily lost in

some species, or that it arose independently numerous times during Araneae

evolution. Deeper phylogenetic sampling in combination with biochemical

characterisation of the nAChR modulators found in spider venom will be required

to discriminate between these possibilities.

2) In this study, nAChR agonists, and specific blockers of the α3* subtype were

only found in the venom of theraphosid spiders, the largest family of mygalomorph

spiders with ~1000 species. Thus, mygalomorph venoms appear to be richer in

nAChR modulators than araneomorph venoms.

From the total of 71 spider venoms used in the present work, 12 belonged to

spiders of the suborder Araneomorphae. The resulting data indicated that

 

68

modulators for vertebrate nAChRs have evolved differently that were leading by

driven forces of natural selection such as geographical isolation and interactions

between preys and predators (Morgenstern, 2011; Zelanis, 2012). Probably, the

reason of absence of modulators in the venom of H. troglodytes is because it lives

in caves and underground drainages (Doran, 2001), and it is unlikely to encounter

vertebrate predators. Furthermore, they do not forage for insects instead they use

their long webs up to 1 metre of diameter to trap insects such as cave crickets.

The cheliceral structure of “modern” spiders evolved into an improved tool that can

be moved sideways, unlike fangs from “primitive” spiders that are moved up and

down 1. This feature in araneomorph spiders allows them grapping easily big preys

such as small vertebrates without using their venoms (Nyffeler and Pusey, 2014).

They attack their prey by crushing their fangs on the neck of preys that could be

small fish and lizards (Nyffeler and Pusey, 2014). In contrast, in mygalomorph

spiders, the position and movement of their cheliceraes do not allow

mygalomorphs to hunt preys bigger than big insects. Instead, they preferentially

use their venoms to immobilise them.

We could hypothesise that appearance of strong, and robust cheliceraes that are

moved sideways as forceps and the reduction of the length of “modern’ spiders

could lead to reduce the synthesis of modulators for vertebrate nAChRs in spider

venom. For instance, spider venoms with no potent blocking effect or no effect on

nAChRs belonged to spiders from the phylogenetic branch RTA clade, although in

the same phylogenetic clade, modulators for nAChRs could randomly appear on

other species. It can be evidenced when no substantial blocking effect on nAChRs

was observed in venoms from Ancylometes sp., V. sylvestriformis (Ctenidae), and

the lack of no modulators in venom from H. jugulans (Sparassidae), while venom

from Megadolomedes australianus contain potent blockers for nAChRs. The two

ctenid spiders studied in this research work have strong and long fangs that may

be used as defence. Moreover, they are found at night at the edge of water

sources in remote areas of the forest or the jungle (Nyffeler and Pusey, 2014)

                                                                                                               1  Most information about the habitat, biology, and behaviour of spiders was obtained from web pages of The Australian Museum and http://www.arachne.org.au..  

 

69

where they may have few contact to humans. Interestingly, M. australianus have

strong and large fangs and also potent blockers for vertebrate nAChRs. We could

speculate that toxins against nAChRs allowed that the spider could prey on fish,

which can be double of its size, tadpoles and insects (Nyffeler and Pusey, 2014).

An important physical adaptation in the spider is the presence of long legs that can

sense the vibrations of water when fish are nearby. Nevertheless, M. australianus

is relatively large because the length of its legs; and it may cause being seen

easily by natural enemies, and toxins against vertebrate nAChRs may be an

advantage.

Interestingly, our data showed that Araneomorph spiders from the araneoidea

family produced venoms with potent antagonists for vertebrate α3* and α7

nAChRs. For instance, two Australasian species of the genus Nephila, N. pilipes

and N. plumites showed to have in their venoms blockers for both α3* and α7

nAChRs. These compounds are likely to be acylpolyamines that have previously

been reported in other Nephila species such as Nephila madagascariencis (for a

review, see (Estrada, 2007)). Their preys are commonly insects, reviewed in

(Harvey, 2007) that are caught in their webs; because they produce a very strong

web between trees, on occasions small birds can get trap. Their natural enemies

are small vertebrates such as hummibirds (Vollrath, 1985). It seems modulators

against vertebrates could be evolved to protect themselves from them. Also,

humans could be considered as predators. Some native tribes from villages and

settlements from Thailand consider spiders of genus Nephila as food (for a review,

see (Harvey, 2007). Sometimes N. pilipes is found in house gardens and N.

plumipes can build their webs against buildings (for a review, see (Harvey, 2007))

and it makes these spiders more exposed to be grape from people; and it might

explain why venoms from N. pilipes and N. plumites contain potent blockers for

human nAChRs.

Mygalomorphae or “primitive” spiders

Because the suborder Mygalomorphae is considered an ancient monophyletic

group (Coddington and Levi, 1991), and its oldest fossil was dated to the Triassic

 

70

(Selden and Gall, 1992), this phylogenetic group can be particularly useful to

estimate the origin of modulators for vertebrate nAChRs.

From the 71 spider species used to initially searching for lead compounds, 59

venoms belonged to spiders of the suborder Mygalomorphae 2. The data showed

that most of the venoms blocked both subtype of mammalian nAChRs, and it is

likely the toxins were type acylpolyamines. Indeed the first acylpolyamine was

discovered in theraphosid spiders (for a review, see (Estrada, 2007)).

The only hexathelid species assayed herein was Macrothele gigas. Its venom

contained blockers for vertebrate α3* and α7 nAChRs. It also might contain an

agonist for α3* but an extra analysis of the active fraction may be carried out to

confirm the agonistic effect. It is likely blockers type acylpolyamines existed in the

venom of primitive spiders since the Triassic period because the ancestor of M.

gigas has been estimated to appear on that era. Interestingly, venoms from

Hexathelidae and Mygalomoprhae contain agonists and antagonists. This

similarity is not surprising because hexathelid and mygalomorph spiders are

closely related. It also suggests that a common ancestor could contain modulators

for vertebrate nAChRs in its venom. They are typically much larger than

araneomorphs, and they are consequently more prone to predation from

vertebrate predators such as birds, rodents and small reptiles.

On the other hand, a potential agonistic activity on nAChR was detected in venom

from Haplopelma hainanum. This spider lives in burrows that can reach up to 1

meter of depth. Its venom is categorized as highly venomous. The data herein

indicates this venom is likely to contain an agonistic component for nAChRs.

Probably, native acetylcholine may be present in the venom in large amounts

because there is a report of small molecules of a related species. For example, in

the venom of Haplopelma lividum was detected the polyamine spermine,

histamine, adenosine; and glutamic acid that was abundant in the venom fraction

of low molecular weight molecules (Moore, 2009). The plasticity of toxins to evolve

in accordance to environmental pressures, such as type of prey and type of

                                                                                                               2  The data is summarised in Figure 2.8.  

 

71

predators have been reported recently in the venom of Haplopelma hainanum

(Zhang, 2015b). It was shown that its intraspecific venom variations of blockers for

vertebrate nAChRs could be attributed to interaction gene-environment. In

addition, the presence of agonist for α7 nAChRs in venoms from Pamphobeteus

sp., Sericoplema rubronites and Xenesthis sp. remain to be confirmed. These

tarantulas are usually trade as pets; and if the presence of agonists for α7

nAChRs is due to the constant interaction with humans when these are in captivity

need to be evaluated. It would be interesting to further study venoms from these

tarantulas that naturally inhabit in the forest and the Amazon in South America.

According to the results of the present thesis, spider venoms from the family

theraphosidae, such as Selenocosmia crassipes, Selenocosmia effera,

Selenocosmia sp (Papua New Guinea), Coremiocnemis tropix, Selenotholus

foelschei contain potent toxins that block preferentially human neuronal α3*

nAChRs. In Australia, Selenocosmia sp. is known to cause severe pain in humans

and death in canines while venoms from Coremiocnemis tropix (spread out in

North Australia) and Selenotholus foelschei (inhabit only in Western Australia) are

shown to have a pre synaptic action at the neuromuscular junction in chicks

(Herzig, 2008). The analysis of our data indicates that these venoms may contain

compounds that preferentially act on the autonomic nervous system where the

“ganglionic” nAChR subtypes are expressed, and mediate the pain observed in

people bite by these spiders instead of targeting the nAChR muscle subtype.

On the other hand, theraphosid spiders from the new world studied herein showed

to contain blockers for vertebrate cholinergic receptors. Scarcely information is

available about venomic, diet and habitat of spiders from the new world, although,

some works have been carried out in some spiders assayed in this work (Bertani

and Fukushima, 2009).

In this study, the data showed that venom from Aphonopelma crinitum had some

blocking effect on α3* nAChRs but it was not significant. The presence of

acylpolyamines cannot be discarded because in a previous work two

acylpolyamines were discovered in the venom of Aphonopelma chalcodes

 

72

(Skinner, 1990). Aphonopelma crinitum, Pseudhapalopus sp. and Homoeomma

sp. may produce few blockers for α3* nAChRs with no effect on α7 nAChRs

because these are found in remote areas where there is little human activity . For

example, Aphonopelma crinitum lives in dry areas or desserts of North Mexico.

The plasticity of toxins to evolve in accordance to environmental pressures have

been reported recently in venoms of several species from Avicularia sp.,

Grammostola, sp., Holothele sp., Nhandu sp., and Psalmopoeus sp. (Zhang,

2015b). The diversity of antagonists for vertebrate nAChRs could be attributed to

interaction gene-environment. The Spiders Euathlus sp. and Thrixopelma sp.

inhabit along the western coast of Chile, and Thrixopelma cyaneolum inhabit the

coast along western Peru, all seemed to contain blockers for the subtype α3*

nAChRs. However, one venom was extracted from a Grammostola grossa species

that inhabits the Eastern Coast of Argentina. Its venom also had a blocking effect

on the subtype α3* nAChRs but the presence of modulators in its venom cannot

be attributed to the type of prey or predators that are found on the geographical

place where this species inhabits (the coast). G. grossa may be the same group of

spiders that originally inhabit the Amazons in Brazil that are spread around

adjacent regions of Brazil. At some point Grammostola species could evolved into

other specie that produce blockers for both subtypes of α3* and α7 nAChRs. This

is the case for Grammosotla rosea that is found in Bolivia, then, there could be a

spread of this species heading down to south America like the case of

Grammostola iheringi and Grammostola porteri that can be found in Chile and

Argentina. In general, few research are carried out about tarantula venoms, in

particular for those species found in Brazil, such as Acanthoscurria paulensis

(Mourão, 2013). Arboreal species Avicularia genus consist of 13 species and

these are found in Central and South America. Scarce studies about venoms from

Avicularia juruensis exist in the scientific literature (Ayroza, 2012). Avicularia

species are threatened for severe deforestation in the rainforest in Brazil and the

illegal trade to collectors (Bertani and Fukushima, 2009). It could explain why

several species of Avicularia has been spread across other regions around Brazil.

Interestingly, all species studied herein that are found in Brazil (in the forests of the

Amazonas) contain blockers for α3* nAChRs.

 

73

The two tarantulas Holothele sp. were collected originally from Colombia and

Peru. Their venoms contain blockers for both subtype nAChRs. Probably both

species are the same and these were spread around Colombia and northern Peru.

These tarantulas live in burrows, and blockers for cholinergic receptors could be

used for protection against small vertebrate predators. Like Holothele sp., the

theraphosid Nhandu vulpinus, Nhandu tripepii and Nhandu chromatus live in

burrows in the savannahs and tropical forests of South Brazil. Their venoms have

low toxicity and could be comparable to bee sting. Their diet is based on crickets,

mealworms and houseflies. It is likely the blocking effect on nAChRs is for

protection against small predators.

It is known from previous studies that nicotinic receptors are densely expressed in

insects and these share similar pharmacological profiles to counterparts

expressed in vertebrates (Sattelle, 2005). It suggests that the effect observed in

human nicotinic receptors caused by some spider venoms from the two major

lineages Mygalomorphae and Araneomorphae could share some similitude to

neuronal nicotinic cholinergic receptors from invertebrates, which known to be

expressed in neurons and cell bodies in insects. It is likely that selective toxins for

invertebrate nAChRs may be found because spiders based most of their diet on

insects.

The mechanisms of toxin evolution and diversity in spiders are currently not well

understood (Zhang, 2015b), although, this thesis provides with important insights

of the evolutionary role of toxins against mammalian cholinergic receptors. The

number of different spider venoms used on the present thesis is small to make

generalised conclusions. However, the results so far indicated that blockers for

vertebrate cholinergic receptors were likely to be present in old ancestor of

mygalomorph spiders. Inhibitors for human neuronal α3* and α7 nAChRs are the

major compounds in “primitive” spiders. These are likely to be acylpolyamines that

have previously been reported in “primitive” spiders (Skinner, 1990; Zhang,

2015b); and it implies that acylpolyamines have existed about 400 million years

ago, and are the simplest toxin to synthesise with high potency, unlike synthesis of

peptides or proteins. Furthermore, the data suggests that modulators for

 

74

vertebrate nAChRs were decreased notoriously when araneomorph spiders

appeared.

Our data also implies that primitive spiders produce a wide range of modulators for

vertebrate nAChRs that could include agonistic modulators on α7. It seems the

large size of mygalomorph spiders and their cheliceral position to subdue their

preys was no advantageous with the appearance of new vertebrate animals. It is

likely that in “primitive” spiders the role of toxins for both subtypes of mammalian

nAChRs is for defence. We could speculate also that in “modern” spiders

synthesis of agonists for α7 is energetically more expensive than no selective

antagonists for α3* and α7 nAChRs. After all, no selective blockers may lead to

the same outcome that warns vertebrate predators away.

To summarize, we can suggest that “modern” spiders (Araneopmorphae)

appeared with new physical characteristics to adapt them to new ecosystems; and

in parallel their venoms evolved with the appearance of different vertebrate

species, including small mammals. Araneomorph spiders retain the presence of

cheliceraes but it seems these were adapted to pinch the preys and avoid they

scape, instead of rely on their venoms to immobilise their preys, like in the case of

Mygalomorph spiders.

The resulting data of this thesis is a started point to further selection of venoms

with potential effect on cholinergic receptors of invertebrates. Extra screenings

using the FLIPR system, and including as positive control neonicotinoids may help

to choose “hit” venoms.

This is the first study of distribution of toxins from spider venoms against human

neuronal nAChRs, and it is hoped that it can serve to the discovery of novel toxins

against invertebrates as well as help to reveal the evolutional process of spider

toxins.

 

75

CHAPTER 3 — NACHR MODULATORS FROM SCORPION VENOM 3.1 INTRODUCTION

Like spiders, scorpion venoms contain a vast number of uncharacterized

compounds, and therefore they represent an untapped source of novel toxins with

potential applications in medicine, agronomy and as research tools (Harrison,

2014; King, 2014; King and Coaker, 2014; Wu, 2014). It has been estimated that

the molecules currently described from scorpion venoms represent less than 1%

of all components predicted to be in their venoms (Quintero-Hernandez, 2013).

Thus, we extended the search for modulators of vertebrate α7 and α3* nAChRs to

five scorpion venoms. As outlined below, this work led to the isolation of a non-

selective agonist of α7 and α3* nAChRs, from the venom of the giant forest

scorpion Heterometrus spinnifer.

3.1.1 Scorpion toxins and their pharmacological action Scorpions (Arthropoda: Chelicerata: Arachnida: Scorpiones) use their venoms to

immobilise prey and/or to defend against predators or competitors. The common

biological function of these venoms is their action on a variety of voltage-gated ion

channels in invertebrates and small vertebrates. In particular, scorpion venoms are

rich in modulators of both insect and vertebrate voltage-gated sodium (for a

review, see (Egea, 2015)) and Potassium (Kv) (Chugunov, 2013; Morales-Lázaro,

2015; Quintero-Hernandez, 2013; Swartz, 2013). The most toxic scorpion venoms

for mammals are from Androctonus sp. and Leirurus quinquestriatus (paleotropical

especies) and Centruroides sp. and Tityus sp. (neotropical especies), and these

venoms have been extensively studied due to their clinical relevance (DeBin,

1993; Diaz, 2009; Kularatne and Senanayake, 2014; Quintero-Hernandez, 2013;

Rjeibi, 2011). Most of the components purified and identified from scorpions to

date are toxins that act on Nav (Forsyth, 2012; Meves, 1984; Zhu, 2012), Kv

(Camargos, 2011; Nirthanan, 2005; Xie, 2012), Cav (Norton and McDonough,

2008; Shahbazzadeh, 2007) and chloride channels (DeBin, 1993; Nie, 2012;

Rjeibi, 2011; Zheng, 2000). In particular, toxins that target Nav channels that are

largely responsible for the symptomatology of mammalian envenomation

 

76

(Caliskan, 2012; Clot-Faybesse, 2000; Quintero-Hernandez, 2013) and paralysis

in insects (Billen, 2008; DeBin, 1993; Forsyth, 2012). However, the effect of

scorpion venom on nAChRs has been scarcely documented.

Androctonin, a disulphide-bridged peptide isolated from the hemolymph, but not

the venom of Androctonus australis, shows some similarity to a nAChR named α-

conotoxin SII, but its effect on nAChRs has not been determined (Ehret-Sabatier,

1996).

A non-toxic venom extract from the scorpion Buthus occitanus tunetanus contains

a component that depolarises muscle cells via an effect on nAChRs (Cheikh,

2007), but the authors did not isolate the active component.

Venoms from scorpions reported as non-threatening to humans have not been

extensively studied. Interestingly, venom from the giant forest scorpion

Heterometrus spinnifer was reported to contain high concentration of acetylcholine

and norepinephrine (Nirthanan, 2002). The authors suggested that acetylcholine

induces muscle contraction in rat nerve-smooth muscle preparations through

activation of muscarinic receptors. However, all of the bioassays were carried out

with a venom fraction that contained all molecules from the venom with molecular

masses less than 3 kDa, and the activity was not compared with pure

acetylcholine.

From our previous screening of 71 arachnid venoms for searching modulators for

nAChR using a high-throughput FLIPR calcium assay, we detected an agonistic

activity elicited from crude venom of H. spinnifer. We continued further to isolate

the “hit” toxin responsible for the activity because venom compounds seem to

have promising applications in therapy (Nie, 2012; Yang, 2011) and likely in

agronomy (Morales-Lázaro, 2015 #7), as mentioned before the venom is devoid of

harm in humans (Kularatne and Senanayake, 2014) and has not been extensively

studied due to the lack of clinical interest (Cheikh, 2007; Nirthanan, 2002).

The agonistic compound was isolated, and identified as a choline ester, named

senecioylcholine. It activates human neuronal nAChRs endogenously expressed

 

77

in SH-SY5Y cells and the synthetic form did not show insecticidal effect. It is

known ester of choline occurs naturally (Erspamer and Glässer, 1957; Erspamer,

1953; Florey, 1963; Keyl, 1957), and mainly purified from hypobranchial gland of

gastropods (Keyl and Whittaker, 1958; Roseghini, 1996). Even when acetylcholine

is a choline ester present in invertebrate nervous system (Florey, 1963),

senecioylcholine has not been before identified in scorpion venoms. Few

pharmacological studies have been done for senecioylcholine and its isomer

named tygloylcholine (Roseghini, 1996). Furthermore, there are discrepancies on

the function of choline ester in the venom of gastropods as mechanism of defence

(Erspamer and Glässer, 1957; Roseghini, 1996) and in the correct identification of

senecioylcholine or tygloylcholine (Baker and Duke, 1976, 1978).

The toxicity of senecioylcholine reported in vertebrates (Roseghini, 1996) and its

non-selective agonistic effect on human neuronal nAChRs suggest its role in the

venom is highly for defence against small vertebrates. The discovery of

senecioylcholine in the venom of a scorpion, which uses its chelae to subdue its

preys, makes a significant contribution in the evolution of venom compounds as

defensive weapons, and it is the first evidence of a convergent evolution between

scorpions and molluscs. To provide with a complete base for future research

works in interactions prey-predator and evolution, this study was extended up to

the pharmacological characterization of senecioylcholine and its isomers

tygloylcholine and angeloylcholine, which could be present in other arachnid

venoms.

3.2 METHODS

We used the high-throughput FLIPR assay outlined in Chapter 2 to screen five

scorpion venoms for modulators of vertebrate α7 and α3* nAChRs. For details of

the procedures used to identify “hit” venoms and purify bioactive venom

components see the Section 2.5 and 3.3.2, respectively.

3.2.1 Figure 3.1 shows an example of the protocol as applied to scorpion venoms

 

78

Figure 3.1A, shows the fluorescent response obtained by stimulation of α3*

nAChRs with nicotine, and inhibition of this response by d-tubocurarine. Figure

3.1B shows that the calcium fluorescence response is evoked mainly by activation

of α3* nAChRs, which was inhibited by d-tubocurarine, and this nicotine response

is independent of activation of Nav channels. On Figure 3.1C is shown the

fluorescent response evoked by crude venom from A. crassicauda, which induces

a robust increase in fluorescence prior to the addition of nicotine. The venom-

induced increase in fluorescence was not substantially inhibited by d-tubocurarine

(Figure 3.1D) but it was notably by TTX (Figure 3.1 E), a non-selective Nav

channel inhibitor (Figure 3.1E), Thus, the robust increase in fluorescence induced

by A. crassicauda venom is likely due to the presence of an agonist of Nav

channels endogenously expressed in SH-SY5Y cells.

 

79

Figure 3.1. A. crassicauda venom can induce calcium responses elicited via activation of Nav channels. (A) Fluorescent responses elicited by stimulation of α3* nAChRs with nicotine, and inhibition of this response by d- tubocurarine. (B) The calcium fluorescent response observed is mediated mostly by activation of α3* nAChRs with nicotine (30 µM). The inhibitor for Nav channels, TTX, does not inhibit the calcium response. (C) Addition of crude venom (2 µg) from A. crassicauda elicited fluorescent response (black trace), prior to the activation of α3* nAChRs with nicotine (blue trace). The venom-induced increase in fluorescence was not substantially inhibited by d-tubocurarine (panel D) but it was significantly inhibited by 2 µM TTX (panel E).

0

1

2

3

4

1 151 301 451 601time (seconds)

A. crassicauda venom 2 µg

TTX 2 µM + A. crassicauda venom 2 µg

E)

Max

imun

cal

cium

resp

onse

(RLU

)

0

1

2

3

1 151 301 451 601

Max

imun

cal

cium

resp

onse

(R

LU)

time (seconds)

nicotine 30 µM d-tubocurarine 30 µM

A)

0

1

2

3

1 151 301 451 601

Max

imun

cal

cium

resp

onse

(R

LU)

time (seconds)

nicotine 30 µM TTX (2 µM)

B)

0

1

2

3

4

1 151 301 451 601

Max

imun

cal

cium

resp

onse

(R

LU)

time (seconds)

A. crassicauda venom 2 µg nicotine 30 µM

C)

0

1

2

3

4

1 151 301 451 601M

axim

un c

alci

um re

spon

se

(RLU

)

time (seconds)

A. crassicauda venom 2 µg d-tubocurarine 30 µM + A. crassicauda venom 2 µg

D)

 

80

3.2.2 Scorpion venoms

Dr Volker Herzig kindly provided five scorpion venoms for screening against

vertebrate α7 and α3* nAChRs. The list of scorpion species used in this study is

given in Appendix I.

3.2.3 Venom fractionation

Crude scorpion venom (1 mg) was dissolved in 1 ml of 5% of buffer B (90%

acetonitrile containing 0.05% TFA) and centrifuged at 5,000 g for 15 min at 4°C.

After centrifugation, the supernatant was loaded onto a Phenomenex C18 reverse-

phase HPLC (RP-HPLC) column Jupiter 250 x 4.60 mm; 5 µm particle size; 300 Å

pore size and fractionation performed using Shimadzu HPLC system. Venom

fractionation was performed using a flow rate of 1 ml/min and the following

gradient of Buffer B in Buffer A (Milli-Q water + 0.05% TFA): 0–5 min: 5% Buffer B;

5–10 min: 5–20% Buffer B; 10–50 min: 20–40% Buffer B; 50–55 min: 40–80%

Buffer B; 55–57 min: 80% Buffer B.

The elution profile was recorded at 214 nm and 280 nm, and fractions were

collected manually based on 214 nm absorbance. At the end of the fractionation

the samples were frozen, lyophilized, dissolved in Milli-Q water, and stored at –

20°C until required.

3.2.4 Discovery of a choline ester from the venom of H. spinnifer

We isolated the active fraction from H. spinnifer venom using two chromatographic

steps of purification using the FLIPR high-throughput calcium assay described in

the previous section. The active fraction from the initial venom fractionation using

a Jupiter C18 RP-HPLC column was further fractionated on a Grace Discovery

Science Vydac® 218TP C18 RP-HPLC column 250 x 4.60 mm; 5 µm particle size;

and 300 Å pore size using a flow rate of 1 ml/min and the following gradient: 0–5

min: 5% Buffer B; 5–10 min: 5–10% Buffer B; 10–50 min: 10–30% Buffer B; 50–55

min: 30–80% Buffer B; 55–57 min: 80% Buffer B.

 

81

3.2.5 Determination of mass and purity

The active component isolated from H. spinnifer venom was dissolved in 5% of

acetonitrile (grade HPLC) and analysed via matrix-assisted laser

desorption/ionization (MALDI) mass spectrometry (MS) performed on an Applied

Biosystems mass spectrometer operating in the positive ion linear mode. The

sample (0.4 µL) was mixed on a MALDI plate with 0.4 µl of a-cyano-4-

hydroxycinnamic acid (CHCA) matrix (6 mg/mL) dissolved in acetonitrile/ MilliQ

water /formic Acid (50:45:5, v/v).

The MALDI-TOF spectra did not reveal any masses higher than 1 kDa. Thus, the

active fraction was further analysed via LCMS using an Agilent 1100 Series

separations module equipped with diode array detector and Agilent 1100 Series

LC/MSD mass detector in both positive and negative ion modes. The sample was

run on an Agilent Zorbax-C8 column 4.6 mm x 150 mm, 5 µm particle size using a

gradient of 10–100% methanol/H2O (with constant 0.05% formic acid modifier)

over 15 min, using a flow rate of 1 mL/min.

3.2.6 Identification of active compound using HRMS and LCMS

High-resolution (HR) electrospray ionisation (ESI) MS measurements were

obtained on a Bruker micrOTOF with an ESI probe by direct infusion in acetonitrile

at 3 µL/min using sodium formate clusters as an internal calibrant. The active

venom yielded m/z 186.1504 [M]+ (calculated for C10H20NO2, 186.1489).

LC/MS was performed on native compound co-injected with synthetic choline

esters using an Agilent 1100 series LC/MS with an Agilent Zorbax-C18 aqua

column 4.6 x 150 mm, 5 µm particle size. Isocratic elution was performed using

4% methanol/H2O (with constant 0.02% formic acid modifier) at a flow rate of 1

mL/min, with single ion extraction at 186 [M+].

 

82

3.2.7 Chemical synthesis of choline esters 3.2.7.1 Synthesis of chlorocholine chloride

Choline chloride (279 mg, 2 mmol) was dried under high vacuum at 200°C for 2 h.

SOCl2 (2 mL) was added and the solution was heated under reflux for 1 h. After

cooling, solvent was evaporated to dryness. Solid residue was redissolved in

methanol (3 x 2 mL), and re-evaporated to dryness. The resultant white solid was

dried under high vacuum at 80°C for 2 h.

3.2.7.2 Synthesis of tigloylcholine

To a solution of tiglic acid (200 mg, 2 mmol) in a dioxane–water (3:2) mixture (10

mL), a solution of K2CO3 (138 mg, 1 mmol in water 10 mL) was added. After

stirring for 10 min, the mixture was evaporated to dryness and again dried under

high vacuum at 70 °C for 1 h. Chlorocholine chloride and anhydrous DMSO were

added and the mixture heated at 60 °C for 48 h. DMSO was removed by vacuum

distillation and the residue was purified by reverse phase column chromatography

to afford tigloylcholine as a light brown hygroscopic solid. 1H NMR (600 MHz,

CD3OD): δ 6.92 (qq, 7.1, 1.3 Hz, H-3), 4.59 (m, H2-2ʹ′), 3.75 (m, H2-1ʹ′), 3.22 (s,

NMe3), 1.85 (dd, 1.3, 1.1 Hz, H3-5), 1.82 (dq, J = 7.1, 1.1 Hz, H3-4). 13C NMR (150

MHz, CD3OD): δ 168.4 (C-1), 140.3 (C-3), 129.1 (C-2), 66.4 (C-2ʹ′), 59.1 (C-1ʹ′),

54.60 (NMe), 54.58 (NMe), 54.55 (NMe), 14.6 (C-4), 12.3 (C-5). HRMS calc. for

C10H20NO2+ 186.1489, found m/z = 186.1491.

ClNMe3

ClHONMe3

Cl

SOCl2

70 o C, 1h

ONMe3OH

O Oi) K2CO3, dioxane/H2O, rt

DMSO, 60 oC, 48 hCl

NMe3 Clii)

1'

2'

Cl

12

3

4

5

 

83

3.2.7.3 Synthesis of senecioylcholine

As described for the synthesis of tigloylcholine, starting with choline chloride (2

mmol, 279 mg) and 2-methyl butenoic acid (2 mmol, 200 mg), senecioylcholine

was synthesized and purified by reverse phase column chromatography, to afford

senecioylcholine as a white hygroscopic solid. 1H NMR (600 MHz, CD3OD): δ 5.76

(qq, 1.2, 1.2 Hz, H-2), 4.54 (m, H2-2ʹ′), 3.73 (m, H2-1ʹ′), 3.22 (s, NMe3), 2.18 (d, 1.2

Hz, H3-4), 1.93 (d, 1.2 Hz, H3-5), 13C NMR (150 MHz, CD3OD): δ 166.8 (C-1),

161.2 (C-3), 115.8 (C-2), 66.4 (C-2ʹ′), 58.1 (C-1ʹ′), 54.66 (NMe), 54.63 (NMe), 54.61

(NMe), 27.6 (C-4), 20.6 (C-5). HRMS calc. for C10H20NO2+ 186.1489, found m/z =

186.1485.

3.2.7.4 Synthesis of angeloylcholine

As described for the synthesis of tigloylcholine, starting with choline chloride (2

mmol, 279 mg) and angelic acid (2 mmol, 200 mg), angeloylcholine was

synthesized and purified by reverse phase column chromatography, to afford

angeloylcholine as a white hygroscopic solid. 1H NMR (600 MHz, δ 6.23 (qq, 7.2,

1.3 Hz, H-3), 4.60 (m, H2-2ʹ′), 3.75 (m, H2-1ʹ′), 3.22 (s, NMe3), 2.00 (dq, 7.2, 1.3 Hz,

H3-4), 1.91 (dq, J = 1.3, 1.3 Hz, H3-5). 13C NMR (150 MHz, CD3OD): δ 167.9 (C-1),

141.7 (C-3), 128.0 (C-2), 66.4 (C-2ʹ′), 58.6 (C-1ʹ′), 54.59 (NMe), 54.56 (NMe), 54.54

(NMe), 20.8 (C-4), 16.2 (C-5). HRMS calc. for C10H20NO2+ 186.1489, found m/z =

186.1483.

ONMe3OH

O Oi) K2CO3, dioxane/H2O, rt

DMSO, 60 oC, 48 hCl

NMe3 Clii)

1'

2'1

23

4

5

Cl

ONMe3OH

O Oi) K2CO3, dioxane/H2O, rt

DMSO, 60 oC, 48 hCl

NMe3 Clii)

Cl

1'

2'1

23

4

5

 

84

3.3 RESULTS

3.3.1 High-throughput FLIPR assay

To speed the venom-based discovery of “hit” biomolecules from five scorpion

venoms a high-throughput FLIPR assay was performed (King, 2014; Vetter, 2011).

Five venoms derived from scorpions of the family Buthidae were screened using

the high-throughput FLIPR assay described in Chapter 2. At 2 µg, none of the

venoms had a significant effect on vertebrate α7 or α3* nAChRs (Figure 3.2).

However, crude venom (1 or 2 µg) from H. spinnifer elicited a transient calcium

response that was potentiated by 50% in the presence of the positive allosteric

modulator PNU-120596 (Figure 3.3A). This response was completely blocked by

d-tubocurarine (Figure 3.3B) indicating that the venom activates neuronal

nAChRs. The total elimination of the calcium response in the presence of PNU-

120596 by d-tubocurarine suggests that the agonistic effect observed is mediated

totally by α3* and α7 nAChRs. Two other concentrations of venoms were tested at

0.02 µg or at 0.2 µg (data not shown), and no effect was observed. The agonistic

effect of this venom is therefore dose-dependent.

 

85

Figure 3.2. FLIPR screening of scorpion venoms against α7 and α3* nAChRs. 1 µg of crude venoms from (A) L. quinquestriatus, (B) A. australis, (C) A. crassicauda, (D) G. grandidieri and (E) H. spinnifer was added to loaded- SH-SY5Y cells in the presence of PNU-120596. Calcium responses were then measured for 300 s, followed by choline addition, and recording of calcium response for a further another 300 s. To show the participation of cholinergic receptors in the calcium responses following venom addition, the cells were treated by d-tubocurarine before starting the assay. There was no effect of d-tubocurarine in the responses caused by venom from (A) L. quinquestriatus (trace in dashed lines and orange colour), (B) A. australis (trace in dashed lines and dark blue colour), and (C) A. crassicauda (trace in red colour). However, d-tubocurarine caused significant inhibition of the calcium response elicited by venoms from (D) G. grandidieri (trace in dashed lines and black colour) and (E) H. spinnifer (trace in blue colour). Traces are the average of at least three wells.

0

1

2

3

4

1 151 301 451 601

Max

imun

cal

cium

resp

onse

(RLU

)

time (seconds)

crude venom G.grandidieri 1 µg + PNU-120596 (10 µM)

crude venom G. grandidieri 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

0

1

2

3

1 151 301 451 601

Calc

ium

resp

onse

(RLU

)

time (seconds)

venom H. spinnifer 1 µg + PNU d-tubocurarine 30 µM

0

1

2

3

4

1 151 301 451 601Max

imun

calci

um re

spon

se (R

LU)

time (seconds)

A. crassicauda crude venom 2 µg d-tubocurarine 30 µM+ venom A. crassicauda 2 µg

C)

0

1

2

3

1 151 301 451 601M

axim

un

cal

ciu

m r

esp

on

se (

RLU

)

time (seconds)

crude venom A. australis 1 µg + PNU-120596 (10 µM) crude venom A. australis 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

0

1

2

3

1 151 301 451 601Ma

xim

un

ca

lciu

m r

esp

on

se

(RLU

)

time (seconds)

crude venom L. quinquestriatus 1 µg + PNU-120596 (10 µM) crude venom L. quinquestriatus 1 µg + PNU-120596 (10 µM) + d-tubocurarine (30 µM)

A)#

D)#

B)# C)#

E)#

 

86

Figure 3.3. Non-selective activation of α7 and α3* nAChRs expressed in SH-SY5Y by crude venom from H. spinnifer. (A) 2 µg of crude venom from H. spinnifer induced an increase of intracellular calcium in SH-SY5Y cells (blue trace) that was potentiated by PNU-120596 (red trace). (B) The calcium response elicited by 1 µg of crude venom from H. spinnifer in the presence of PNU-120596 (red trace) was totally blocked by d-tubocurarine (black trace in black colour). Traces are the average of at least three wells.

0  

1  

2  

3  

4  

5  

6  

1   51   101   151   201   251  Calcium  re

spon

se  (R

LU)  

Tme  (seconds)  

2  µg  venom  

2  µg  venom  +  PNU  

A)

0  

1  

2  

3  

1   51   101   151   201   251   301  

Calcium  re

spon

se  (R

LU)  

Tme  (seconds)  

1  µg  venom  +  PNU120596  

1  µg  venom  +  PNU120596  +  d-­‐tubocurarine  

B)

 

87

3.3.2 Isolation of the active compound from H. spinnifer venom

1 mg of crude venom from H. spinnifer was separated by RP-HPLC (Figure 3.4)

and fractions were manually collected. Each resulting fraction at 0.42 µg of total

crude venom was evaluated using the FLIPR assay to evaluate its impact on

activation of nAChRs using nicotine or choline plus the positive allosteric

modulator PNU-120596 to stimulate the heteromeric subtypes α3β2/α3β4 and the

homomeric subtype α7 nAChR, respectively. Only fraction 10, which eluted at

9.49% acetonitrile in the C18 RP-HPLC separation (inset, Figure 3.4), activated

the α3* subtype as its activity was substantially inhibited (66%) by d-tubocurarine

(30 µM) (Figure 3.5). Furthermore, the increased response in the presence of

PNU-120596 (Figure 3.5) indicates that α7 nAChRs are also activated by fraction

10. Thus, fraction 10 contains the agonistic molecule that is responsible for the

calcium response elicited by crude H. spinnifer venom.

Figure 3.4. HPLC fractionation of crude venom from H. spinnifer. Chromatogram resulting from fractionation of crude venom via C18 RP-HPLC. The fraction highlighted in black (F10) activated α7 and α3* nAChRs in the FLIPR assay. The inset shows an expansion of the region containing F10, which eluted at ∼ 13.6 min.

20 40 60

0

1

2

3

Time (min)

Abs

orba

nce

214

nm

12 14 16

0

1

2

3

Time (min)

Abs

orba

nce

214

nm

Fraction 10

 

88

Figure 3.5. Activity of F10 from fractionation of crude venom from H. spinnifer. Active fraction 10 (F10) (1% from initial fractionation of 1 mg of crude venom) was assayed against vertebrate nAChRs using the FLIPR. F10 induced a maximum fluorescent response similar to that evoked by nicotine. The response was inhibited by d-tubocurarine and potentiated by PNU-120596. The difference are significant with a P < 0.05. One-way ANOVA was used to compare the means of each experimental treatment in relation to the control response (nicotine response). The values are averages of three independent experiments with n = 3. Dotted lines represent the control response in the absence of venoms. Fraction 10 (72 µg) was further fractionated using an analytical RP-HPLC

(GRACE-Vydac C18 column RP-HPLC) (Figure 3.6) and the resulting fractions

collected manually. Each of the active fractions were then analysed using the

FLIPR assay, for activity against vertebrate nAChRs. Only two fractions were

active in the FLIPR assay, which eluted at 11.1 min (fraction 4) and 13.6 min

(fraction 7) (Figure 3.6). Unfortunately, further study of fraction 7 was not possible

due to its low abundance as evidenced by its very low absorbance at 214 nm.

Thus, further studies focused on fraction 4. Assay of fraction 4 (77.35 µM) in the

FLIPR assay revealed that it induced a fluorescent response that was completely

inhibited by 30 µM d-tubocurarine and potentiated by 10 µM PNU-120596 (Figure

3.7). We therefore focussed attention on identification of the active component in

this fraction.

F10 (4

.2 µg)

F10 (4

.2 µg)

d-tuboc

urarin

e (30

µM)

F10 (4

.2 µg)

PNU1205

96 (1

0 µM

)0

100

200

300%

FM

ax

****

****

****

 

89

Figure 3.6. Purification of the “hit’ toxin by RP-HPLC. Chromatogram resulting from separation of fraction 10 (72 µg) on an analytical C18 RP-HPLC. The “hit” toxin (fraction 4) eluted at 11.1 min (10.35% Buffer B), and fraction 7 eluted at 13.6 min (12.9% Buffer B).

Figure 3.7. Agonistic activity of the “hit” toxin. Fraction 4 (77 µM) from analytical HPLC induced a fluorescent response that was totally blocked by d-tubocurarine and by PNU-120596. This suggests that fraction 4 contains the “hit” toxin that is primarily responsible for the non-selective agonism of α3* and α7 nAChRs by H. spinnifer venom. The difference are significant with a P < 0.05. One-way ANOVA was used to compare the means of each experimental treatment in relation to the control response (nicotine response). The values are averages of three independent experiments with n = 3. Dotted lines represent the control response in the absence of venoms.

d-tuboc

urarin

e (30

µM)

Native

senec

ioylch

oline (

77 µM

)

Native

senec

ioylch

oline (

77 µM

)

d-tuboc

urarin

e (30

µM)

Native

senec

ioylch

oline (

77 µM

)

PNU1205

96 (1

0 µM

)

0

50

100

150

200

250

% F

Max

****

****

****

****

0 20 40

0

100

200

300

Time (min)

Abs

orba

nce

(214

nm

)

Frac

tion

4

11.1

Frac

tion

7

 

90

3.3.3 Identification of the active compound by high-resolution mass spectrometry

Analysis of fraction 4 using MALDI-TOF-MS revealed no masses larger than 1

kDa; it is difficult to detect masses smaller than this via MALDI-TOF-MS due to the

noise caused by the matrix (CHCA). Thus, the sample was further analysed using

HR-MS; this revealed a single compound with m/z 186.1489. Database searching

revealed that the mass formula of C10H20N1O2 corresponds to a previously

described choline ester named senecioylcholine (Keyl, 1957; Roseghini, 1996;

Whittaker, 1960). However, there are two isomers of senecioylcholine,

tigloylcholine and angeloylcholine, with identical mass formula and it is impossible

to determine, which of these isomers corresponds to the native venom-derived

compound based on HR-MS data alone. Thus, all three isomers were chemical

synthesized and LC-MS co-elution studies were performed to identify the native

compound. As shown in Figure 3.8, senecioylcholine but neither of the two

isomers, co-elutes with the active nAChR agonist purified from H. spinnifer venom.

Thus, we conclude that the α3*/α7 nAChR agonist in H. spinnifer venom is

senecioylcholine.

 

91

Figure 3.8. Co-elution of bioactive compound from H. spinnifer venom and synthetic choline esters. LC-MS chromatograms of synthetic choline esters (top three panels), the α3*/α7 nAChR agonist from H. spinnifer venom (bottom panel). LC was performed using a Zorbax C18-aqua column (4.6 × 150 mm), 5 µm particle size with a gradient of methanol + 0.02% formic acid at a flow rate of 1 ml/min.

0 5000

10000 15000

0 2500 5000 7500 10000 12500

0

2000 4000 6000 8000

10000

1000 2000 3000 4000 5000

1 2 3 4 5 6 7 8 9 0

5000 10000 15000

0

synthetic angeloylcholine

synthetic tigloylcholine

synthetic senecioylcholine

native senecioylcholine

co-injection of native + synthetic senecioylcholine

Retention time (min)

tota

l ion

cou

nt

 

92

3.4 DISCUSSION

H. spinnifer is a scorpion from the Scorpionidae family. It is widely distributed in

South-eastern Asia but its venom has been poorly studied due to its innocuous

effect on humans. However, it is still a rich source of mostly uncharacterised

compounds that could be useful as novel modulators of nAChRs with clinical or

agronomic relevance. In this study, we used activity-guided fractionation to isolate

a novel agonist of vertebrate nAChRs from the venom of H. spinnifer. By using

HR-MS in combination with chemical synthesis, we identified this agonist as

senecioylcholine, a choline ester. We found that senecioylcholine is a non-

selective agonist of both α3β2/α3β4 and α7 nAChR subtypes endogenously

expressed in SH-SY5Y cells.

Choline esters are secreted by the hypobranchial gland of predatory marine

gastropods from the family Muricidae (phylum Mollusca), for a review, see

(Benkendorff, 2013) where they are postulated to play a role in predation and/or

defence against predators. They have also been detected in the ventral nerve cord

of the American lobster Homarus americanus (phylum Arthropoda, class

Crustacea) (Keyl, 1957). The main choline ester identified in Mollusks is murexine

(Erspamer and Glässer, 1957) although senecioylcholine has also been detected

in significant quantities in the hypobranchial gland of Dicathais orbita. A more

recent study identified tigloylcholine, an isomer of senecioylcholine, muricids of the

genus Thais (Shiomi, 1998). Murexine and tigloylcholine are both lethal to mice,

with intravenous administration leading to LD50 values of 8.5 mg/kg and 0.92

mg/kg (Shiomi, 1998), respectively, for a review, see (Benkendorff, 2013). Like

murexine, senecioylcholine is also a neuromuscular blocking compound with

nicotine action on vertebrates (Roseghini, 1996). Interestingly, however, even high

doses of senecioylcholine have no effect on the fruit fly Drosophila melanogaster

(data not shown). Thus, it is likely that senecioylcholine is used by H. spinnifer for

defence against vertebrate predators.

A few bioactive compounds have previously been isolated from H. spinnifer venom

k-KTx1.3, a non-toxic peptidic modulator of Kv channels (Nirthanan, 2005), and six

novel antimicrobial peptides (Nie, 2012; Wu, 2014). H. spinnifer venom was also

 

93

shown to contain high concentrations of acetylcholine (∼80 µM) and this was

proposed to be responsible for the reversible venom-evoked contracture of chick

biventer cervicis muscle that was blocked by d-tubocurarine (Nirthanan, 2002).

However, all the bioassays were carried out using a venom fraction that contained

all molecules with molecular masses less than 3 kDa. Thus, it is likely that

senecioylcholine, which was identified in H. spinnifer venom in the current study,

mighty be at least partially responsible for the observed contracture of chick

biventer cervicis muscle. The discovery of senecioylcholine in both scorpion

venom (phylum Arthropoda) and predatory marine gastropods (phylum Mollusca)

suggests that these phylogenetically unrelated organisms have convergently

evolved the use of choline esters to defend themselves against vertebrate

predators. It remains to be seen whether choline esters are ubiquitous in scorpion

venoms or restricted to specific families or genera. However, the fact that only 2 of

the scorpion venoms tested in this study appear to contain agonists of human

α3β2/α3β4 and α7 nAChRs suggests that senecioylcholine and related choline

esters are not widely distributed in scorpion venoms.

 

94

CHAPTER 4 — CONCLUSIONS Venomous animals use their venom to hunt preys and/or protect themselves

against predators or competitors (Olsen, 2011). Arachnid venoms are complex

mixtures of salts, small organic molecules, peptides and proteins that target a wide

range of neuronal receptors and ion channels (Billen, 2008; Estrada, 2007;

Morales-Lázaro, 2015; Quintero-Hernandez, 2013). A large number of modulators

of voltage-gated ion channels have been isolated from the venom of spiders and

scorpions, but there are very few reports of arachnid venom components

modulating the activity of nAChRs. This is surprising given that neonicotinoid

insecticides are one of the most successful classes of chemical insecticides, which

suggests that targeting of insect nAChRs would be a useful pharmacology for

arachnid venoms. Thus, the present work was carried out to survey the prevalence

of nAChR modulators in arachnid venoms.

Until recently, most characterizations of arachnid venoms used traditional

biological assays that require large amounts of venom or venom fraction, which

hampered studies of small venomous animals such as arachnids. In the current

study we used a high-throughput FLIPR assay that enabled screens to be rapidly

performed against human α3* and α7 nAChRs using only a few micrograms of

venom. This approach allowed simultaneous screening of 76 arachnid venoms at

four different concentrations.

The data were reproducible even when different pools of venoms were used. This

suggests that nAChR modulators in arachnid venoms are maintained even when

they are kept in captivity. Our data revealed that spider venoms are a rich source

of nAChR modulators, and that at least some scorpion venoms contain choline

esters that agonize vertebrate nAChRs.

We propose that these nAChR modulators are primarily used for defence against

vertebrate predators. It would be of great interest in future to perform high-

throughput screens of arachnid venoms against insect nAChRs. Predation of

insects is the primary role of arachnid venoms and therefore we might expect to

find an even greater abundance and variety of compounds in these venoms that

 

95

modulate the activity of invertebrate nAChRs. Nevertheless, the current results

strongly support the view that arachnid venoms are a novel source of modulators

of ligand-gated ion channels with potential biotechnological applications.

SUMMARY AND FUTURE DIRECTIONS

The current work revealed, for the first time, that many spider venoms (65 of 71

tested in this study) contain modulators of vertebrate nAChRs. Moreover, 31 of the

65 active venoms contained non-selective modulators that affected both α3* and

α7 nAChRs, whereas a smaller number (25) were selective modulators of

vertebrate α3* nAChRs. This suggests that during the course of evolution, non-

selective modulators of vertebrate nAChRs were selected for the purposes of

defence against vertebrate predators. However, future studies aimed at screening

a taxonomically broader range of spiders against a larger panel of vertebrate

nAChR subtypes will be required to address this hypothesis.

The current study also identified a non-selective agonist of vertebrate nAChRs in

the venom of the giant forest scorpion Heterometrus spinnifer (Scorpionidae). In

contrast with most channel modulators found in arachnid venoms, this nAChR

modulator was identified as senecioylcholine, a choline ester. Senecioylcholine

was previously identified in the hypobranchial gland of some predatory marine

gastropods (family Muricidae) but this is the first report of it being present in

scorpion venom. H. spinnifer is one of the largest scorpion species but its venom

is no lethal to humans. However, envenomation of humans by H. spinnifer leads to

intense pain and inflammation, and senecioycholine may contribute to these

symptoms. Thus, this study provides the first evidence for convergent evolution of

a non-peptide toxin directed against vertebrate nAChRs. It would be interesting in

future studies to examine whether senecioylcholine and related choline ester are

found more broadly in scorpion venoms, particularly in species that are not

relevant in clinic.

While the current study focussed on vertebrate nAChRs, it would be of great

interest in future to examine whether arachnid venoms also target nAChR

 

96

subtypes found in their major prey, namely insects. Compounds that specifically

targeted insect, but not vertebrate, nAChRs would be of significant interest as

bioinsecticide leads. In this respect, it might therefore be interesting to examine

venoms that were shown in this study to be inactive against vertebrate nAChRs,

such as those from the araneomorph spider Heteropoda jugulans and the

mygalomorph spider Aphonopelma crinitum.

Most venom research to date has focussed on animals of medical relevance, but

this study has highlighted the value of studying venoms with non-lethal effects on

humans when searching for novel compounds. The continued development of

high-throughput screening methods and analytical tools that require only small

amounts of venom will provide more opportunities in future for exploring the vast

pharmacological repertoire encoded within the venoms of small venomous animals

such as spiders and scorpions.

 

97

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APPENDIX I Table I. List of spiders whose venoms were used in this work

Araneomorphae (“modern” spiders)

Abbreviations

No effect on α3* and α7 nAChR

Austrochiloidea family

1. Hickmania troglodytes H. troglodytes

Blocking effect on α3* nAChR

Araneoidea family

2. Cyrtophora moluccensis C. moluccensis

Blocking effect on α3* and α7

nAChR

Araneoidea family

3. Eriophora transmarina E. transmarina

Genus Nephila

4. Nephila pilipes N. pilipes

5. Nephila plumipes N. plumites

No effect on α3* and α7 nAChR

RTA Clade

Superfamily Ctenoidea

6. Ancylometes sp. Ancylometes sp.

7. Viridasius sp. (sylvestriformis) Viridasius sp.

Sparassidae family

8. Heteropoda jugulans H. jugulans

Partial blocking effect on α3* and α7 nAChR

RTA Clade

Superfamily Lycosoidea

9. Lycosidae sp. Lycosidae sp.

Sparassidae family

10. Sparassidae sp. 1 Sparassidae sp. 1

11. Sparassidae sp. 2 Sparassidae sp. 2

 

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Blocking effect on α3* and α7

nAChR

RTA Clade

Pisauridae family

12. Megadolomedes M. australianus

Mygalomorphae (“primitive” spiders) Abbreviations

Blocking effect on neuronal α3*

and α7 nAChR

1. Macrothele gigas M. gigas

2. Acanthoscurria cf insubtilis A. insubtilis

3. Ceratogyrus bechunanicus C. bechunanicus

4. Ceratogyrus marshalli C. marshalli

5. Ceratogyrus ezendami C. ezendami

6. Eupalaestrus campestratus E. campestratus

7. Ephebopus uatuman E. uatuman

8. Chilobrachys sp. 2 Chilobrachys sp. 2

9. Grammostola porteri G. porteri

10. Grammostola rosea G. rosea

11. Grammostola sp. (Chilean

North) Grammostola sp. 1

12. Grammostola sp. (Concepcion,

Chile) Grammostola sp. 2

13. Grammostola sp. (Maule River,

Chile) Grammostola sp. 3

14. Holothele sp. (Carabobo,

Colombia) Holothele sp. 1

15. Holothele sp. (Peru) Holothele sp. 2

16. Idiothele nigrofulva I. nigrofulva

17. Lasiodorides striatus L. striatus

18. Lasiodora striatipes L. striatipes

19. Nhandu chromatus N. chromatus

20. Nhandu tripepii N. tripepii

 

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21. Nhandu vulpinus N. vulpinus

22. Phormictopus cancerides P. cancerides

23. Pamphobeteus cf petersi Pamphobeteus cf petersi

24. Psalmopoeus cambridgei P. cambridgei

25. Psalmopoeus pulcher P. pulcher

26. Psalmopoeus langenbucheri P. langenbucheri

27. Theraphosinae sp. (Chile) Theraphosinae sp.

Potent blocking effect on α3*

nAChR

28. Avicularia metallica A. metallica

29. Avicularia ulrichea A. ulrichea

30. Avicularia sp. (Amazonas

Purple, Peru) Avicularia sp. 1

31. Coremiocnemis tropix C. tropix

32. Chilobrachys sp. (Penang,

Malaysia) Chilobrachys sp. 1

33. Euathlus sp. (green, chile) Euathlus sp. 1

34. Euathlus sp. (“red”=”fire”,

Chile) Euathlus sp. 2

35. Euathlus vulpinus (crème,

Chile highland) E. vulpinus

36. Grammostola grossa G. grossa

37. Grammostola iheringi G. iheringi

38. Orphnaecus philippinus O. philippinus

39. Orphnaecus sp. (‘treedweller’,

Paqnai-Island, Phillippines)

Orphnaecus sp.

40. Paraphysa scrofa P. scrofa

41. Pelinobius muticus P. muticus

42. Poecilotheria striatus P. striatus

43. Pterinochilus murinus P. murinus

44. Selenocosmia crassipes S. crassipes

45. Selenocosmia effera S. effera

46. Selenocosmia sp. (Papua Selenocosmia sp. 1

 

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New-Guinea) 47. Selenocosmia sp. Selenocosmia sp. 2

48. Selenotholus foelschei S. foelschei

49. Thrixopelma cyaneolum

(PERU) T. cyaneolum

50. Thrixopelma sp. (Chile) Thrixopelma sp.

51. Thrixopelma lagunas sp.

(Peru) T. lagunas

Potential agonistic effect on α7 nAChR. Further analysis is required to

confirm the venom effect on subtype α7.

52. Haplopelma hainanum H. hainanum

53. Pamphobeteus sp.

(platyomma) Pamphobeteus sp.

54. Sericopelma rubronites S. rubronites

55. Xenesthis sp. (‘white’,

Colombia) Xenesthis sp.

No effect on α3* and α7 nAChR

56. Aphonopelma crinitum A. crinitum

57. Avicularia sp. (Rio Madre,

Bolivia) Avicularia sp. 2

58. Homoeomma sp. (blue) Homoeomma sp.

59. Pseudhapalopus sp.

(langhaarig) Pseudhapalopus sp.

Table II. List of scorpions whose venoms were used in this work

Abbreviations

Buthidae family

1. Androctonus australis A. australis

2. Androctonus crassicauda A. crassicauda

3. Leirurus quinquestriatus L. quinquestriatus

4. Grosphus grandidieri G. grandidieri

Scorpionidae family

 

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Agonistic effect on α3* and α7

nAChR

1. Heterometrus spinnifer H. spinnifer