Publications by authors named "Jeffrey R McArthur"

21 Publications

  • Page 1 of 1

Pathophysiological metabolic changes associated with disease modify the proarrhythmic risk profile of drugs with potential to prolong repolarisation.

Br J Pharmacol 2021 Nov 26. Epub 2021 Nov 26.

Victor Chang Cardiac Research Institute, Sydney, Australia.

Background And Purpose: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat COVID-19. While concern already existed around their proarrhythmic potential there is little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles.

Experimental Approach: Potency of hERG block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals.

Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC of 1.47±0.07 μM and 3.78±0.17 μM respectively, indicating proarrhythmic risk at concentrations effective against SARS-CoV-2 in vitro. Hypokalaemia and hypermagnesemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure, and identified specific genetic backgrounds associated with emergence of arrhythmia.

Conclusion And Implications: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation.
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http://dx.doi.org/10.1111/bph.15757DOI Listing
November 2021

Analgesic α-conotoxins modulate native and recombinant GIRK1/2 channels via activation of GABA receptors and reduce neuroexcitability.

Br J Pharmacol 2022 Jan 25;179(1):179-198. Epub 2021 Nov 25.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.

Background And Purpose: Activation of GIRK channels via G protein-coupled GABA receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABA receptors resulting in inhibition of Ca 2.2 and Ca 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K currents and on neuronal excitability.

Experimental Approach: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons.

Key Results: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA receptors. GABA receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABA receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability.

Conclusions And Implications: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo.
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http://dx.doi.org/10.1111/bph.15690DOI Listing
January 2022

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Stem Cells Transl Med 2021 08 18;10(8):1157-1169. Epub 2021 Mar 18.

Department of Biomedical Engineering, The University of Melbourne, Parkville, Australia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.
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http://dx.doi.org/10.1002/sctm.20-0334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284774PMC
August 2021

Small-molecule mimicry hunting strategy in the imperial cone snail, .

Sci Adv 2021 Mar 12;7(11). Epub 2021 Mar 12.

Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT 84112, USA.

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Using the model polychaete worm , we demonstrate that venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.
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http://dx.doi.org/10.1126/sciadv.abf2704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954447PMC
March 2021

Spider Venom Peptide Pn3a Inhibition of Primary Afferent High Voltage-Activated Calcium Channels.

Front Pharmacol 2020 28;11:633679. Epub 2021 Jan 28.

Discipline of Pharmacology, University of Sydney, Sydney, NSW, Australia.

Despite potently inhibiting the nociceptive voltage-gated sodium (Na) channel, Na1.7, -theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses >3,000-fold greater than its Na1.7 by a yet undefined mechanism. Na channels are structurally related to voltage-gated calcium (Ca) channels, Ca1 and Ca2. These channels mediate the high voltage-activated (HVA) calcium currents ( ) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch clamp recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA- and 60-80% of Ca1.2, Ca1.3, Ca2.1, and Ca2.2 mediated currents in HEK293 cells, with no inhibition of Ca2.3. As a major DRG component, Ca2.2 inhibition by Pn3a ( = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA- inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Cas at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA- by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA- at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.
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http://dx.doi.org/10.3389/fphar.2020.633679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875911PMC
January 2021

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons.

Front Cell Neurosci 2020 4;14:600895. Epub 2020 Dec 4.

Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.
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http://dx.doi.org/10.3389/fncel.2020.600895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761588PMC
December 2020

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds.

Mar Drugs 2020 Mar 20;18(3). Epub 2020 Mar 20.

College of Engineering and Technology, American University of the Middle East, Kuwait.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.
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http://dx.doi.org/10.3390/md18030173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143316PMC
March 2020

Ketamine inhibits synaptic transmission and nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglia in situ.

Neuropharmacology 2020 03 3;165:107932. Epub 2020 Jan 3.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, 2522, Australia. Electronic address:

The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-clamp modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107932DOI Listing
March 2020

Structural basis of the potency and selectivity of Urotoxin, a potent Kv1 blocker from scorpion venom.

Biochem Pharmacol 2020 04 25;174:113782. Epub 2019 Dec 25.

Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW 2522, Australia; Electrophysiology Facility for Cell Phenotyping and Drug Discovery, IHMRI, Wollongong, NSW 2522, Australia. Electronic address:

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1-1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin's structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin.
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http://dx.doi.org/10.1016/j.bcp.2019.113782DOI Listing
April 2020

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

Mar Drugs 2019 Aug 29;17(9). Epub 2019 Aug 29.

Department of Physiology & Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10 to 10 M), we estimated an IC = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.
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http://dx.doi.org/10.3390/md17090510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780087PMC
August 2019

Analgesic transient receptor potential vanilloid-1-active compounds inhibit native and recombinant T-type calcium channels.

Br J Pharmacol 2019 07 16;176(13):2264-2278. Epub 2019 May 16.

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.

Background And Purpose: T-type calcium (Ca 3) and transient receptor potential vanilloid-1 (TRPV1) channels play central roles in the control of excitability in the peripheral nervous system and are regarded as potential therapeutic pain targets. Modulators that either activate or inhibit TRPV1-mediated currents display analgesic properties in various pain models despite opposing effects on their connate target, TRPV1. We explored the effects of TRPV1-active compounds on Ca 3-mediated currents.

Experimental Approach: Whole-cell patch clamp recordings were used to examine the effects of TRPV1-active compounds on rat dorsal root ganglion low voltage-activated calcium currents and recombinant Ca 3 isoforms in expression systems.

Key Results: The classical TRPV1 agonist capsaicin as well as TRPV1 antagonists A-889425, BCTC, and capsazepine directly inhibited Ca 3 channels. These compounds altered the voltage-dependence of activation and inactivation of Ca 3 channels and delayed their recovery from inactivation, leading to a concomitant decrease in T-type current availability. The TRPV1 antagonist capsazepine potently inhibited Ca 3.1 and 3.2 channels (K  < 120 nM), as demonstrated by its slow off rate. In contrast, neither the TRPV1 agonists, Palvanil and resiniferatoxin, nor the TRPV1 antagonist AMG9810 modulated Ca 3-mediated currents.

Conclusions And Implications: Analgesic TRPV1-active compounds inhibit Ca 3 currents in native and heterologous systems. Hence, their analgesic effects may not be exclusively attributed to their actions on TRPV1, which has important implications in the current understanding of nociceptive pathways. Importantly, our results highlight the need for attention in the experimental design used to address the analgesic properties of Ca 3 channel inhibitors.
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http://dx.doi.org/10.1111/bph.14676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555866PMC
July 2019

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

J Gen Physiol 2019 02 26;151(2):186-199. Epub 2018 Dec 26.

Department of Physiology & Pharmacology and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.
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http://dx.doi.org/10.1085/jgp.201812278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363421PMC
February 2019

NMR Structure of μ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced Na Channel Potency.

Molecules 2018 Oct 22;23(10). Epub 2018 Oct 22.

Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia.

μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of Na1.4 channels, but the same Na selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.
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http://dx.doi.org/10.3390/molecules23102715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222493PMC
October 2018

Novel analgesic ω-conotoxins from the vermivorous cone snail Conus moncuri provide new insights into the evolution of conopeptides.

Sci Rep 2018 09 7;8(1):13397. Epub 2018 Sep 7.

IMB Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca2.2 in fluorimetric assays and rat Ca2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA (I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.
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http://dx.doi.org/10.1038/s41598-018-31245-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128854PMC
September 2018

Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

Br J Pharmacol 2018 06 21;175(12):2284-2295. Epub 2017 Jul 21.

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.

Background And Purpose: Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Ca 2.2) and T-type (Ca 3.1, Ca 3.2 and Ca 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy.

Experimental Approach: In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Ca 1.2, Ca 1.3, Ca 2.1, Ca 2.2, Ca 2.3, Ca 3.1, Ca 3.2 and Ca 3.3.

Key Results: MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCa 3.1, hCa 3.2 and hCa 3.3 (IC : 3.3 ± 0.3, 1.7 ± 0.1 and 7.2 ± 0.3 μM, respectively) than N-type calcium channel, hCa 2.2 (IC : 34.0 ± 3.6 μM). It interacted with L-type calcium channels Ca 1.2 and Ca 1.3 with significantly lower potency (IC  > 100 μM) and did not inhibit hCa 2.1 or hCa 2.3 channels at concentrations as high as 100 μM. State- and use-dependent inhibition of hCa 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCa 3.1 channels suggesting a different mode of action between these two channels.

Conclusions And Implications: Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Ca channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCa 2.2 and hCa 3.1 channels. Such Ca channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies.

Linked Articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
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http://dx.doi.org/10.1111/bph.13910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980596PMC
June 2018

Analgesic conopeptides targeting G protein-coupled receptors reduce excitability of sensory neurons.

Neuropharmacology 2017 Dec 19;127:116-123. Epub 2017 May 19.

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, New South Wales, 2522, Australia. Electronic address:

Conotoxins (conopeptides) are a diverse group of peptides isolated from the venom of marine cone snails. Conus peptides modulate pain by interacting with voltage-gated ion channels and G protein-coupled receptors (GPCRs). Opiate drugs targeting GPCRs have long been used, nonetheless, many undesirable side effects associated with opiates have been observed including addiction. Consequently, alternative avenues to pain management are a largely unmet need. It has been shown that various voltage-gated calcium channels (VGCCs) respond to GPCR modulation. Thus, regulation of VGCCs by GPCRs has become a valuable alternative in the management of pain. In this review, we focus on analgesic conotoxins that exert their effects via GPCR-mediated inhibition of ion channels involved in nociception and pain transmission. Specifically, α-conotoxin Vc1.1 activation of GABA receptors and inhibition of voltage-gated calcium channels as a novel mechanism for reducing the excitability of dorsal root ganglion neurons is described. Vc1.1 and other α-conotoxins have been shown to be analgesic in different animal models of chronic pain. This review will outline the functional effects of conopeptide modulation of GPCRs and how their signalling is translated to downstream components of the pain pathways. Where available we present the proposed signalling mechanisms that couples metabotropic receptor activation to their downstream effectors to produce analgesia. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'
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http://dx.doi.org/10.1016/j.neuropharm.2017.05.020DOI Listing
December 2017

Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation.

J Gen Physiol 2014 Apr;143(4):465-79

Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia.

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba(2+) currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5'-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.
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http://dx.doi.org/10.1085/jgp.201311104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971658PMC
April 2014

Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors.

ACS Chem Biol 2013 Aug 17;8(8):1815-21. Epub 2013 Jun 17.

School of Chemistry, Monash University, Clayton 3800, Victoria, Australia.

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide's innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide's potent analgesic activity.
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http://dx.doi.org/10.1021/cb4002393DOI Listing
August 2013

Conotoxins targeting neuronal voltage-gated sodium channel subtypes: potential analgesics?

Toxins (Basel) 2012 Nov 8;4(11):1236-60. Epub 2012 Nov 8.

Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia.

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.
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http://dx.doi.org/10.3390/toxins4111236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509706PMC
November 2012

ω-Conotoxin GVIA mimetics that bind and inhibit neuronal Ca(v)2.2 ion channels.

Mar Drugs 2012 Oct 22;10(10):2349-68. Epub 2012 Oct 22.

CSIRO Materials Science and Engineering, Bag 10, Clayton South, Victoria 3169, Australia.

The neuronal voltage-gated N-type calcium channel (Ca(v)2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²⁵I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca(v)2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²⁵I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.
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http://dx.doi.org/10.3390/md10102349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497028PMC
October 2012

Modulation of KvAP unitary conductance and gating by 1-alkanols and other surface active agents.

Biophys J 2010 Mar;98(5):762-72

Hotchkiss Brain Institute and Department of Physiology and Pharmacology, University of Calgary, Alberta, Canada.

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.
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http://dx.doi.org/10.1016/j.bpj.2009.10.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830433PMC
March 2010
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