Comparative venomic profiles of three spiders of the genus Phoneutria.

Background: Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.

Mapping of Brain Activity in the Analgesia Induced by Phα1ß and Morphine.

Preclinical evidence suggests the potential of Phα1ß, a toxin obtained from the venom of spider Phoneutria nigriventer, as a new analgesic drug. Molecular brain imaging techniques have afforded exciting opportunities to examine brain processes in clinical pain conditions. This paper aims to study the brain regions involved in the analgesic effects of Phα1ß compared with Morphine, in a model of acute pain induced by formalin in Sprague Dawley rats. We used 18F-fluorodeoxyglucose as a metabolic radiotracer to perform brain imaging of rats pretreated with Phα1ß or Morphine in a model of acute inflammatory pain caused by intraplantar injection of formalin. The rats' hind paw's formalin stimulation resulted in a brain metabolic increase at the bilateral motor cortex, visual cortex, somatosensory cortex, thalamus, and cingulate cortex.In rats treated with Phα1ß, selective inhibition of unilateral motor cortex and cingulate cortex was observed. Morphine treatment leads to small and selective inhibition at the bilateral amygdala striatum and accumbens. Our results indicate that the analgesic effect of Phα1ß and Morphine possesses a differential profile of central processing in the pain state.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus.

The aim of this study was to investigate the effect of spider toxins on brain injury induced by oxygen deprivation and low glucose (ODLG) insult on slices of rat hippocampus. After ODLG insult cell viabilility in hippocampal slices was assessed by confocal microscopy and epifluorescence using the live/dead kit containing calcein-AM and ethidium homodimer and CA1 population spike amplitude recording during stimulation of Schaffer collateral fibers. Spider toxins Tx3-3 or Tx3-4 and conus toxins, omega-conotoxin GVIA or omega-conotoxin MVIIC are calcium channel blockers and protected against neuronal damage in slices subjected to ODLG insult. Confocal imaging of CA1 region of rat hippocampal slices subject to ischemic insult treated with Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC showed a decrease in cell death that amounted to 68 +/- 4.2%, 77 +/- 3.8%, 32 +/- 2.3%, and 46 +/- 2.9%, respectively. This neuroprotective effect of Tx3-4 was corroborated by eletrophysiological recordings of population spikes amplitudes in CA1. The neuroprotection promoted on hippocampal slices by Tx3-3 or Tx3-4 was also observed when the toxins were applied 10, 20, 30, 60, 90, or 120 min after induction of the ODLG injury. During the ischemic insult, glutamate release from slices was increased by 71% (from 7.0 +/- 0.3 nM/mg of protein control slices not subjected to ischemia to 12 +/- 0.4 nM/mg of protein in slices exposed to ischemia). Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC inhibited the ischemia-induced increase on glutamate release by 54, 72, 60, and 70%, respectively. Thus Tx3-3 and Tx3-4 provided robust ischemic neuroprotection showing potential as a novel class of agent that exerts neuroprotection in an in vitro model of brain ischemia.

Immunodetection of toxins in historesin-embedded sections of Phoneutria nigriventer venom glands using laser confocal scanning microscopy.

In the last decades, main advances were achieved in the identification, structural and pharmacological characterization of Phoneutria nigriventer toxins. However, studies on the venom-producing apparatus are rare. Presently, we applied immunolabeling to historesin-embedded cross-sections of P. nigriventer venom glands. Toxins and toxin-secreting cells were successfully located in situ, using laser confocal scanning microscopy. The methodological strategy was successful and may be applied in future studies on venom glands and other secreting tissues, in general.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system.

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.

Effect of the spider toxin Tx3-3 on spinal processing of sensory information in naive and neuropathic rats: an in vivo electrophysiological study.

INTRODUCTION: Drugs that counteract nociceptive transmission in the spinal dorsal horn preferentially after nerve injury are being pursued as possible neuropathic pain treatments. In a previous behavioural study, the peptide toxin Tx3-3, which blocks P/Q- and R-type voltage-gated calcium channels, was effective in neuropathic pain models. OBJECTIVES: In the present study, we aimed to investigate the effect of Tx3-3 on dorsal horn neuronal responses in rats under physiological conditions and neuropathic pain condition induced by spinal nerve ligation (SNL). METHODS: In vivo electrophysiological recordings of dorsal horn neuronal response to electrical and natural (mechanical and thermal) stimuli were made in rats under normal physiological state (naive rats) or after the SNL model of neuropathic pain. RESULTS: Tx3-3 (0.3-100 pmol/site) exhibited greater inhibitory effect on electrical-evoked neuronal response of SNL rats than naive rats, inhibiting nociceptive C-fibre and Aδ-fibre responses only in SNL rats. The wind-up of neurones, a measurement of spinal cord hyperexcitability, was also more susceptible to a dose-related inhibition by Tx3-3 after nerve injury. Moreover, Tx3-3 exhibited higher potency to inhibit mechanical- and thermal-evoked neuronal response in conditions of neuropathy. CONCLUSION: Tx3-3 mediated differential inhibitory effect under physiological and neuropathic conditions, exhibiting greater potency in conditions of neuropathic pain.

Comparative venomic profiles of three spiders of the genus Phoneutria

Abstract Background: Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Characterization of the antinociceptive effect of PhTx3-4, a toxin from Phoneutria nigriventer, in models of thermal, chemical and incisional pain in mice.

Venom-derived peptides constitute a unique source of drug prototypes for the pain management. Many of them can modulate voltage-gated calcium channels that are central in the processing of pain sensation. PhTx3-4 is a peptide isolated from Phoneutria nigriventer venom, which blocks high voltage-activated calcium channels with low specificity, thereby leading to neuroprotection in models of ischemia in vitro. The aim of the present work was evaluating the potential of intrathecal PhTx3-4 in the reversal of different nociceptive states in mice, furthermore assessing the potential of PhTx3-4 in triggering motor side effects. We found that bellow 100 pmol/site, PhTx3-4 did not cause major motor side effects. By comparison, ω-conotoxin MVIIA and ω-conotoxin MVIIC triggered motor side effects at the doses of 10 and 100 pmol/site, respectively. Also, PhTx3-4 (30 pmol/site) caused no significant alterations in the forced locomotor activity test (rotarod) and in the exploratory activity test (versamax). In a model of inflammatory persistent pain (formalin test), PhTx3-4 reversed nociceptive behavior both pre or post-administered, although this effect was observed only at the inflammatory phase of the test and not at the neurogenic phase. Comparatively, ω-conotoxin MVIIC was effective only when post-administered in the formalin test. Nonetheless, PhTx3-4 treatment was devoid of action in acute nociceptive thermal model (hotplate test), whereas morphine showed efficacy in this test. Efficacy of PhTx3-4 in the formalin test was associated with inhibition of formalin-induced glutamate release in the cerebrospinal fluid. PhTx3-4, but not ω-conotoxin MVIIC, reversed NMDA-induced nociceptive behavior indicating a putative role of PhTx3-4 at ionotropic glutamate receptors. Finally, we observed efficacy of PhTx3-4 in ameliorating mechanical hypersensitivity induced by paw incision, a post-operative and more clinically relevant pain model. Taken together, our data show that PhTx3-4 possesses antinociceptive effect in different models of pain in mice, suggesting that this toxin may serve as drug prototype for pain control.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system

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)

The selective A-type K+ current blocker Tx3-1 isolated from the Phoneutria nigriventer venom enhances memory of naïve and Aß(25-35)-treated mice.

Potassium channels regulate many neuronal functions, including neuronal excitability and synaptic plasticity, contributing, by these means, to mnemonic processes. In particular, A-type K(+) currents (IA) play a key role in hippocampal synaptic plasticity. Therefore, we evaluated the effect of the peptidic toxin Tx3-1, a selective blocker of IA currents, extracted from the venom of the spider Phoneutria nigriventer, on memory of mice. Administration of Tx3-1 (i.c.v., 300 pmol/site) enhanced both short- and long-term memory consolidation of mice tested in the novel object recognition task. In comparison, 4-aminopyridine (4-AP; i.c.v., 30-300 pmol/site), a non-selective K(+) channel blocker did not alter long-term memory and caused toxic side effects such as circling, freezing and tonic-clonic seizures. Moreover, Tx3-1 (i.c.v., 10-100 pmol/site) restored memory of Aß25-35-injected mice, and exhibited a higher potency to improve memory of Aß25-35-injected mice when compared to control group. These results show the effect of the selective blocker of IA currents Tx3-1 in both short- and long-term memory retention and in memory impairment caused by Aß25-35, reinforcing the role of IA in physiological and pathological memory processes.

Developmental biology of the Brazilian 'Armed' spider Phoneutria nigriventer (Keyserling, 1891): microanatomical and molecular analysis of the embryonic stages.

Phoneutria (Ctenidae) is among the most dangerous venomous spiders in Brazil. Its venom is composed of a mixture of pharmacologically active components, some of which have been quite extensively studied due to their potentiality as models for new pharmaceutical drugs. Nevertheless, literature data on the venom-producing glands are very limited. In the present study, we follow the biological development of intra-cocoon stages of Phoneutria nigriventer spiders, mainly regarding the formation of the venomous apparatus and venom production. The results showed that the venom glands of Phoneutria are already present in the early 1st pre-larva stage. The venomous apparatus is completely formed in the larva, a stage that precedes the spider eclosion from the cocoon. At embryo stages, transcripts of a vertebrate-active neurotoxin (PhTx1) were shown to be present, as well as, unidentified venom proteins that were immunolabeled by anti-venom antibodies. It seems that venom toxins play roles in the protection and survival of those early developmental stages of Phoneutria spiders.

Analgesic effect in rodents of native and recombinant Ph alpha 1beta toxin, a high-voltage-activated calcium channel blocker isolated from armed spider venom.

Calcium influx through neuronal voltage-sensitive calcium channels (VSCC S) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCC S blockers, such as omega-conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Ph alpha 1beta, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Ph alpha 1beta showed higher efficacy and long-lasting analgesia in a thermal model of pain, when compared with omega-conotoxin MVIIA. Moreover, Ph alpha 1beta was more effective and potent than omega-conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca2+-evoked release of pro-nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Ph alpha 1beta had a therapeutic index wider than omega- conotoxin MVIIA. Finally, recombinant Ph alpha 1beta expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Ph alpha 1beta have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.