RESUMO

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.

RESUMO

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.(AU)

Assuntos

Animais , Filogenia , Aranhas/classificação , Aranhas/genética , Venenos de Aranha/toxicidade , Complexo IV da Cadeia de Transporte de Elétrons/análise , Código de Barras de DNA Taxonômico/veterinária , México

RESUMO

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Assuntos

Miniproteínas Nó de Cistina , Venenos de Aranha , Aranhas , Toxinas Biológicas , Ratos , Animais , Canal de Cátion TRPA1/genética , Aranhas/química , Neurotoxinas , Espectroscopia de Ressonância Magnética , Dissulfetos , Venenos de Aranha/farmacologia , Venenos de Aranha/química

RESUMO

Peptides are molecules that have emerged as crucial candidates for the development of anticancer drugs. Spider venoms are a rich source of peptides (venom peptides - VPs) with biological effects. VPs have been tested as adjuvants in the activation of cells of the immune system with the aim of improving immunotherapies for the treatment of neoplasms. In the present study, the effects of SNX-482, a peptide from the African tarantula Hysterocrates gigas, on macrophages were described. The results showed that the peptide activated M0-macrophages, increasing costimulatory molecules (CD40, CD68, CD80, CD83, CD86) involved in antigen presentation, and also augmenting the checkpoint molecules PD-L1, CTLA-4 and FAS-L; these effects were not concentration-dependent. SNX-482 also increased the release of IL-23 and upregulated the expression of ccr4, ifn-g, gzmb and pdcd1, genes important for the anticancer response. The pretreatment of macrophages with the peptide did not interfere in the modulation of T cells, and macrophages previously polarized to M1 and M2 profile did not respond to SNX-482. These findings represent the expansion of knowledge about the use of VPs in drug discovery, pointing to a potential new candidate for anticancer immunotherapy. Considering that most immunotherapies target the adaptive system, the modulation of macrophages (an innate immune cell) by SNX-482 is especially relevant.

Assuntos

Adjuvantes Imunológicos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Venenos de Aranha/química , Aranhas/química , Animais , Antígenos CD/imunologia , Linhagem Celular Tumoral , Polaridade Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Ativação de Macrófagos/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Venenos de Aranha/farmacologia

RESUMO

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels - NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 - Ap2, Ap3 and Ap5 inhibited 20-30 % of CaV2.1 current at 1 µM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.

Assuntos

Agatoxinas/isolamento & purificação , Agatoxinas/farmacologia , Venenos de Aranha/química , Agatoxinas/química , Animais , Células CHO , Canais de Cálcio Tipo N/metabolismo , Cricetulus , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Aranhas , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo

RESUMO

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that NaV channel subtypes NaV1.7-NaV1.9 are involved in nociception. More recently, NaV1.1, NaV1.3 and NaV1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived NaV inhibitors into smaller, more "drug-like" molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. We designed a series of small, stable and novel NaV probes displaying NaV subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to NaV subtype modulation.

Assuntos

Fragmentos de Peptídeos/farmacologia , Venenos de Escorpião/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Venenos de Escorpião/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Xenopus laevis

RESUMO

Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (∼40%) and outward (∼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.

Assuntos

Canais de Potássio de Abertura Dependente da Tensão da Membrana , Venenos de Aranha , Aranhas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Moscas Domésticas , Humanos , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Domínios Proteicos , Ratos , Ratos Wistar , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/toxicidade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade , Canais de Sódio Disparados por Voltagem/química

RESUMO

BACKGROUND: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. METHODS: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). RESULTS: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 µg of PnTx4(5-5) injection in rat paw. CONCLUSION: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

RESUMO

Background:The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated.Methods:The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2).Results:PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw.Conclusion:The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.(AU)

Assuntos

Animais , Ratos , Nociceptividade , Analgésicos/análise , Peptídeos , Venenos de Aranha/uso terapêutico , Glutamatos

RESUMO

The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.(AU)

Assuntos

Venenos de Aranha , Dinoprostona , Fármacos Atuantes sobre Aminoácidos Excitatórios , Analgésicos/síntese química

RESUMO

Epilepsy is considered as one of the major disabling neuropathologies. Almost one third of adult patients with temporal lobe epilepsy (TLE) do not respond to current antiepileptic drugs (AEDs). Additionally, most AEDs do not have neuroprotective effects against the inherent neurodegenerative process underlying the hippocampal sclerosis on TLE. Dysfunctions in the GABAergic neurotransmission may contribute not only to the onset of epileptic activity but also constitute an important system for therapeutic approaches. Therefore, molecules that enhance GABA inhibitory effects could open novel avenues for the understanding of epileptic plasticity and for drug development. Parawixin2, a compound isolated from Parawixia bistriata spider venom, inhibits both GABA and glycine uptake and has an anticonvulsant effect against a wide range of chemoconvulsants. The neuroprotective potential of Parawixin2 was analyzed in a model of TLE induced by a long-lasting Status Epilepticus (SE), and its efficiency was compared to well-known neuroprotective drugs, such as riluzole and nipecotic acid. Neuroprotection was assessed through histological markers for cell density (Nissl), astrocytic reactivity (GFAP) and cell death labeling (TUNEL), which were performed 24 h and 72 h after SE. Parawixin2 treatment resulted in neuroprotective effects in a dose dependent manner at 24 h and 72 h after SE, as well as reduced reactive astrocytes and apoptotic cell death. Based on these findings, Parawixin2 has a great potential to be used as a tool for neuroscience research and as a probe to the development of novel GABAergic neuroprotective agents.

Assuntos

Epilepsia do Lobo Temporal/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Venenos de Aranha/uso terapêutico , Ureia/análogos & derivados , Animais , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/patologia , Hipocampo/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos Wistar , Ureia/uso terapêutico

RESUMO

The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the toxin δ-CNTX-Pn1c (PnTx2-6), isolated from the venom of the spider Phoneutria nigriventer. Behavioural tests suggest that PnPP-19 induces antinociception by activation of CB1, µ and δ opioid receptors. Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation, the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the subtype selectivity for µ-, δ- and/or κ-opioid receptors. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion neurons, and analyzed whether this modulation was opioid-mediated. PnPP-19 selectively activates µ-opioid receptors inducing indirectly inhibition of calcium channels and hereby impairing calcium influx in dorsal root ganglion (DRG) neurons. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce ß-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates µ-opioid receptors. The lack of ß-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists.

Assuntos

Canais de Cálcio/fisiologia , Peptídeos/farmacologia , Receptores Opioides mu/fisiologia , Venenos de Aranha/farmacologia , Animais , Gânglios Espinais/fisiologia , Células HEK293 , Humanos , Neurônios/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Ratos Wistar , Xenopus laevis

RESUMO

(1) Background: Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. It is also the one with the highest percentage of drug-resistance to the current available anti-epileptic drugs (AED). Additionaly, most antiepileptic drugs are only able to control seizures in epileptogenesis, but do not decrease the hippocampal neurodegenerative process. TLE patients have a reduced population of interneuronal cells, which express Parvalbumin (PV) proteins. This reduction is directly linked to seizure frequency and severity in the chronic period of epilepsy. There is therefore a need to seek new therapies with a disease-modifying profile, and with efficient antiepileptic and neuroprotective properties. Parawixin2, a compound isolated from the venom of the spider Parawixia bistriata, has been shown to inhibit GABA transporters (GAT) and to have acute anticonvulsant effects in rats. (2) Methods: In this work, we studied the effects of Parawixin2 and Tiagabine (an FDA- approved GAT inhibitor), and compared these effects in a TLE model. Rats were subjected to lithium-pilocarpine TLE model and the main features were evaluated over a chronic period including: (a) spontaneous recurrent seizures (SRS), (b) neuronal loss, and (c) PV cell density in different regions of the hippocampus (CA1, CA3, DG and Hilus). (3) Results: Parawixin2 treatment reduced SRS frequency whereas Tiagabine did not. We also found a significant reduction in neuronal loss in CA3 and in the hilus regions of the hippocampus, in animals treated with Parawixin2. Noteworthy, Parawixin2 significantly reversed PV cell loss observed particularly in DG layers. (4) Conclusions: Parawixin2 exerts a promising neuroprotective and anti-epileptic effect and has potential as a novel agent in drug design.

Assuntos

Anticonvulsivantes/uso terapêutico , Epilepsia do Lobo Temporal/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Inibidores da Captação de Neurotransmissores/uso terapêutico , Venenos de Aranha/uso terapêutico , Ureia/análogos & derivados , Animais , Anticonvulsivantes/farmacologia , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Hipocampo/efeitos dos fármacos , Lítio , Masculino , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Inibidores da Captação de Neurotransmissores/farmacologia , Ácidos Nipecóticos/farmacologia , Ácidos Nipecóticos/uso terapêutico , Pilocarpina , Ratos Wistar , Venenos de Aranha/farmacologia , Tiagabina , Ureia/farmacologia , Ureia/uso terapêutico

RESUMO

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Assuntos

Cisteína Endopeptidases/genética , Proteína SUMO-1/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/toxicidade , Plaquetas/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Cisteína Endopeptidases/metabolismo , Desintegrinas/genética , Desintegrinas/toxicidade , Escherichia coli/genética , Humanos , Fosfolipase D/genética , Fosfolipase D/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Proteína SUMO-1/metabolismo , Venenos de Aranha , Aranhas

RESUMO

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Assuntos

Analgésicos/farmacologia , GMP Cíclico/metabolismo , Canais KATP/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Peptídeos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Pé/fisiopatologia , Masculino , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico Sintase Tipo III/análise , Manejo da Dor , Sistema Nervoso Periférico/efeitos dos fármacos , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Venenos de Aranha

RESUMO

PnTx4(6-1), henceforth renamed δ-Ctenitoxin-Pn1a (δ-CNTX-Pn1a), a peptide from Phoneutria nigriventer spider venom, initially described as an insect toxin, binds to site 3 of sodium channels in nerve cord synaptosomes and slows down sodium current inactivation in isolated axons in cockroaches (Periplaneta americana). δ-CNTX-Pn1a does not cause any apparent toxicity to mice, when intracerebroventricularly injected (30 µg). In this study, we evaluated the antinociceptive effect of δ-CNTX-Pn1a in three animal pain models and investigated its mechanism of action in acute pain. In the inflammatory pain model, induced by carrageenan, δ-CNTX-Pn1a restored the nociceptive threshold of rats, when intraplantarly injected, 2 h and 30 min after carrageenan administration. Concerning the neuropathic pain model, δ-CNTX-Pn1a, when intrathecally administered, reversed the hyperalgesia evoked by sciatic nerve constriction. In the acute pain model, induced by prostaglandin E2, intrathecal administration of δ-CNTX-Pn1a caused a dose-dependent antinociceptive effect. Using antagonists of the receptors, we showed that the antinociceptive effect of δ-CNTX-Pn1a involves both the cannabinoid system, through CB1 receptors, and the opioid system, through µ and δ receptors. Our data show, for the first time, that δ-Ctenitoxin-Pn1a is able to induce antinociception in inflammatory, neuropathic and acute pain models.

Assuntos

Dor Aguda/tratamento farmacológico , Analgésicos/uso terapêutico , Proteínas de Artrópodes/uso terapêutico , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Peptídeos/uso terapêutico , Dor Aguda/metabolismo , Analgésicos/farmacologia , Animais , Proteínas de Artrópodes/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Carragenina , Dinoprostona , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Masculino , Antagonistas de Entorpecentes/farmacologia , Neuralgia/induzido quimicamente , Neuralgia/metabolismo , Peptídeos/farmacologia , Ratos Wistar , Receptores de Canabinoides/metabolismo , Receptores Opioides/metabolismo , Nervo Isquiático/lesões , Venenos de Aranha/química , Aranhas

RESUMO

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1­S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

Assuntos

Peptídeos , Rhodnius , Venenos de Aranha , Inseticidas