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1.
Adv Exp Med Biol ; 1131: 27-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646506

RESUMO

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ 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 Gq-coupled GPCRs.


Assuntos
Bioensaio , Canais Iônicos , Receptores Acoplados a Proteínas-G , Animais , Bioensaio/tendências , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Humanos , Canais Iônicos/análise , Receptores Acoplados a Proteínas-G/análise
2.
Pain ; 160(8): 1766-1780, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31335646

RESUMO

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.

3.
J Pain ; 2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31260808

RESUMO

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.

4.
J Physiol ; 597(14): 3751-3768, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087362

RESUMO

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 ß-scorpion toxin, Cn2, is selective for NaV 1.6 in dorsal root ganglion neurons. NaV 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. NaV 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 NaV isoforms in pain generation. ABSTRACT: Peripheral sensory neurons express multiple voltage-gated sodium channels (NaV ) 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): NaV 1.7, NaV 1.8 and NaV 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 NaV 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV 1.6 channels in peripheral sensory neurons. The potent and NaV 1.6 selective ß-scorpion toxin Cn2 was used to assess the effect of NaV 1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV 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, NaV 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 NaV 1.6 channels in sensory neurons. Activation of NaV 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 NaV 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.

5.
Pain ; 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30908355

RESUMO

Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, post-surgical 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 post-surgical 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 anti-allodynic 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. Additionally, we found super-additive antinociceptive effects of sub-therapeutic 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 post-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.

6.
PLoS One ; 14(3): e0213751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856233

RESUMO

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.

7.
Structure ; 27(2): 315-326.e7, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30554841

RESUMO

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-SLPTX15-Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/ß fold (CSαß). This class, which we have named the two-disulfide CSαß fold (2ds-CSαß), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαß peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαß 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.

8.
Neurosci Lett ; 679: 4-14, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-29425731

RESUMO

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.

9.
J Med Chem ; 61(4): 1730-1736, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29378414

RESUMO

We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel NaV1.6. 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 NaV1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is antinociceptive in mouse models of NaV1.6-mediated pain.

10.
Mol Immunol ; 94: 68-74, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29274925

RESUMO

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.

11.
Pain Med ; 19(4): 708-734, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29036469

RESUMO

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.

12.
Sci Signal ; 10(508)2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29208680

RESUMO

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.


Assuntos
Ansiedade/tratamento farmacológico , Receptores de Ocitocina/agonistas , Animais , Células COS , Cercopithecus aethiops , Química Farmacêutica , Condicionamento (Psicologia)/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Infusões Intraventriculares , Ligantes , Masculino , Camundongos , Ratos
13.
PLoS One ; 12(9): e0182848, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28880874

RESUMO

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.


Assuntos
Analgésicos/farmacologia , Venenos de Aranha/química , Aranhas/química , Analgésicos/química , Animais , Sítios de Ligação/efeitos dos fármacos , Canais de Cálcio Tipo N/metabolismo , Eletrofisiologia , Fluorometria , Humanos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ratos
14.
Front Mol Neurosci ; 10: 284, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28932184

RESUMO

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.

16.
Br J Pharmacol ; 174(15): 2528-2544, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28542706

RESUMO

BACKGROUND AND PURPOSE: Naturally occurring dysfunction of voltage-gated sodium (NaV ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. EXPERIMENTAL APPROACH: We used high-throughput screens to identify a NaV 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 NaV inhibitor named µ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (CaV 3) currents but had no activity against the voltage-gated potassium (KV 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV 1.1-1.7). Df1a also slowed fast inactivation of NaV 1.1, NaV 1.3 and NaV 1.5 and modified the voltage-dependence of activation and inactivation of most of the NaV 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 NaV 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.


Assuntos
Analgésicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/tratamento farmacológico , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Analgésicos/química , Analgésicos/isolamento & purificação , Animais , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/induzido quimicamente , Venenos de Escorpião/administração & dosagem , Aranhas , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação
17.
Angew Chem Int Ed Engl ; 56(29): 8495-8499, 2017 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-28513074

RESUMO

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 Ca2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC50 ) 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.

18.
Adv Pharmacol ; 79: 67-116, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28528674

RESUMO

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.


Assuntos
Peptídeos/farmacologia , Canais de Sódio/metabolismo , Peçonhas/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Canais de Sódio Disparados por Voltagem/metabolismo
19.
Neuropharmacology ; 127: 87-108, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28416444

RESUMO

Toxins and venom components that target voltage-gated sodium (NaV) 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. NaV 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 NaV channels and are important tools due to their at times exquisite subtype-selectivity. We review the pharmacology of NaV 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.'


Assuntos
Peptídeos/farmacologia , Peçonhas/química , Agonistas do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia
20.
PLoS One ; 12(3): e0173551, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28301520

RESUMO

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.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Animais , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Conformação Proteica , Proteínas Recombinantes/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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