Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels.
ABSTRACT The μO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC₅₀ 95.9±74.3 nM) and Na(v)1.4 (IC₅₀ 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC₅₀ 431-6203 nM; Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management.
- SourceAvailable from: Oliver Knapp[Show abstract] [Hide abstract]
ABSTRACT: Voltage-gated sodium channels (VGSC) are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.Toxins 01/2012; 4(11):1236-60. · 2.13 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ciguatera, the most common form of non-bacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon-reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin-induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH-SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin-induced Ca(2+) responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin-sensitive ciguatoxin-evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin-induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin-induced cold allodynia did not correlate with their potency to inhibit ciguatoxin-induced responses in SH-SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7 or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera.Pain 06/2013; · 5.64 Impact Factor
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ABSTRACT: Cold allodynia, pain in response to cooling, occurs during or within hours of oxaliplatin infusion and is thought to arise from a direct effect of oxaliplatin on peripheral sensory neurons. To characterize the pathophysiological mechanisms underlying acute oxaliplatin-induced cold allodynia, we established a new intraplantar oxaliplatin mouse model that rapidly developed long-lasting cold allodynia mediated entirely through tetrodotoxin-sensitive Nav pathways. Using selective inhibitors and knockout animals, we found that Nav1.6 was the key isoform involved, while thermosensitive transient receptor potential channels were not involved. Consistent with a crucial role for delayed-rectifier potassium channels in excitability in response to cold, intraplantar administration of the K(+)-channel blocker 4-aminopyridine mimicked oxaliplatin-induced cold allodynia and was also inhibited by Navl.6 blockers. Intraplantar injection of the Nav1.6-activator Cn2 elicited spontaneous pain, mechanical allodynia and enhanced 4-aminopyridine-induced cold allodynia. These findings provide behavioural evidence for a crucial role of Nav1.6 in multiple peripheral pain pathways including cold allodynia.Pain 05/2013; · 5.64 Impact Factor
Isolation, characterization and total regioselective synthesis of the novel
mO-conotoxin MfVIA from Conus magnificus that targets voltage-gated
Irina Vettera,1, Zoltan Dekana,1, Oliver Knappb, David J. Adamsb, Paul F. Alewooda, Richard J. Lewisa,*
aDivision of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia
bHealth Innovations Research Institute, RMIT University, Melbourne, Australia
Voltage-gated sodium channels (Nav) are primarily found in
excitable cells, including central and peripheral neurons as well as
in skeletal and cardiac muscle, where they play a pivotal role in the
initiation and propagation of action potentials. The nine pore-
forming a subunits, termed Nav1.1–Nav1.9, are differentially
expressed in various tissues. Nav1.4 expression is restricted to
skeletal muscle and Nav1.5 is found primarily in cardiac muscle
[1,2]. All other isoforms are expressed, in a temporally and spatially
restricted manner, in central or peripheral neurons, where they
have been proposed as pharmacological targets for a variety of
neurological diseases including epilepsy and pain [2–4].
The tetrodotoxin-resistant (TTX-R) isoform Nav1.8 is expressed
predominantly in small nociceptive afferent fibres, where it
contributes to action potential initiation and repetitive firing
[2,5–7]. In addition, Nav1.8 is essential for maintaining excitability
at noxious cold temperatures and the development of cold pain .
A variety of inflammatory mediators have been shown to increase
Nav1.8 activity in sensory neurons by increasing protein expres-
sion and/or modulating channel kinetics . Similarly, Nav1.8
protein expression is increased locally after nerve injury [9,10],
Biochemical Pharmacology 84 (2012) 540–548
A R T I C L E
I N F O
Received 6 March 2012
Accepted 9 May 2012
Available online 16 May 2012
Regioselective disulfide bond synthesis
Voltage-gated sodium channels
A B S T R A C T
The mO-conotoxins are notable for their unique selectivity for Nav1.8 over other sodium channel
isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the
discovery of a novel mO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput
screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence
RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to mO-conotoxin MrVIB. To
overcome the synthetic challenges posed by mO-conotoxins due to their hydrophobic nature and
difficult folding, we developed a novel regioselective approach for the synthesis of mO-conotoxins.
Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully
protected peptide allowed manipulations in organic solvents with no chromatography required between
steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields.
Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysio-
logical recording techniques. MfVIA preferentially inhibits Nav1.8 (IC5095.9 ? 74.3 nM) and Nav1.4 (IC50
81 ? 16 nM),
Nav1.5 > 1.6 ? 1.7 ? 1.3 ? 1.1 ? 1.2). This improved approach to mO-conotoxin synthesis will facilitate
the optimization of mO-conotoxins as novel analgesic molecules to improve pain management.
Crown Copyright ? 2012 Published by Elsevier Inc. All rights reserved.
Abbreviations: Nav, voltage-gated sodium channel; Acm, acetamidomethyl; Boc,
tertbutyloxycarbonyl; CHO, Chinese hamster ovarian; DCM, dichloromethane;
DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DRG, dorsal root
ganglion; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid; HF, anhydrous hydrogen fluoride; HFIP, 1,1,1,3,3,3-
hexafluoroisopropanol; Meb, 4-methylbenzyl; Pbf, 2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl; RP-HPLC, reversed-phase high-performance liquid chro-
matography; tBu, tertbutyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; Trt,
triphenylmethyl; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive; ESI-
MS, electrospray ionization mass spectrometry; FBS, foetal bovine serum; RPMI,
Roswell Park Memorial Institute; DMEM, Dulbecco’s Modified Eagle’s Medium;
MALDI-TOF, Matrix-assisted laser desorption/ionization-time of flight mass
spectrometry; CHCA, a-cyano-4-hydroxycinnamic acid; HBTU, 2-(1H-Benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; FLIPR, fluorescent
imaging plate reader; EDTA, ethylenediaminetetraacetic acid; HBSS, Hank’s
balanced salt solution; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.
* Corresponding author at: Institute for Molecular Bioscience, The University of
Queensland, St Lucia QLD 4072, Australia. Tel.: +61 7 3346 2374;
fax: +61 7 3346 2984.
E-mail address: firstname.lastname@example.org (R.J. Lewis).
1These authors contributed equally to this research.
Contents lists available at SciVerse ScienceDirect
jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m
0006-2952/$ – see front matter. Crown Copyright ? 2012 Published by Elsevier Inc. All rights reserved.
suggesting a prominent role of this Navsubtype in neuropathic
pain. Indeed, knockout and gene deletion studies have confirmed
Nav1.8 as an attractive analgesic target in inflammatory and
neuropathic pain [9,11,12].
While peptides have yielded some of the most subtype-
selective Navinhibitors known, few Nav1.8-selective modulators
have been described to date. The mO-conotoxins MrVIA and
MrVIB, from the venom of Conus marmoreus, are of particular
interest because they potently inhibit Nav1.8 in mammalian
sensory neurons with >10-fold selectivity over tetrodotoxin-
sensitive (TTX-S) Navisoforms [13,14]. Accordingly, both peptides
display analgesic activity in a variety of animal models of pain and
decrease mechanical and thermal pain in inflammatory and
neuropathic pain models [14,15]. Importantly, selectivity over
motor side effects was more than 30-fold greater than for
lidocaine , making the mO-conotoxins promising leads for the
development of novel, Nav1.8-selective molecules with analgesic
Here, we report the isolation, synthesis and pharmacological
characterization of a novel mO-conotoxin, MfVIA, isolated from the
venom of Conus magnificus. MfVIA is selective for Nav1.8 and
Nav1.4 over other mammalian Navsubtypes and displays weak
activity at the centrally expressed Nav1.2. To facilitate the study of
mO-conotoxins, we have developed a novel approach to synthe-
sizing MfVIA, resulting in improved yields of the correctly folded
2.1. Isolation of MfVIA
Crude venom was isolated from two specimens of Conus
magnificus by stripping the venom duct contents. Stripped venom
was dissolved in 30% acetonitrile/0.1% formic acid, vortexed and
centrifuged at 10,000 g for 5 min to remove insoluble components.
Crude venom (500 mg) was fractionated into 60 ? 1 min fractions
using a Vydac 218TP C18 column (250 ? 4.6 mm, 5 mm) eluted at a
flow rate of 0.7 mL/min with 5–100% solvent B over 60 min
(solvent A, H2O/0.1% formic acid; solvent B, 90% acetonitrile/0.1%
Native peptide purified by reversed-phase high-performance
liquid chromatography (RP-HPLC) was analyzed on a MALDI-TOF
mass spectrometer (4700 Proteomics Analyzer, Applied Biosys-
tems, Mulgrave, VIC) using a-cyano-4-hydroxycinnamic acid
(CHCA) (5 mg/mL) as the matrix. The sequence of the purified
peptide was determined by Edman degradation at the Australian
Proteome Research Facility. In brief, the peptide was dissolved in
urea (4 M) in ammonium bicarbonate (50 mM) and reduced
with dithiothreitol (100 mM) at 56 8C for 1 h under argon.
The sample was then alkylated using acrylamide (220 mM)
for 0.5 h in the dark. The reaction was quenched by the addition
of excess dithiothreitol. After desalting by RP-HPLC, the
collected fraction was loaded onto pre-cycled bioprene discs
and subjected to 35 cycles of Edman N-terminal sequencing
using an Applied Biosystems 494 Procise Protein Sequencing
2.2. Peptide synthesis
MfVIA was assembled using standard Fmoc protocols on
preloaded H-Val-2-chlorotrityl resin on a 0.1 mmol scale
(0.61 mmol/g resin loading) on a Symphony (Protein Technologies
Inc.) automated peptide synthesizer. Couplings were performed
using 5 equivalents (relative to resin loading) of amino
acid:HBTU:DIEA (1:1:1) for 2 ? 20 min. Fmoc deprotections
were accomplished using 30% piperidine/dimethylformamide
(DMF) (1 ? 1 min then 1 ? 3 min). The cysteine side-chain
protecting groups used were 4-methylbenzyl (Meb) for Cys 1
and 4, acetamidomethyl (Acm) for Cys 2 and 5, and triphenyl-
methyl (Trt) for Cys 3 and 6. The remaining side-chain
functionalities were protected as Asp(OtBu), Glu(OtBu), Lys(Boc),
Gln(Trt), Arg(Pbf), Trp(Boc) and Tyr(tBu). After removal of the
final Fmoc group, the fully side-chain protected peptide was
cleaved from the resin using 10% AcOH/10% trifluoroethanol (TFE)
in dichloromethane (DCM) for 1 h. The resin was then filtered off,
the solvent evaporated under vacuum and triturated with hexane
(3x) to remove traces of AcOH giving a white powder (386 mg).
ESI-MS (m/z) calculated (average) 1822.6 [M + 3H]3+, observed
Selective oxidative deprotection of the Cys 3–6 (Trt) groups was
based on the method described by Kamber et al. . The fully
protected peptide (386 mg) was dissolved in neat 1,1,1,3,3,3-
hexafluoroisopropanol (HFIP) (5 mL) and added drop-wise over
5 min to a solution of I2(8 eq) in 20 mL of 10% HFIP/DCM, then
stirred at room temperature for an additional 5 min. The solution
was then poured into 0.2 M ascorbic acid/0.5 M NaOAc. The
aqueous layer was extracted with 10% MeOH/CHCl3(2x) then DCM
(1x). The organic layer was washed with H2O (1x) and dried over
MgSO4. The solvent was removed to obtain a sticky solid (357 mg).
ESI-MS (m/z) calculated (average) 1660.7 [M + 3H]3+, observed
To oxidise the Cys 2–5 (Acm) pair, the obtained product was
redissolved in DMF (10 mL) and added drop-wise to a stirred
solution of I2(8 eq) in DMF (20 mL) at room temperature over
20 min. After stirring for another 20 min, the solution was diluted
with DCM (150 mL) and washed with 0.2 M ascorbic acid/0.5 M
NaOAc. The aqueous layer was extracted with 10% MeOH/CHCl3
(2x) and DCM (1x), then the organic layer was washed with H2O
(1x) and dried over MgSO4. Most of the solvent was removed under
vacuum and the product was precipitated with cold Et2O and
hexane. The precipitate was collected by filtration and dried to give
an off-white powder (295 mg). ESI-MS (m/z) calculated (average)
1612.4 [M + 3H]3+, observed 1612.5.
To remove the remaining protecting groups (except Cys(Meb)),
the peptide was treated with 95% trifluoroacetic acid (TFA)/2.5%
triisopropylsilane/2.5% H2O (20 mL) for 2 h at room temperature.
Most of the solvent was removed under vacuum. The product was
precipitated with Et2O, collected by filtration and lyophilized from
0.05% TFA in MeCN/H2O (1:1) to give a white powder (230 mg).
ESI-MS (m/z) calculated (average) 1287.2 [M + 3H]3+, observed
1287.1 (Fig. 3A).
The Cys 1–4 (Meb) groups were removed using 90% hydrogen
fluoride (HF)/10% p-cresol at 0 8C for 1 h. The product was
precipitated with Et2O and lyophilized from 0.05% TFA in MeCN/
H2O (1:1) to give a white powder (180 mg). ESI-MS (m/z)
calculated (average) 1217.8 [M + 3H]3+, observed 1217.7 (Fig. 3B).
To oxidize the obtained Cys 1–4 free thiols, the crude product
(180 mg) was redissolved in 0.1% TFA in MeCN/H2O (1:1) at a
concentration of ?0.3 mg/mL. A solution of I2(4 eq) in MeCN was
added and stirred for 20 min before the addition of ascorbic acid to
obtain a colorless solution (Fig. 3C). This solution was purified by
preparative HPLC (6 separate runs) on a Vydac C18 218TP1022
column, running at 16 mL/min using a gradient of 30–70% B over
60 min. The combined fractions were lyophilized to give a total of
15 mg white powder. The overall yield was 4%, as calculated from
the initial resin loading. ESI-MS (m/z) calculated 1217.1 [M + 3H]3+,
Analytical HPLC of the synthetic intermediates was performed
on a Shimadzu LC-20AT system using a Thermo Hypersil GOLD C18
2.1 ? 100 mm column at a flow rate of 0.3 mL/min with a gradient
of 25 to 70% B over 30 min. Solvent A consisted of 0.05% TFA/H2O,
solvent B contained 0.043% TFA in 90% MeCN/H2O.
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
2.3. Biological activity
2.3.1. Cell culture and transfection
Activity-guided isolation of MfVIA was carried out using a FLIPR
assay assessing veratridine-stimulated
activity of Navendogenously expressed in the human neuroblas-
toma cell line SH-SY5Y . SH-SY5Y human neuroblastoma cells
were a gift from Victor Diaz (Max Planck Institute for Experimental
Medicine, Goettingen, Germany). Cells were maintained at 37 8C/
5% CO2 in Roswell Park Memorial Institute (RPMI) medium
containing 15% foetal bovine serum (FBS) and 2 mM L-glutamine.
Human embryonic kidney 293 (HEK293) cells and ND7/23 cells (all
American Tissue Culture Collection, Manassas, VA, USA) were
maintained at 37 8C in a 5% humidified CO2incubator in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing 10% FBS, 2 mM L-
glutamine, pyridoxine and 110 mg/mL sodium pyruvate (Invitro-
gen). Chinese hamster ovarian (CHO) cells stably expressing
hNav1.4, hNav1.6, and hNav1.7 a-subunits (Genionics, Schlieren,
Switzerland) were grown in F-12 Medium (Invitrogen, Mulgrave,
VIC), supplemented with 9% fetal bovine serum (Lonza, Mt
Waverley, VIC), 0.9% penicillin/streptomycin (Invitrogen) and
100 mg/mL hygromycin B (Invitrogen). HEK293 cells stably
transfected with the hNav1.5 a-subunit (Genionics) were grown
in Dulbecco’s Modified Eagle Medium (Invitrogen), 0.9% penicillin/
streptomycin and 100 mg/mL zeocin (Invitrogen). All cells were
split every 3–6 days in a ratio of 1:5, using 0.25% trypsin/EDTA
(ethylenediaminetetraacetic acid) (Invitrogen).
ND7/23 cells were plated on T75 tissue culture flasks 24 h prior to
transfection with plasmid DNA of rNav1.8 cloned into pCMV-Script
(K. Zimmermann, University of Erlangen) using Fugene (Roche, Dee
Why, NSW). Nav1.2 and Nav1.5 were transfected in HEK293 cells
using a commercial BacMam transfection system (Invitrogen),
following the manufacturer’s instructions. In brief, cells were
incubated with virus solution in phosphate-buffered saline for 3 h
at room temperature, then incubated with enhancer solution in
complete medium for 2 h at 37 8C. BacMam-transfected cells were
plated on 384-well plates after an overnight incubation in normal
complete growth medium at 37 8C in a 5% humidified CO2incubator.
They were then assayed after incubation for another 24 h. hNav1.1,
hNav1.3, hNav1.5, hNav1.6 and hNav1.7 EZCells were purchased from
ChanTest Corp (Cleveland, OH, USA), plated on 384-well plates
immediately after thawing, and assayed after incubation for another
24 h at 37 8C in a 5% humidified CO2incubator.
2.4. FLIPR Ca2+and membrane potential assays
Ca2+responses were assessed using the FLIPRTETRA(Molecular
Devices, Sunnyvale, CA) plate reader, as described previously .
SH-SY5Y cells were plated at a density of 50,000 cells/well on 384-
well black-walled imaging plates (Corning), 48 h before the assay.
Cells were loaded with the Calcium 4 No-wash dye (Molecular
Devices) by diluting the lyophilized dye in physiological salt
solution, composition: 140 mM NaCl, 11.5 mM glucose, 5.9 mM
KCl, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 1.8 mM
CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic ac-
id (HEPES), pH 7.4, and incubated for 30 min at 37 8C in a 5%
humidified CO2incubator. Fluorescence responses were recorded
every second for 300 s after adding crude venom or venom
fractions (excitation, 470–495 nm; emission, 515–575 nm). A
further 300 fluorescence reads were recorded at 1 s intervals after
adding veratridine (50–100 mM).
To assess changes in membrane potential, HEK293, ND7/23 and
CHO cells expressing Nav1.1–Nav1.8 isoforms were loaded with
membrane potential dye (red, proprietary composition; Molecular
Devices) for 30 min at 37 8C in a 5% humidified CO2incubator. After
addition of MfVIA using the FLIPRTETRA, cells were incubated a
further 30 min before stimulating Navsubtypes using veratridine
(Nav1.1–Nav1.7) or deltamethrin (100 mM, Nav1.8) . Fluores-
cence reads (excitation 510–545 nm; emission 565–625 nm) were
acquired every second for 300 s after adding agonists.
Sub-confluent HEK293 and CHO cells were detached from the
cell culture flask with 0.25% trypsin (Invitrogen), washed with
extracellular solution and pelleted by centrifugation at 300 g for
5 min. After agitating the cells to break cell clusters and an
additional centrifugation step, we resuspended the pellet in
extracellular solution to get optimal concentration of 5 ? 105–
5 ? 107cells/mL for electrophysiological experiments.
Depolarization-activated Na+currents from Nav1.4, Nav1.5,
Nav1.6 and Nav1.7 were recorded using a Port-a-Patch system
(Nanion, Munich, Germany) in the whole-cell mode. Cells were
held at ?80 mV in the whole-cell recording configuration and
depolarized every 20 s–0 mV. The experiments were carried out at
room temperature (22–23 8C). Concentration-response curves
were obtained by adding an external solution containing various
concentrations of MfVIA to the recording chamber and Na+
currents were recorded using a HEKA EPC10 patch-clamp amplifier
(HEKA Elektronik GmbH, Lambrecht/Pfalz, Germany). The external
solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, 5 mM D-glucose and 10 mM HEPES, pH 7.4 (NaOH). The
internal solution contained 50 mM CsCl, 10 mM NaCl, 60 mM CsF,
2 mM MgCl2, 20 mM EGTA, and 10 mM HEPES, pH 7.2 (CsOH).
2.6. Oocyte preparation, RNA injection and oocyte electrophysiology
Stage V–VI oocytes were harvested from sexually mature
female Xenopus laevis anesthetized with 0.1% tricaine (3-amino-
benzoic acid ethyl ester), following protocols approved by the
RMIT University Animal Ethics Committee. The oocytes were
defolliculated in collagenase (1.5 mg/mL) and dissolved in calci-
um-free solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and
5 mM HEPES-NaOH, at pH 7.4) before RNA injection. The
constructs encoding rNav1.2 and hNav1.8 (cloned into pcDNA3.1)
were linearized and cRNA was synthesized using a T7 RNA
polymerase in vitro transcription kit (mMESSAGE mMachine;
Ambion, Foster City, CA). cRNA concentration was measured
spectrophotometrically and adjusted to 2.5 ng rNav1.2 and 5 ng
hNav1.8 for injection in Xenopus laevis oocytes. Injected oocytes
were incubated for 2–5 days at 18 8C in ND98 solution containing
96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2and 5 mM
HEPES-NaOH, at pH 7.4 supplemented with pyruvate (5 mM), and
gentamicin (50 mg/ml).
Oocytes were transferred to a 50 mL volume recording chamber
and gravity perfused at 1.5–2.5 mL/min with ND96 solution
(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2and 5 mM
HEPES, at pH 7.4 (NaOH)). Two-electrode (virtual ground circuit)
voltage-clamp experiments were performed at room temperature
(21–23 8C) using a GeneClamp 500B amplifier (Molecular Devices)
and pCLAMP 8 software (Molecular Devices). Peak Na+current was
elicited at a test potential of +10 mV for Nav1.8 and ?10 mV for
Nav1.2. Micropipettes pulled from glass capillaries (3-000-203 GX,
Drummond Scientific Co.) and filled with 3 M KCl had open-tip
resistances of 0.3–1.5 M V. Whole-cell Na+currents were low-pass
filtered at 1 kHz and digitized at 10 kHz. Leak currents were
subtracted online using a-P/6 protocol.
2.7. Dorsal root ganglion (DRG) neuron preparation
DRG neurons were enzymatically dissociated from ganglia of 6
to 14-day-old Wistar rats according to standard protocols. Rats
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
were killed by cervical dislocation, as approved by the RMIT
University Animal Ethics Committee. Ganglia of all areas of the
spinal cord were collected in ice cold Hank’s Balanced Salt Solution
(HBSS; Invitrogen) and incubated in 100 mg/mL collagenase
(Sigma Aldrich) in HBBS for 30 min at 37 8C. Ganglia were
triturated with a fine bore glass pipette and resuspended in warm
Neurobasal media containing B27 supplement (both Invitrogen),
0.5 mM L-glutamine and 1% Penicillin/Streptomycin. They were
then seeded onto poly-D-lysine-coated multi-well plates or glass
cover slips. The cells were incubated at 37 8C in a 5% humidified
CO2incubator and used after 2-4 days.
Depolarization-activated Na+currents were recorded using the
whole-cell patch-clamp technique with an Axopatch 200B amplifier
(Molecular Devices). The patch recording electrodes had resistances
of 1.5–3 M V. Under the voltage-clamp recording configuration,
cells were depolarized to +10 mV from a holding potential of
?80 mV every 20 s to elicit voltage-gated Na+channel currents.
Membrane currents were generated using pClamp 9.2 software
(Molecular Devices), filtered at 2 kHz and sampled at 10 kHz by a
Digidata 1322A interface (Molecular Devices). Sampled data was
stored digitally on a personal computer for analysis. Na+currents
were recorded in the presence of TTX (300 nM) using patch pipettes
filled with an internal solution containing 10 mM NaCl, 130 mM CsF,
10 mM CsCl, 10 mM EGTA, 10 mM HEPES, pH 7.2 (CsOH). The bath
solution contained 50 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM
CaCl2, 10 mM glucose, 90 mM tetraethylammonium (TEA)-Cl,
10 mM HEPES, pH 7.3 (TEA-OH). Series resistance was routinely
compensated by 70–80%. Capacitive and leakage currents were
digitally subtracted using a-P/6 pulse protocol.
MfVIA was diluted to the appropriate final concentration and
applied via perfusion in the bath solution. The toxin solution
contained ?0.01% dimethyl sulfoxide (DMSO). DMSO at this
concentration had no effect on Na+currents. Peak current amplitude
in response to the depolarizing pulse was acquired once we achieved
a steady-state current (?5–10 min). The current amplitudes
recorded in the presence of the drug were normalized by dividing
by the current amplitude from control conditions (I/Imax).
2.8. Data analysis
Unless otherwise stated, all data are expressed as the mean
? standard error of the mean (SEM) determined from at least n = 3
replicates and are representative of at least 3–5 independent
experiments. For measurement of membrane potential, responses
after addition of veratridine or deltamethrin were plotted against
MfVIA concentration. A 4-parameter Hill equation with variable Hill
slope was fitted to the data using GraphPad Prism (Version 4.00, San
Diego, California). Potency of MfVIA is reported as the mean ? SEM of
3–5 separate experiments. Electrophysiological data were analyzed
using PatchMaster v2.20 (HEKA Elektronik GmbH, Lambrecht/Pfalz,
Germany). Concentration-response curves were analyzed using
SigmaPlot 10 (Systat, Chicago USA) and fitted with a 4-parameter
logistic curve. Oocyte electrophysiological data were analyzed with
Clampfit (v 10.2, MDS Analytical Technologies) and Sigma Plot (v 11,
Systat Software Inc.) software. Hill slopes were determined to be
statistically significantly different from ?1 if the 95% confidence
intervals calculated using GraphPad Prism (Version 4.00, San Diego,
California) did not include ?1.
For other data, statistical significance was determined using a
one-way ANOVA with Tukey’s post hoc test and defined as p < 0.05
unless otherwise stated.
2.9. Chemicals and reagents
We sourced veratridine from Ascent Scientific (Bristol, UK).
Fmoc amino acids, resins and HBTU were obtained from Iris
Biotech GmbH (Marktredwitz, Germany). All other reagents, unless
otherwise stated, were sourced from Sigma–Aldrich (Castle Hill,
3.1. Isolation of a novel mO-conotoxin from Conus magnificus venom
Crude venom isolated from Conus magnificus inhibited veratri-
dine-induced Nav responses in SH-SY5Y cells at ?300 mg/mL.
Activity-guided fractionation revealed activity corresponding to a
single peak eluting at ?64% solvent B (Fig. 1A). N-terminal Edman
degradation of the purified native peptide corresponding to this
activity revealed a novel 32-residue mO-conotoxin homologous to
MrVIA and MrVIB (Table 1) with the following sequence:
RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV (Table 1).
Compared with MrVIB, this novel peptide has an extended
N-terminus and the residue substitutions A1D, S3Q and K4E
Fig. 1. Isolation of MfVIA from Conus magnificus crude venom. (A) Crude venom isolated from Conus magnificus (500 mg) was fractionated on a Vydac 218TP C18 column with a
two-step linear acetonitrile/0.1% formic acid gradient (5–50% B/10 min, 50–100% B/50 min; blue line). Green circles: activity of 1 min fractions on Navresponses in SH-SY5Y
cells. The fractions containing MfVIA (arrow; elution at ?64% B) fully inhibited veratridine-induced Navresponses. Inset; Conus magnificus shell. (B) Native peptide purified by
RP-HPLC was analyzed on a MALDI-TOF mass spectrometer. The observed mass (M + H, 3646.7 m/z) was consistent with the predicted mass of MfVIA. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of the article).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
(Table 1). The observed mass for MfVIA (M + H 3646.7 Da) was
consistent with the predicted mass of the peptide sequence
obtained by Edman degradation (Fig. 1B). Identity of the peptide
was further confirmed by co-elution of the native and synthetic
peptide (Fig. 3D).
3.2. An improved approach for regioselective synthesis of mO-
mO-conotoxins like MfVIA have until now posed significant
synthetic challenges. Their highly hydrophobic nature leads to
aggregation in solution and poor recovery. In addition, difficulties
with oxidative folding of the three disulfide bonds to the native
isomer have hindered the efficient synthesis, structure–activity
studies and further optimization of these molecules. Thus, we
developed a simple and more efficient, fully regioselective protocol
for the synthesis of MfVIA requiring only a single HPLC purification
step (Fig. 2).
Peptide assembly was performed using Fmoc chemistry on the
highly acid-labile 2-chlorotrityl resin, with cysteine side-chains
protected as 1,4(Meb), 2,5(Acm) and 3,6(Trt). Cleavage from the
resin using AcOH left all side-chain protecting groups intact,
enabling subsequent manipulations to be performed in organic
solvents. This circumvented the poor aqueous solubility and
chromatographic problems we previously encountered with
MrVIB. Based on the solvent-dependent rate difference between
the reactions of S-Trt and S-Acm groups towards iodine , the
Cys3–6 and Cys2–5 disulfide bonds were formed selectively by
iodine oxidation of the 3,6(Trt) cysteine pair in a HFIP/DCM
mixture, followed by oxidation of the 2,5(Acm) pair by a second
treatment with iodine in DMF [16,20]. The third protecting group
for the Cys1-4 pair required more optimization. Our initial trials
with the p-methoxybenzyl group, which was expected to be stable
to iodine treatment  and is removable by other oxidative
methods [21,22], resulted in loss of regioselectivity at the iodine-
DMF oxidation step. This was most likely because of the reaction of
p-methoxybenzyl thioether with the sulfenyl iodide intermediates
[16,23] formed during deprotection of the Acm groups. The p-
methylbenzyl (Meb) group proved to be completely stable under
these conditions and was used to complete the synthesis. The
remaining side-chain protecting groups, except Cys 1–4 (Meb),
were removed by treating the Cys 3–6 and Cys 2–5 oxidized
peptides with TFA (Fig. 3A). The Meb groups were cleaved using HF
to obtain the free thiols in the 1–4 positions (Fig. 3B), which were
then oxidized by iodine treatment in acidic aqueous acetonitrile
solution (Fig. 3C) to give the fully folded product following
purification by HPLC. Identity of the product was confirmed by co-
elution with the native peptide (Fig. 3D).
Fig. 2. An improved approach for regioselective synthesis of mO-conotoxins. Schematic representation of the approach for regioselective synthesis of the disulfide bonds of
Peptide sequences of mO-conotoxins.
GECLGWSNYCTSHSI CCSGE CILSY CDIW
WPCKVAGSPCGLVSE CCGT CNVLRNRCV
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
3.3. Navsubtype selectivity of MfVIA
The biological activity of synthetic MfVIA was assessed using a
membrane potential assay in mammalian cells over-expressing
Navisoforms. Consistent with previously reported activity of mO-
expressed Nav1.8 (IC50529 ? 50 nM, Hill slope ?2.9 ? 0.37) over
the TTX-S and TTX-R Navisoforms Nav1.3 (IC502.2 ? 0.3 mM, Hill
slope ?1.7 ? 0.23), Nav1.5 (IC503.9 ? 0.9 mM, Hill slope ?1.5 ? 0.42),
Nav1.6 (IC504.6 ? 0.5 mM, Hill slope ?1.2 ? 0.12) and Nav1.1 (IC50
3.3 ? 0.1 mM, Hill slope ?1.6 ? 0.27) (Fig. 4A and B). Nav1.7 and
Nav1.2 were relatively resistant to inhibition by MfVIA (IC50
5.5 ? 0.9 mM, Hill slope ?2.3 ? 1.0 and 6.3 ? 0.7 mM, Hill slope
?4.4 ? 1.36, respectively) (Fig. 4A and B).
These results were confirmed using Xenopus oocytes expressing
Nav1.2 and Nav1.8 (Fig. 5) and cell lines (HEK and CHO)
heterologously expressing Nav1.4, Nav1.5, Nav1.6 and Nav1.7 a-
subunits (Fig. 6). MfVIA inhibited depolarization-activated Nav1.4
currents with an IC50 of 81 ? 16 nM, consistent with previous
reports that mO-conotoxins display relatively high affinity for the
skeletal muscle Navsubtype. Heterologously expressed Nav1.8 was
also potently inhibited with an IC50of 157.6 ? 63.8 nM. In contrast,
(IC50= 431 ? 146 nM), Nav1.6
Nav1.7 (IC50= 2.3 ? 0.3 mM) were much less sensitive to inhibition
by MfVIA. Similar to the results using membrane potential
measurement, Nav1.2 was the subtype most resistant to block by
MfVIA (IC50= 5.1 ? 0.5 mM). With the exception of Nav1.2 (Hill slope
?2.3 ? 0.3), the Hill slopes of MfVIA-mediated inhibition of Nav1.4
(IC50= 1.2 ? 0.2 mM)
Fig. 3. Analytical HPLC traces of synthetic MfVIA and intermediates. (A) Analytical HPLC traces of crude product obtained after TFA cleavage with oxidized Cys3–6 and Cys2–5
and Meb protected Cys 1 and 4 (B) crude product obtained after HF cleavage of the Meb groups to give the free thiols of Cys 1 and 4, and (C) crude product obtained after iodine
oxidation of Cys1-4. The retention time of the major peak corresponds to that of native MfVIA. (D) Co-elution of native and synthetic MfVIA. Analytical HPLC traces of native
and synthetic MfVIA show identical retention time and peak width.
Fig. 4. Navsubtype selectivity of MfVIA assessed using membrane potential assays. (A and B) Biological activity of synthetic MfVIA on veratridine-evoked Navresponses was
assessed using a membrane potential assay in mammalian cells overexpressing Nav1.1 (light blue; IC50= 3.3 ? 0.1 mM), Nav1.2 (purple; IC50= 6.3 ? 0.7 mM), Nav1.3 (light
green; IC50= 2.2 ? 0.3 mM), Nav1.5 (magenta, IC50= 3.9 ? 0.9 mM), Nav1.6 (dark green; IC50= 4.67 ? 0.5 mM), Nav1.7 (dark blue; IC50= 5.5 ? 0.9 mM) and Nav1.8 (orange;
IC50= 529 ? 50 nM). (B) MfVIA was significantly (p < 0.05) more potent at heterologously expressed Nav1.8 (pIC506.28 ? 0.05) than the TTX-S and TTX-R Navisoforms Nav1.3 (pIC50
5.68 ? 0.09), Nav1.5 (pIC505.55 ? 0.14), Nav1.6 (pIC505.65 ? 0.04) and Nav1.1 (pIC505.47 ? 0.02). Nav1.7 and Nav1.2 were relatively resistant to inhibition by MfVIA (pIC50
5.36 ? 0.13 and 5.21 ? 0.05, respectively). Data are presented as mean ? SEM from at least 3 independent experiments. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
(Hill slope ?0.8 ? 0.11), Nav1.5 (Hill slope ?1.0 ? 0.3), Nav1.6 (Hill
slope ?3.3 ? 1.98), Nav1.7 (Hill slope ?1.9 ? 0.6) and Nav1.8 (Hill
slope ?1.2 ? 0.28) were not statistically different from ?1. Finally,
the effects of MfVIA on whole-cell TTX-R currents in rat DRG neurons
were assessed in the presence of TTX under conditions that inactivate
Nav1.9, but not Nav1.8. In this assay, MfVIA (1 mM) potently inhibited
native rat Nav1.8 (IC5095 ? 74 nM) (Fig. 7).
mO-Conotoxins belong to the O superfamily of conopeptides
that inhibit Navby interfering with the voltage sensors in domain II
of Nav[24,25]. Despite their selectivity for Nav1.8 over other TTX-S
isoforms, and resultant analgesic activity in a variety of animal
models of pain [13–15], little is known of the structure-activity
relationships that define their subtype specificity. In addition, only
four native mO-conotoxins have been described to date, including
the recently identified LtVIIA and LtVIC [26,27] which were
classified as mO-conotoxins based on sequence homology and
Fig. 5. Inhibition of depolarization-activated Nav1.2 and Nav1.8 currents by
synthetic MfVIA. (A) Voltage protocols and superimposed inward Na+currents
evoked by a voltage step are shown in the absence (control) and presence of MfVIA
(1 mM) for Nav1.2 and Nav1.8 expressed in Xenopus oocytes. For ease of comparison,
data are presented as I/Imax. Peak current amplitudes were ?2.0 nA (Nav1.2, 10%
inhibition by 1 mM MfVIA to ?1.8 nA) and ?0.4 nA (Nav1.8, 63% inhibition by 1 mM
MfVIA to ?0.15 nA). (B) Concentration-response relationships obtained for MfVIA
inhibition of Na+currents mediated by Nav1.2 (closed circle; IC50= 5.1 ? 0.5 mM)
and Nav1.8 (open circle; IC50= 157.6 ? 63.8 nM).
Fig. 6. Inhibition of depolarization-activated Nav1.4, Nav1.5, Nav1.6 and Nav1.7
currents by synthetic MfVIA. (A) Voltage protocols and superimposed inward Na+
currents evoked by a voltage step are shown in the absence (control) and presence
of MfVIA (1 mM) for Nav1.4, Nav1.5, Nav1.6 and Nav1.7 a-subunits expressed in
mammalian cell lines (HEK and CHO). For ease of comparison, data are presented as
I/Imax. Peak current amplitudes were ?5.1 nA (Nav1.4, 79% inhibition by 1 mM
MfVIA to ?1.1 nA); ?5.0 nA (Nav1.5, 76% inhibition by 1 mM MfVIA to ?1.2 nA);
?1.0 nA (Nav1.6, 24% inhibition by 1 mM MfVIA to ?0.76 nA) and ?1.6 nA (Nav1.7,
relationships obtained for MfVIA inhibition of Na+currents mediated by Nav1.4
(closed circle; IC50= 81 ? 16 nM), Nav1.5 (open square; IC50= 431 ? 146 nM), Nav1.6
(closed triangle; IC50= 1.2 ? 0.2 mM) and Nav1.7 (open rhombus; IC50= 2.3 ? 0.3 mM).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
pharmacological activity. However, MrVIA and MrVIB remain the
only mO-conotoxins for which mammalian Navsubtype selectivity
data is available, albeit not across all Nav subtypes. Here, we
describe the isolation of the novel mO-conotoxin MfVIA from the
venom of the molluscivorous Conus magnificus using a high-
throughput Navassay . MfVIA is a hydrophobic 32-residue
GLICGPFVCV) with highest sequence homology to mO-conotoxin
The pharmacological activity of synthetic mO-conotoxin MfVIA
was characterized across mammalian Nav1.1–Nav1.8 subtypes.
Despite relatively few changes in the sequence of MfVIA compared
with MrVIA and MrVIB, we found surprising changes in potency
and mammalian Nav subtype selectivity. Relative to Nav1.8
potency, MfVIA was found to have 3-fold enhanced Nav1.4 potency
and 5-fold reduced Nav1.2 potency (see Table 2). Interestingly,
MfVIA was ?5-fold more potent at native rNav1.8 expressed in
DRG neurons than rNav1.8 heterologously expressed in ND7/23
cells. This observation is consistent with the decreased affinity of
other mO-conotoxins, in particular MrVIB, for heterologously
expressed Nav1.8 compared with native Nav1.8-mediated TTX-R
currents in DRG neurons (Table 2). This effect has been suggested
to occur due to the presence of auxiliary b subunits, and perhaps
other accessory proteins, in neurons, which particularly affect the
on-rate of MrVIB and can thus profoundly affect the pharmacology
of mO-conotoxins .
The difference in potency between native and heterologous
expression systems was less pronounced for MfVIA than the ?15-
fold difference in potency reported for MrVIB [28,29]. This may be
due to the presence of endogenous b subunits in the ND7/23 cell
line we used for heterologous expression, as b subunits have been
reported to affect the Kdof mO-conotoxins at Nav1.8 [28,29]. The
potency of MfVIA may also be enhanced by the prolonged pre-
incubation protocol used in the present study, as b subunits have
been reported to particularly affect the on-rate of inhibition by
mO-conotoxins  and electrophysiological experiments gen-
erally preclude prolonged pre-incubation protocols. With the
exception of Nav1.4 and Nav1.8, MfVIA inhibited all other Nav
subtypes with significantly lower affinity (IC50 431–6203 nM;
Nav1.5 > 1.6 ? 1.7 ? 1.3 ? 1.1 ? 1.2). This includes Nav1.1 and
Nav1.6, for which mO-conotoxin activity has not previously been
The simplest model of mO-conotoxin-mediated inhibition of
Navchannels, where a single molecule of MfVIA is sufficient for full
channel block, is consistent with MfVIA block of all sodium channel
subtypes except Nav1.2. At this subtype, the concentration-
dependence of inhibition was signficantly steeper than a Hill
slope of ?1, indicative of positive cooperativity which may be more
apparent at Nav1.2. This might arise if mO-conotoxins have more
than a single binding site on sodium channels, bind to the channel
after first partitioning into the plasma membrane, or have slow on-
rates that make it difficult to reach equilibrium at low concentra-
tions of peptide. Although not directly assessed in this study, the
effects of membrane potential (Vm) on the interaction of mO-
conotoxins with Nav have been assessed systematically in the
literature [24,28]. These studies have found that inhibition of Nav
currents by MrVIA was voltage-dependent and strongly dimin-
ished after depolarizing voltage steps, consistent with mO-
conotoxins acting as gating modifiers. While MrVIA and MrVIB
are analgesic in various animal models of pain, the analgesic
activity of MfVIA remains to be determined.
The mO-conotoxins MfVIA, MrVIA and MrVIB are notable for
their unique selectivity profile for blocking Nav channels ,
making these peptides attractive drug leads for development of
improved analgesic molecules [14,30]. Unfortunately, these
peptides are also notable for the synthesis challenges they have
posed since their discovery [31,32]. These challenges arise due to
their highly hydrophobic nature and the difficulties of forming
their three disulfide bonds to the native isomer, which to date
could not be done through the oxidative folding of fully reduced
precursors. These properties have largely hindered the efficient
synthesis, structure-activity studies and further optimization of
Fig. 7. Effect of mO-conotoxin MfVIA on transient Na+currents in rat DRG neurons in
the presence of 300 nM TTX. (A) Voltage protocol and superimposed inward Na+
currents evoked by a voltage step to +10 mV from a holding potential of–80 mV are
shown in the absence (control) and presence of MfVIA (1 mM). (B) Concentration-
response relationships obtained for MfVIA inhibition of Na+currents mediated by
TTX-R currents in rat DRG neurons. MfVIA inhibition of native TTX-R currents in
DRG neurons gave an IC50of 95.9 ? 74.3 nM.
Navsubtype selectivity of mO-conotoxins MrVIA, MrVIB and MfVIA.
nr – not reported; nd – not determined.
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
While replacement of cysteines with selenocysteines has been
used to assist with the folding of MrVIB , the synthesis of the
native molecule currently relies on a semi-selective approach
[15,31] with a low overall yield (? 1%) and possibly requiring
labour-intensive re-optimizations for the preparation of non-
native analogues. Fully regioselective strategies that involve
chromatographic purifications between disulfide forming steps
are not well suited to hydrophobic peptides. In addition to being
time consuming, these approaches tend to produce even lower
yields of the mO-conotoxins due to poor recovery from RP-HPLC
purification steps. In these cases, an efficient one-pot oxidation
strategy would be ideal. However, using the described methods
 failed to produce correctly folded mO-conotoxins during our
To facilitate the study of mO-conotoxins, we developed an
improved, fully regioselective protocol for the synthesis of the novel
mO-conotoxin MfVIA. To confirm its usability, we successfully
applied this approach to the synthesis of MrVIB, which also required
only a single HPLC purification step (data not shown). Our approach
resulted in sufficient yields to enable the production of toxin for
detailed subtype selectivity analysis. This new synthetic approach is
expected to facilitate the first structure-activity relationship studies
and pharmacophore mapping of mO-conotoxins.
This research was facilitated by access to the Australian
Proteome Analysis Facility, supported under the Australian
Government’s National Collaborative Research Infrastructure
Strategy (NCRIS). We thank the Australian Proteome Research
Facility staff, in particular Bernie McInerney, for N-terminal
sequencing. This research was funded by a National Health and
Medical Research Council (NHMRC) Australian Postdoctoral
Fellowship (IV), NHMRC Research Fellowship (RJL), ARC Australian
Professorial Fellowship (DJA) and NHMRC Program grant (RJL, DJA
and PFA). The FLIPR was supported by an Australian Research
Council LIEF equipment grant.
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