Publications by authors named "Jennifer R Deuis"

45 Publications

Venom chemistry underlying the painful stings of velvet ants (Hymenoptera: Mutillidae).

Cell Mol Life Sci 2021 May 10. Epub 2021 May 10.

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

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.
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http://dx.doi.org/10.1007/s00018-021-03847-1DOI Listing
May 2021

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1β release.

J Exp Med 2021 May;218(5)

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.
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http://dx.doi.org/10.1084/jem.20201452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933984PMC
May 2021

Discovery, Pharmacological Characterisation and NMR Structure of the Novel µ-Conotoxin SxIIIC, a Potent and Irreversible Na Channel Inhibitor.

Biomedicines 2020 Oct 2;8(10). Epub 2020 Oct 2.

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

Voltage-gated sodium (Na) channel subtypes, including Na1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent Na channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human Na1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from . Here we present the isolation of native peptide, chemical synthesis, characterisation of human Na channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique Na channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 > 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human Na1.7 putative pore blockers (IC 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for Na channel pore blocker selectivity and subsequently important for chronic pain drug development.
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http://dx.doi.org/10.3390/biomedicines8100391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599555PMC
October 2020

Neurotoxic peptides from the venom of the giant Australian stinging tree.

Sci Adv 2020 Sep 16;6(38). Epub 2020 Sep 16.

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

Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. -induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.
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http://dx.doi.org/10.1126/sciadv.abb8828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494335PMC
September 2020

Recombinant production, bioconjugation and membrane binding studies ofPn3a, a selective Na1.7 inhibitor.

Biochem Pharmacol 2020 11 12;181:114148. Epub 2020 Jul 12.

Centre for Advanced Imaging, The University of Queensland, Australia. Electronic address:

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Nas) play a critical role in this process. In particular, the Na subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.
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http://dx.doi.org/10.1016/j.bcp.2020.114148DOI Listing
November 2020

Mapping the Molecular Surface of the Analgesic Na1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions.

ACS Pharmacol Transl Sci 2020 Jun 19;3(3):535-546. Epub 2020 Feb 19.

Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St. Lucia, Queensland 4072, Australia.

Compelling human genetic studies have identified the voltage-gated sodium channel Na1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na1.4, Na1.5, and Na1.6. The Pn3a[D8N] mutant retained analgesic activity , significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na1.7 in pain.
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http://dx.doi.org/10.1021/acsptsci.0c00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296542PMC
June 2020

Characterization of Synthetic Tf2 as a Na1.3 Selective Pharmacological Probe.

Biomedicines 2020 Jun 11;8(6). Epub 2020 Jun 11.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

Na1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of , has been reported to selectively activate Na1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for Na1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of Na1.3 activation in the hyperpolarizing direction by -11.3 mV, with no activity at Na1.1, Na1.2, and Na1.4-Na1.8. Additional activity was found at Na1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by -6.3 mV. In an attempt to convert Tf2 into an Na1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at Na1.3, although it still caused a small -5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the Na1.1/1.3 inhibitor ICA-121431 nor in Na1.9 mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at Na1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.
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http://dx.doi.org/10.3390/biomedicines8060155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345637PMC
June 2020

Pharmacological activity and NMR solution structure of the leech peptide HSTX-I.

Biochem Pharmacol 2020 11 7;181:114082. Epub 2020 Jun 7.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia; National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States. Electronic address:

The role of voltage-gated sodium (Na) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na1.8 and Na1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na1.8 and mouse Na1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na1.8 and Na1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.
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http://dx.doi.org/10.1016/j.bcp.2020.114082DOI Listing
November 2020

Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7.

J Biol Chem 2020 04 5;295(15):5067-5080. Epub 2020 Mar 5.

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

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and activity at the voltage-gated sodium channel subtype 1.7 (Na1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for Na1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human Na1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for Na1.7, examined alongside off-target Nas, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between potency at Na1.7 and affinity for lipid bilayers. In a murine Na1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved selectivity for the pain target Na1.7 and with an efficacy similar to that of native HwTx-IV.
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http://dx.doi.org/10.1074/jbc.RA119.012281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152767PMC
April 2020

Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of Na1.7.

Biomedicines 2020 Feb 19;8(2). Epub 2020 Feb 19.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula that modulate Na channels. Pme1a is a 35 residue peptide that inhibits Na1.7 peak current (IC 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na1.7 peak current (EC > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na1.7 (IC 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na channels in the future.
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http://dx.doi.org/10.3390/biomedicines8020037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167818PMC
February 2020

Enzymatic Ligation of a Pore Blocker Toxin and a Gating Modifier Toxin: Creating Double-Knotted Peptides with Improved Sodium Channel Na1.7 Inhibition.

Bioconjug Chem 2020 01 16;31(1):64-73. Epub 2019 Dec 16.

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

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na1.7 inhibitors-PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain-with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage sensing domain and the pore domain.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00744DOI Listing
January 2020

High-Throughput Fluorescence Assays for Ion Channels and GPCRs.

Adv Exp Med Biol 2020 ;1131:27-72

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia.

Ca, Na and K- permeable ion channels as well as GPCRs linked to Ca release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and G-coupled GPCRs.
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http://dx.doi.org/10.1007/978-3-030-12457-1_3DOI Listing
October 2019

Antiallodynic effects of the selective NaV1.7 inhibitor Pn3a in a mouse model of acute postsurgical pain: evidence for analgesic synergy with opioids and baclofen.

Pain 2019 08;160(8):1766-1780

Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia.

Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, postsurgical pain continues to be a frequent and undermanaged condition. Here, we investigate the analgesic potential of pharmacological NaV1.7 inhibition in a mouse model of acute postsurgical pain, based on incision of the plantar skin and underlying muscle of the hind paw. We demonstrate that local and systemic treatment with the selective NaV1.7 inhibitor μ-theraphotoxin-Pn3a is effectively antiallodynic in this model and completely reverses mechanical hypersensitivity in the absence of motor adverse effects. In addition, the selective NaV1.7 inhibitors ProTx-II and PF-04856264 as well as the clinical candidate CNV1014802 also reduced mechanical allodynia. Interestingly, co-administration of the opioid receptor antagonist naloxone completely reversed analgesic effects of Pn3a, indicating an involvement of endogenous opioids in the analgesic activity of Pn3a. In addition, we found superadditive antinociceptive effects of subtherapeutic Pn3a doses not only with the opioid oxycodone but also with the GABAB receptor agonist baclofen. Transcriptomic analysis of gene expression changes in dorsal root ganglia of mice after surgery did not reveal any changes in mRNA expression of endogenous opioids or opioid receptors; however, several genes involved in pain, including Runx1 (Runt related transcription factor 1), Cacna1a (CaV2.1), and Cacna1b (CaV2.2), were downregulated. In summary, these findings suggest that pain after surgery can be successfully treated with NaV1.7 inhibitors alone or in combination with baclofen or opioids, which may present a novel and safe treatment strategy for this frequent and poorly managed condition.
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http://dx.doi.org/10.1097/j.pain.0000000000001567DOI Listing
August 2019

Inflammatory and Neuropathic Gene Expression Signatures of Chemotherapy-Induced Neuropathy Induced by Vincristine, Cisplatin, and Oxaliplatin in C57BL/6J Mice.

J Pain 2020 Jan - Feb;21(1-2):182-194. Epub 2019 Jun 29.

Centre for Pain Research, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, Australia; School of Pharmacy, The University of Queensland, Woolloongabba, Queensland, Australia. Electronic address:

Vincristine, oxaliplatin, and cisplatin are commonly prescribed chemotherapeutic agents for the treatment of many tumors. However, a main side effect is chemotherapy-induced peripheral neuropathy (CIPN), which may lead to changes in chemotherapeutic treatment. Although symptoms associated with CIPN are recapitulated by mouse models, there is limited knowledge of how these drugs affect the expression of genes in sensory neurons. The present study carried out a transcriptomic analysis of dorsal root ganglia following vincristine, oxaliplatin, and cisplatin treatment with a view to gain insight into the comparative pathophysiological mechanisms of CIPN. RNA-Seq revealed 368, 295, and 256 differential expressed genes induced by treatment with vincristine, oxaliplatin, and cisplatin, respectively, and only 5 shared genes were dysregulated in all 3 groups. Cell type enrichment analysis and gene set enrichment analysis showed predominant effects on genes associated with the immune system after treatment with vincristine, while oxaliplatin treatment affected mainly neuronal genes. Treatment with cisplatin resulted in a mixed gene expression signature. PERSPECTIVE: These results provide insight into the recruitment of immune responses to dorsal root ganglia and indicate enhanced neuroinflammatory processes following administration of vincristine, oxaliplatin, and cisplatin. These gene expression signatures may provide insight into novel drug targets for treatment of CIPN.
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http://dx.doi.org/10.1016/j.jpain.2019.06.008DOI Listing
June 2019

Na 1.6 regulates excitability of mechanosensitive sensory neurons.

J Physiol 2019 07 13;597(14):3751-3768. Epub 2019 May 13.

IMB Centre for Pain Research, Institute for Molecular Bioscience, 306 Carmody Rd (Building 80), University of Queensland, Brisbane, Queensland, 4072, Australia.

Key Points: Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The β-scorpion toxin, Cn2, is selective for Na 1.6 in dorsal root ganglion neurons. Na 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. Na 1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted Na isoforms in pain generation.

Abstract: Peripheral sensory neurons express multiple voltage-gated sodium channels (Na ) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): Na 1.7, Na 1.8 and Na 1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests Na 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of Na 1.6 channels in peripheral sensory neurons. The potent and Na 1.6 selective β-scorpion toxin Cn2 was used to assess the effect of Na 1.6 channel activation in the PNS. The multidisciplinary approach included Ca imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates Na 1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, Na 1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on Na 1.6 channels in sensory neurons. Activation of Na 1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing Na 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.
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http://dx.doi.org/10.1113/JP278148DOI Listing
July 2019

Development of a high-throughput fluorescent no-wash sodium influx assay.

PLoS One 2019 11;14(3):e0213751. Epub 2019 Mar 11.

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

Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 μM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213751PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411159PMC
December 2019

A Centipede Toxin Family Defines an Ancient Class of CSαβ Defensins.

Structure 2019 02 13;27(2):315-326.e7. Epub 2018 Dec 13.

Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

Disulfide-rich peptides (DRPs) play diverse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX-Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/β fold (CSαβ). This class, which we have named the two-disulfide CSαβ fold (2ds-CSαβ), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαβ peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαβ is widely distributed outside centipedes and is likely an ancient fold predating the split between prokaryotes and eukaryotes. Our results provide insights into the ancient evolutionary history of a widespread DRP fold and highlight the usefulness of 3D structures as evolutionary tools.
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http://dx.doi.org/10.1016/j.str.2018.10.022DOI Listing
February 2019

Toxins as tools: Fingerprinting neuronal pharmacology.

Neurosci Lett 2018 07 6;679:4-14. Epub 2018 Feb 6.

IMB Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia. Electronic address:

Toxins have been used as tools for decades to study the structure and function of neuronal ion channels and receptors. The biological origin of these toxins varies from single cell organisms, including bacteria and algae, to complex multicellular organisms, including a wide variety of plants and venomous animals. Toxins are a structurally and functionally diverse group of compounds that often modulate neuronal function by interacting with an ion channel or receptor. Many of these toxins display high affinity and exquisite selectivity, making them valuable tools to probe the structure and function of neuronal ion channels and receptors. This review article provides an overview of the experimental techniques used to assess the effects that toxins have on neuronal function, as well as discussion on toxins that have been used as tools, with a focus on toxins that target voltage-gated and ligand-gated ion channels.
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http://dx.doi.org/10.1016/j.neulet.2018.02.001DOI Listing
July 2018

The E15R Point Mutation in Scorpion Toxin Cn2 Uncouples Its Depressant and Excitatory Activities on Human Na1.6.

J Med Chem 2018 02 3;61(4):1730-1736. Epub 2018 Feb 3.

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

We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel Na1.6. In an attempt to decouple channel activation from channel binding, we also synthesized the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin's excitatory and depressant activities, effectively resulting in a Na1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is antinociceptive in mouse models of Na1.6-mediated pain.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01609DOI Listing
February 2018

Role of complement anaphylatoxin receptors in a mouse model of acute burn-induced pain.

Mol Immunol 2018 02 21;94:68-74. Epub 2017 Dec 21.

IMB Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia; School of Pharmacy, Pharmacy Australia Centre of Excellence, Woolloongabba, Australia. Electronic address:

The complement system is an essential component of the innate immune response. The anaphylatoxins C3a and C5a are key drivers of the complement system, acting through the receptors C3aR, C5aR1 and C5aR2 to regulate inflammation. While a role for C5a activation of C5aR1 in inflammatory and neuropathic pain has been established, the role of the complement system in burn-induced pain has not been investigated. To address this gap, we assessed the role of complement receptors C3aR, C5aR1 and C5aR2 in a mouse model of acute burn-induced pain. Superficial burn injury was induced in C57BL/6 mice by firm application of left hind paw plantar surface to a hot plate set at 52.5 °C for 25 s. Development of burn-induced mechanical allodynia, thermal allodynia, weight bearing changes and edema was assessed in C3aR, C5aR1 and C5aR2 mice and compared to their wild type controls over three days. Burn-induced mechanical allodynia, thermal allodynia and weight bearing changes developed normally C3aR, C5aR1 and C5aR2 mice. However, burn-induced edema was significantly reduced in C5aR2 male mice, but not C5aR2 female mice. These results suggest that the complement system has a limited role in the development of acute burn-induced pain.
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http://dx.doi.org/10.1016/j.molimm.2017.12.016DOI Listing
February 2018

Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species.

Sci Signal 2017 Dec 5;10(508). Epub 2017 Dec 5.

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

Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues.
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http://dx.doi.org/10.1126/scisignal.aan3398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892705PMC
December 2017

Burn Pain: A Systematic and Critical Review of Epidemiology, Pathophysiology, and Treatment.

Pain Med 2018 04;19(4):708-734

Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, Australia.

Objective: This review aims to examine the available literature on the epidemiology, pathophysiology, and treatment of burn-induced pain.

Methods: A search was conducted on the epidemiology of burn injury and treatment of burn pain utilizing the database Medline, and all relevant articles were systemically reviewed. In addition, a critical review was performed on the pathophysiology of burn pain and animal models of burn pain.

Results: The search on the epidemiology of burn injury yielded a total of 163 publications of interest, 72 of which fit the inclusion/exclusion criteria, with no publications providing epidemiological data on burn injury pain management outcomes. The search on the treatment of burn pain yielded a total of 213 publications, 14 of which fit the inclusion/exclusion criteria, highlighting the limited amount of evidence available on the treatment of burn-induced pain.

Conclusions: The pathophysiology of burn pain is poorly understood, with limited clinical trials available to assess the effectiveness of analgesics in burn patients. Further studies are needed to identify new pharmacological targets and treatments for the effective management of burn injury pain.
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http://dx.doi.org/10.1093/pm/pnx228DOI Listing
April 2018

Methods Used to Evaluate Pain Behaviors in Rodents.

Front Mol Neurosci 2017 6;10:284. Epub 2017 Sep 6.

IMB Centre for Pain Research, Institute for Molecular Bioscience, The University of QueenslandSt. Lucia, QLD, Australia.

Rodents are commonly used to study the pathophysiological mechanisms of pain as studies in humans may be difficult to perform and ethically limited. As pain cannot be directly measured in rodents, many methods that quantify "pain-like" behaviors or nociception have been developed. These behavioral methods can be divided into stimulus-evoked or non-stimulus evoked (spontaneous) nociception, based on whether or not application of an external stimulus is used to elicit a withdrawal response. Stimulus-evoked methods, which include manual and electronic von Frey, Randall-Selitto and the Hargreaves test, were the first to be developed and continue to be in widespread use. However, concerns over the clinical translatability of stimulus-evoked nociception in recent years has led to the development and increasing implementation of non-stimulus evoked methods, such as grimace scales, burrowing, weight bearing and gait analysis. This review article provides an overview, as well as discussion of the advantages and disadvantages of the most commonly used behavioral methods of stimulus-evoked and non-stimulus-evoked nociception used in rodents.
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http://dx.doi.org/10.3389/fnmol.2017.00284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592204PMC
September 2017

Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi.

PLoS One 2017 7;12(9):e0182848. Epub 2017 Sep 7.

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

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn't act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1-1.2 and Nav1.7-1.8 (IC50 0.1-6.9 μM) but not Nav1.3-1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182848PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589098PMC
October 2017

Modulatory features of the novel spider toxin μ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus.

Br J Pharmacol 2017 08 27;174(15):2528-2544. Epub 2017 Jun 27.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.

Background And Purpose: Naturally occurring dysfunction of voltage-gated sodium (Na ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel Na modulators, we investigated spider venoms for new inhibitors of Na channels.

Experimental Approach: We used high-throughput screens to identify a Na modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model.

Key Results: We identified a potent Na inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by Na 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (Ca 3) currents but had no activity against the voltage-gated potassium (K 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the Na channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all Na subtypes tested (hNa 1.1-1.7). Df1a also slowed fast inactivation of Na 1.1, Na 1.3 and Na 1.5 and modified the voltage-dependence of activation and inactivation of most of the Na subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the Na activator OD1.

Conclusion And Implications: μ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.
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http://dx.doi.org/10.1111/bph.13865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513869PMC
August 2017

Sodium Channels and Venom Peptide Pharmacology.

Adv Pharmacol 2017 8;79:67-116. Epub 2017 Apr 8.

Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; School of Pharmacy, The University of Queensland, Brisbane, QLD, Australia. Electronic address:

Venomous animals including cone snails, spiders, scorpions, anemones, and snakes have evolved a myriad of components in their venoms that target the opening and/or closing of voltage-gated sodium channels to cause devastating effects on the neuromuscular systems of predators and prey. These venom peptides, through design and serendipity, have not only contributed significantly to our understanding of sodium channel pharmacology and structure, but they also represent some of the most phyla- and isoform-selective molecules that are useful as valuable tool compounds and drug leads. Here, we review our understanding of the basic function of mammalian voltage-gated sodium channel isoforms as well as the pharmacology of venom peptides that act at these key transmembrane proteins.
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http://dx.doi.org/10.1016/bs.apha.2017.01.004DOI Listing
December 2017

Δ-Myrtoxin-Mp1a is a Helical Heterodimer from the Venom of the Jack Jumper Ant that has Antimicrobial, Membrane-Disrupting, and Nociceptive Activities.

Angew Chem Int Ed Engl 2017 07 13;56(29):8495-8499. Epub 2017 Jun 13.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia.

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca influx in SH-SY5Y cells with a half maximal effective concentration (EC ) of 4.3 μm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 μm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity.
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http://dx.doi.org/10.1002/anie.201703360DOI Listing
July 2017

The pharmacology of voltage-gated sodium channel activators.

Neuropharmacology 2017 Dec 14;127:87-108. Epub 2017 Apr 14.

Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Qld 4072, Australia; School of Pharmacy, The University of Queensland, Woolloongabba, Qld 4102, Australia. Electronic address:

Toxins and venom components that target voltage-gated sodium (Na) channels have evolved numerous times due to the importance of this class of ion channels in the normal physiological function of peripheral and central neurons as well as cardiac and skeletal muscle. Na channel activators in particular have been isolated from the venom of spiders, wasps, snakes, scorpions, cone snails and sea anemone and are also produced by plants, bacteria and algae. These compounds have provided key insight into the molecular structure, function and pathophysiological roles of Na channels and are important tools due to their at times exquisite subtype-selectivity. We review the pharmacology of Na channel activators with particular emphasis on mammalian isoforms and discuss putative applications for these compounds. 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.2017.04.014DOI Listing
December 2017

The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.

PLoS One 2017 16;12(3):e0173551. Epub 2017 Mar 16.

Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia.

Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354290PMC
September 2017