RESUMO
The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.
Assuntos
Nociceptores/efeitos dos fármacos , Papio/metabolismo , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Aranhas/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Gânglios Espinais/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Canais Iônicos/metabolismo , Camundongos , Dor/tratamento farmacológico , Tetrodotoxina/farmacologiaRESUMO
Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.
Assuntos
Venenos de Artrópodes/química , Proteínas de Insetos/química , Lepidópteros/química , Neuropeptídeos/química , Dor/genética , Animais , Venenos de Artrópodes/genética , Evolução Molecular , Proteínas de Insetos/genética , Mariposas/química , Neuropeptídeos/genética , Peptídeos/química , Peptídeos/genética , Proteômica , Venenos de Aranha/química , Venenos de Aranha/genética , Transcriptoma/genéticaRESUMO
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.
Assuntos
Canais de Cálcio Tipo T , Modelos Animais de Doenças , Hiperalgesia , Dor Pós-Operatória , Venenos de Escorpião , Animais , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/química , Camundongos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/metabolismo , Cálcio/metabolismo , Masculino , Humanos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/químicaRESUMO
Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Venenos de Aranha/farmacologia , Estresse Mecânico , Animais , Modelos Animais de Doenças , Feminino , Gânglios Sensitivos/citologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Síndrome do Intestino Irritável/metabolismo , Masculino , Bainha de Mielina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/química , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Oócitos/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Aranhas/química , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.
Assuntos
Analgésicos Opioides/farmacologia , Proteínas Fúngicas/farmacologia , Oligopeptídeos/farmacologia , Penicillium/química , Receptores Opioides mu/agonistas , Analgésicos Opioides/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas Fúngicas/química , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Ligação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismoRESUMO
Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.
Assuntos
Venenos de Cnidários/química , Peptídeos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Venenos de Cnidários/genética , Venenos de Cnidários/metabolismo , Evolução Molecular , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Conformação Proteica , Dobramento de Proteína , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , TranscriptomaRESUMO
The µO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay.
Assuntos
Analgésicos/farmacologia , Membrana Celular/metabolismo , Conotoxinas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Dor/prevenção & controle , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Cristalografia por Raios X , Eletrofisiologia , Células HEK293 , Humanos , Lipossomos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.8/química , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Dor/induzido quimicamente , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two ß-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85â µm) while inhibiting apoptosis (EC50 2.2â µm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.
Assuntos
Apoptose/efeitos dos fármacos , Conotoxinas/farmacologia , Cisteína/química , Granulinas/farmacologia , Mimetismo Molecular , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Conotoxinas/química , Dissulfetos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer Aâ chain and a 23-mer Bâ 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 4.3â µm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25â nm). Intraplantar injection (10â µm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Hiperalgesia/tratamento farmacológico , Dor/tratamento farmacológico , Peptídeos/farmacologia , Peçonhas/química , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Formigas , Cálcio/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptídeos/administração & dosagem , Peptídeos/químicaRESUMO
Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, µ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 µM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.
Assuntos
Analgésicos/farmacologia , Dor/tratamento farmacológico , Venenos de Aranha/farmacologia , Aranhas/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Analgésicos/química , Analgésicos/isolamento & purificação , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Modelos Animais de Doenças , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/induzido quimicamente , Venenos de Escorpião , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Aranhas/classificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificaçãoRESUMO
A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4â nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.
Assuntos
Compostos Benzidrílicos/química , Cisteína/química , Dissulfetos/química , Hepcidinas/síntese química , Sequência de Aminoácidos , Hepcidinas/química , Humanos , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , EstereoisomerismoRESUMO
A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.
Assuntos
Transporte Biológico/efeitos dos fármacos , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Integrinas/metabolismo , Norepinefrina/metabolismo , Engenharia de Proteínas/métodos , Animais , Conotoxinas/química , Conotoxinas/farmacocinética , Humanos , Integrinas/antagonistas & inibidores , Modelos Moleculares , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Oligopeptídeos , Peptídeos Cíclicos/metabolismo , Estabilidade Proteica , RatosRESUMO
Inhibition of T-type calcium channels (CaV3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of CaV3. However, subtype-specific CaV3 inhibitors for therapeutic purposes or for studying the physiological roles of CaV3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. ("Amazonas Purple", Peru) tarantula venom inhibited specific T-type CaV channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 µM) and synthetic ω-Avsp1a (10 µM) inhibited 90% of CaV3.1 and CaV3.3, but only 25% of CaV3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive CaV3.2 and more sensitive CaV3.3. Our results suggest that domain-1 of CaV3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.
RESUMO
α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 310-helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC50) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.
Assuntos
Conotoxinas , Receptores Nicotínicos , Sequência de Aminoácidos , Conotoxinas/farmacologia , Cisteína , Dissulfetos , Músculos/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismoRESUMO
The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2SâO) to a 3-fold increase (3SâO(B)) in potencies.
Assuntos
Conotoxinas/química , Cistationina/química , Antagonistas Nicotínicos/química , Sequência de Aminoácidos , Animais , Caramujo Conus/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V1aR, V1bR, V2R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.
RESUMO
Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of ß-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50-20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50-15.5 ± 1.8 mV). At a concentration of 10 µM, Eo1a has varying effects on the peak current and channel gating of NaV1.1-NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8.
RESUMO
Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of µ-theraphotoxin-Pme1a and µ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 µM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 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 NaV channels in the future.
RESUMO
Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula, exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus, the fruit fly Drosophila melanogaster, and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure-activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types.
RESUMO
BACKGROUND AND PURPOSE: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs). EXPERIMENTAL APPROACH: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology. KEY RESULTS: Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4ß2, α7, and α3ß2 nAChRs which it blocked with IC50 values of 1.8, 7, and 12 µM respectively. CONCLUSIONS AND IMPLICATIONS: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists.