RESUMO
Erythromelalgia (EM), is a familial pain syndrome characterized by episodic 'burning' pain, warmth, and erythema. EM is caused by monoallelic variants in SCN9A, which encodes the voltage-gated sodium channel (NaV) NaV1.7. Over 25 different SCN9A mutations attributed to EM have been described to date, all identified in the SCN9A transcript utilizing exon 6N. Here we report a novel SCN9A missense variant identified in seven related individuals with stereotypic episodes of bilateral lower limb pain presenting in childhood. The variant, XM_011511617.3:c.659G>C;p.(Arg220Pro), resides in the exon 6A of SCN9A, an exon previously shown to be selectively incorporated by developmentally regulated alternative splicing. The mutation is located in the voltage-sensing S4 segment of domain I, which is important for regulating channel activation. Functional analysis showed the p.Arg220Pro mutation altered voltage-dependent activation and delayed channel inactivation, consistent with a NaV1.7 gain-of-function molecular phenotype. These results demonstrate that alternatively spliced isoforms of SCN9A should be included in all genomic testing of EM.
Assuntos
Eritromelalgia , Humanos , Eritromelalgia/genética , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/genética , Mutação , Éxons/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
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.
Assuntos
Neurotoxinas , Peptídeos , Toxinas Biológicas , Urticaceae , Humanos , Neurotoxinas/química , Dor , Técnicas de Patch-Clamp , Peptídeos/química , Peptídeos/toxicidade , Toxinas Biológicas/química , Urticaceae/química , Canais de Sódio Disparados por Voltagem/efeitos dos fármacosRESUMO
Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype NaV1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker µ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6]Sx) was shown to be the best combination as native ProTx-II potency at hNaV1.7 was conserved following ligation. At hNaV1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV1.7.
Assuntos
Peptídeos , Humanos , Peptídeos/farmacologiaRESUMO
OBJECTIVE: N-methyl-d-aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1-3b[I481V]), p.Ala666Ser (GluN1-3b[A666S]), and p.Tyr668His (GluN1-3b[Y668H]). METHODS: Wild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy. RESULTS: Our data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single-receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors. SIGNIFICANCE: Our study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder.
Assuntos
Ketamina , Transtornos do Neurodesenvolvimento , Humanos , Memantina/farmacologia , Maleato de Dizocilpina/farmacologia , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células HEK293 , Glutamatos , Transtornos do Neurodesenvolvimento/genética , Glicina , Proteínas do Tecido Nervoso/metabolismoRESUMO
Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3ß4 (IC50 15.7 ± 3.0 µm) and α7 (IC50 77.1 ± 0.05 µm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equilibrium between conformational states that are the result of a key Ser4-Pro5cis-trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds (cis-trans) plays an important role in receptor subtype selectivity. The cis conformation at the Cys6-Pro7 peptide bond was essential for α3ß4 nAChR subtype allosteric selectivity. In summary, we have identified a unique single-disulfide conopeptide with a noncompetitive, potentially allosteric inhibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for allosteric nAChR modulation. This new paradigm in the "conotoxinomic" structure-function space provides an impetus to screen venom from other Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.
Assuntos
Caramujo Conus/química , Dissulfetos/química , Neurotoxinas , Peptídeos , Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa7 , Regulação Alostérica , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Neurotoxinas/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Relação Estrutura-Atividade , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
The 27-amino acid (aa)-long d-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 mM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.
Assuntos
Conotoxinas/farmacologia , Animais , Canais de Cálcio Tipo T , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ratos , Peixe-ZebraRESUMO
Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.
Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Caramujo Conus/química , Sequência de Aminoácidos , Animais , Conotoxinas/síntese química , Dissulfetos/química , Dissulfetos/farmacologia , Humanos , Conformação Molecular , Venenos de Moluscos/química , Neurofisinas/antagonistas & inibidores , Precursores de Proteínas/antagonistas & inibidores , Receptores de Ocitocina/efeitos dos fármacos , Receptores de Vasopressinas/efeitos dos fármacos , Relação Estrutura-Atividade , Transcriptoma , Vasopressinas/antagonistas & inibidores , Peixe-ZebraRESUMO
T-type calcium channel (CaV3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson's disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved CaV3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual CaV3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two CaV3.2 (2, IC50 = 18.24 µM; 3, IC50 = 6.59 µM) and CaV3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring CaV3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of CaV3.1 activated current (2, IC50 = 5.60 µM; 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.
Assuntos
Organismos Aquáticos/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Pseudallescheria/química , Alcaloides/química , Animais , Células CHO , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/síntese química , Linhagem Celular Tumoral , Cricetulus , Células HEK293 , Humanos , Concentração Inibidora 50 , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Triptofano/químicaRESUMO
Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs.
Assuntos
Alanina/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Cricetinae , Modelos Moleculares , Conformação Molecular , Mutagênese , Polimorfismo de Nucleotídeo Único , Prazosina/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey ß(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/química , Aminoácidos/química , Peptídeos/química , Receptores Adrenérgicos alfa 1/química , Antagonistas de Receptores Adrenérgicos alfa 1/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Simulação por Computador , Cricetinae , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Relação Estrutura-Atividade , PerusRESUMO
Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earlier-eluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfide-stabilized peptides can be dramatically susceptible to disruption.
RESUMO
Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.
Assuntos
Toxinas Biológicas , Urtica dioica , Austrália , Dor , Peptídeos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismoRESUMO
The natural product indole-3-carbinol (I3C) and its major digestive product 3,3'-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3'-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.
RESUMO
QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.
Assuntos
Anestésicos Locais , Lidocaína , Anestésicos Locais/farmacologia , Células HEK293 , Humanos , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma. EXPERIMENTAL APPROACH: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). KEY RESULTS: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. CONCLUSION AND IMPLICATIONS: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.
Assuntos
Antineoplásicos , Melanoma , Trifosfato de Adenosina , Animais , Antineoplásicos/farmacologia , Cálcio , Linhagem Celular Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Mutação , Octopodiformes/química , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/uso terapêutico , RNA Mensageiro , Espécies Reativas de Oxigênio , Taquicininas/genética , Taquicininas/uso terapêutico , Peixe-Zebra/genéticaRESUMO
Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.
Assuntos
Canais de Cálcio Tipo N/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Transfecção/métodos , Potenciais de Ação/efeitos dos fármacos , Analgésicos/farmacologia , Animais , Células CHO , Canais de Cálcio Tipo N/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Microscopia de Fluorescência , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/metabolismo , Dor/patologia , Técnicas de Patch-ClampRESUMO
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
The human norepinephrine transporter (hNET) is a member of the neurotransmitter/sodium symporter family, which also includes the neuronal monoamine transporters for serotonin (SERT) and dopamine (DAT). Its involvement in chronic pain and many neurological disorders underlies its pharmaceutical importance. Using the X-ray crystal structures of the human serotonin transporter (hSERT) (PDB 5I6X) and Drosophila melanogaster dopamine transporter (dDAT) (PDB 4M48 and PDB 4XPA) as templates, we developed molecular models for norepinephrine (NE) bound to its high affinity binding site (S1) in the hNET. Our model suggests that the S1 site for NE is deeply buried between transmembrane helices (TMHs) 1, 3, 6, and 8 and overlaps the binding site for leucine in the bacterial leucine transporter (LeuT) and dopamine (DA) in dDAT. Mutational studies identified the functional binding pocket for NE comprised residues A73, A77, N78, V148, N153, I156, G320, F329, N350, S420, G423, and M424, which all influenced NE affinity and/or transport. These effects support a NE-hNET docking model where A73, A77, G320, S420, G423, and M424 form H-bond interactions with NE, V148, I156, and F329 form hydrophobic interactions with NE, whereas N78 affects NE transport and N350 affects NE affinity and transport via an influence on the octahedral co-ordination of the Na1 + ion. Consistent with a conserved structure-function amongst sodium-dependent neurotransmitter transporters, S1 residues A73, A77 (G100 in hSERT), N78, V148 (I150 in hSERT), N153, G320, F329 (Y331 in d DAT), N350, and G423 are conserved in DAT and SERT, indicating they likely play conserved functional roles.