Bioactive peptides from scorpion venoms: therapeutic scaffolds and pharmacological tools / 中国天然药物

Evolution and natural selection have endowed animal venoms, including scorpion venoms, with a wide range of pharmacological properties. Consequently, scorpions, their venoms, and/or their body parts have been used since time immemorial in traditional medicines, especially in Africa and Asia. With respect to their pharmacological potential, bioactive peptides from scorpion venoms have become an important source of scientific research. With the rapid increase in the characterization of various components from scorpion venoms, a large number of peptides are identified with an aim of combating a myriad of emerging global health problems. Moreover, some scorpion venom-derived peptides have been established as potential scaffolds helpful for drug development. In this review, we summarize the promising scorpion venoms-derived peptides as drug candidates. Accordingly, we highlight the data and knowledge needed for continuous characterization and development of additional natural peptides from scorpion venoms, as potential drugs that can treat related diseases.

Construction of three different recombinant scorpion fusion proteins with bifunctional activity.

This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B.

Efeitos da injeção intra-hipocampal de toxinas isoladas do veneno de escorpião Tityus bahiensis em ratos

No Brasil, os escorpiões Tityus serrulatus e o Tityus bahiensis são os de importância médica. Porém, poucos são os estudos sobre os efeitos do veneno do T. bahiensis principalmente no sistema nervoso central. Os venenos escorpiônicos são compostos por neurotoxinas, poliptídeos de baixo peso molecular. Em estudos prévios, o veneno do Tityus bahiensis mostrou-se convulsivo e com características distintas das do Tityus serrulatus. Suas frações foram estudadas e quatro delas apresentaram resultados interessantes nos parâmetros observados (alterações comportamentais e etroencefalográficas e perda neuronal). O objetivo deste trabalho foi estudar os efeitos hipocampais de toxinas que compõem estas frações em ratos. As frações selecionadas para o estudo foram recromatografadas em HPLC de coluna fase-reversa e os picos de maior rendimento foram utilizados nos experimentos. Cânulas e eletrodos foram implantados no hipocampo de ratos machos Wistar. Cada grupo de animais recebeu 1ul da solução de toxina (1 ou 2ug/ul) ou solução Ringer (grupo controle) e foi submetido à análise comportamentais, em intensidades diferentes. As toxinas 3-IV, 1-V, 24-V e 28-V causaram lesão neuronal significante em CA4 ipsi e contralateral. A toxina Tb 4-V induziu efeito epileptogênico mais pronunciado e por isso foi estudada pela técnica de microdiálise para dosagem de aminoácidos neurotransmissores e mobilização de cálcio intracelular por microscopia confocal. Sua administração provocou aumento diferencial do glutamato nos indivíduos observados e aumento da mobilização de cálcio citoplásmico em algumas fatias hipocampais. Além disso, a toxina 4-V, principalmente, apresentou marcado efeito "tudo ou nada". Portanto, as toxinas provenientes do veneno do Tityus bahiensis podem ser úteis ferramentas para o estudo da fisiologia dos canais iônicos e hodologia neural.

Efeitos da injeção intra-hipocampal de toxinas isoladas do veneno de escorpião Tityus bahiensis em ratos / Effects of intra-hippocampal infection of toxins isolated of Tityus bahiensis scorpion venom in rats

No Brasil, os escorpiões Tityus serrulatus e o Tityus bahiensis são os de importância médica. Porém, poucos são os estudos sobre os efeitos do veneno do T. bahiensis principalmente no sistema nervoso central. Os venenos escorpiônicos são compostos por neurotoxinas, poliptídeos de baixo peso molecular. Em estudos prévios, o veneno do Tityus bahiensis mostrou-se convulsivo e com características distintas das do Tityus serrulatus. Suas frações foram estudadas e quatro delas apresentaram resultados interessantes nos parâmetros observados (alterações comportamentais e etroencefalográficas e perda neuronal). O objetivo deste trabalho foi estudar os efeitos hipocampais de toxinas que compõem estas frações em ratos. As frações selecionadas para o estudo foram recromatografadas em HPLC de coluna fase-reversa e os picos de maior rendimento foram utilizados nos experimentos. Cânulas e eletrodos foram implantados no hipocampo de ratos machos Wistar. Cada grupo de animais recebeu 1ul da solução de toxina (1 ou 2ug/ul) ou solução Ringer (grupo controle) e foi submetido à análise comportamentais, em intensidades diferentes. As toxinas 3-IV, 1-V, 24-V e 28-V causaram lesão neuronal significante em CA4 ipsi e contralateral. A toxina Tb 4-V induziu efeito epileptogênico mais pronunciado e por isso foi estudada pela técnica de microdiálise para dosagem de aminoácidos neurotransmissores e mobilização de cálcio intracelular por microscopia confocal. Sua administração provocou aumento diferencial do glutamato nos indivíduos observados e aumento da mobilização de cálcio citoplásmico em algumas fatias hipocampais. Além disso, a toxina 4-V, principalmente, apresentou marcado efeito "tudo ou nada". Portanto, as toxinas provenientes do veneno do Tityus bahiensis podem ser úteis ferramentas para o estudo da fisiologia dos canais iônicos e hodologia neural (AU).

Vasosensory responses elicited by Indian red scorpion venom last longer than capsaicin-induced responses.

The present study was conducted to compare the time-related cardiorespiratory changes occurring after the injection of Mesobuthus tamulus (BT; 1 mg/kg) venom and capsaicin (1.2 ng/kg) in the peripheral end of femoral artery in urethane anaesthetised rats. Blood pressure (BP), electrocardiogram (for heart rate; HR) and respiratory movements were recorded for 60 min after venom/capsaicin intra-arterially. Minute ventilation (MV) was computed by using appropriate calibrations. After intraarterial injection of BT venom, there was immediate (within 2 sec) increase in respiratory rate (RR) and MV which reached to 40% within 30 sec, followed by a 40% decrease in RR without any change in MV. Further, there was sustained increase in RR (50%) and MV (65%) up to 60 min. The BP began to increase at 40 sec, peaking at 5 min (50%) and remained above the initial level up to 60 min. The bradycardiac response began after 5 min which peaked (50% of the initial) at 25 min and remained at that level up to 60 min. In capsaicin treated group, there was immediate hyperventilatory (increase in RR and MV) changes within 2 sec which returned to the initial level within 2 min and remained at that level up to 60 min. The capsaicin-induced hypotensive response began within 5 sec which returned to the initial level by 5 min and remained at that level throughout. Capsaicin did not produce any change in HR. These observations suggest that intraarterial injection of BT venom produces prolonged cardiorespiratory alterations as compared to the capsaicin-induced responses.

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction. Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.

O íon magnésio no tratamento de acidentes causados pelos escorpiöes Tityus serrulatus, T. bahiensis e Centruroides sculpturatus: estudo experimental / Magnesium in the treatment of accidents caused by the scorpions Tityus serrulatus, T. bahiensis and Centruroides sculpturatus: an experimental study

Os efeitos produzidos pelas peçonhas escorpiônicas säo consequentes, em sua maioria, à liberaçäo de acetilcolina (ACh) e catecolaminas. A verificaçäo de que o magnésio (Mg2+) inibe a liberaçäo de ACh em razäo de bloquear o influxo de cálcio nas terminaçöes nervosas, levou-nos a investigar a açäo deste cátion sobre os distúrbios produzidos pelas peçonhas escorpiônicas. Relatamos na presente comunicaçäo a açäo do Mg2+ sobre os efeitos induzidos pelas peçonhas dos escorpiöes Tityus serrulatus, T. bahiensis e Centruroides sculpturatus nas preparaçöes isoladas nervo frênico-diafragma, íleo, canal deferente e átrios de rato e in vivo, em ratos anestesiados com registro da pressäo arterial e do eletrocardiograma. Os efeitos da peçonha dos escorpiöes nas preparaçöes isoladas foram abolidos ou muito atenuados pelo Mg2+. O Mg2+, no entanto, somente antagonizou os efeitos da peçonha de C. sculpturatus no íleo de rato. Em ratos anestesiados, a hipertensäo e arritmias provocadas pela peçonha de T. serrulatus foram revertidas com exclusäo de bradicardia pela injeçäo do Mg2+. A peçonha de C. sculpturatus na maioria das experiências causou hipotensäo e arritmias de pequena gravidade. O Mg2+ reverteu as arritmias, mas causou quedas acentuadas da pressäo arterial. Os resultados da pesquisa sugerem o emprego do Mg2+ em acidentes graves na ausência de hipotensäo e bradicardia, produzidos por T. serrulatus e T. bahiensis. Parece contra-indicado nos acidentes causados por C. sculpturatus em vista de seu efeito acima referido na pressäo arterial.

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37 degrees Celsius with a 1 per cent (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 mug of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 mug) was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice.

Augmentation of phenyldiguanide-induced bradycardia by Buthus tamulus venom in adult rats.

Intravenous injection of phenyldiguanide (PDG) in anaesthetized rats produced dose-dependent (1-10 micrograms/kg) decrease in heart rate for a period of time (time-response area). The maximum response occurred at 10 micrograms/kg PDG. Administration of B. tamulus (BT) venom as low as 20 micrograms/kg augmented the PDG-induced bradycardia response by 2.5 times the initial PDG (10 micrograms/kg) response. The maximal augmentation was observed after 60 min of venom injection. Increasing the BT venom concentration to 40 micrograms/kg failed to enhance the reflex response (1.7 times the initial response). The threshold concentration of BT venom was 4 micrograms/kg. BT venom (100 micrograms/kg) alone, decreased the heart rate significantly only after 90 min. Results indicate that, even the sublethal concentrations of BT venom sensitize the reflexes elicited by PDG.

Protección contra el alacranismo / Protectión agains scorpionism

En este capítulo se revisan algunos aspectos del problema del alacranismo en México. Se discuten algunos datos obtenidos con una vacuna experimental animal, para la cual el antígeno fue generado por polimerización de extractos solubles de glándulas venenosas de alacranes mexicanos. Se da énfasis a la discusión de los procedimientos y datos bioquímicos más importantes obtenidos en el fraccionamiento del veneno soluble extraído por estimulación eléctrica de telsons de alacranes vivos. También se mencionan algunas propiedades inmunológicas de los extractos de las glándulas, del veneno soluble, de sus fracciones cromatográficas y de sus polipéptidos altamente purificados. Finalmente, se presentan datos sobre la inmunización de ratones con péptidos sintéticos, diseñados con base en la secuencia de aminoácidos de las toxinas de alacranes. Las perspectivas futuras de este trabajo se enfocan a la determinación de los epítopos protectores de las toxinas del veneno y a la clonación y modificación por ingeniería genética de genes que codifican para estos péptidos tóxicos. No obstante el vasto trabajo experimental realizado, todavía no existe una vacuna disponible contra los efectos neurotóxicos del veneno de alacrán. Desde el punto de vista médico, el problema del envenenamiento por piquete de alacrán debe ser tratado mediante el uso del suero antialacrán, única medicina de elección para el tratamiento de los casos severos de accidentes con estos arácnidos

Brazilian scorpion venoms: pharmacological aspects

This review focuses on clinical, epidemiological and therapeutic aspects of envenomation caused by scorpions in Brazil. In addition, it presents studies with the crude venom and neurotoxins isolated from Tityus serrulatus scorpion venom. Such studies show the pharmacological effects of the venom and isolated toxins in experiments that use the scorpion venom and neurotoxins as neurobiological tools to study receptor sites in sodium channels and neurotransmitter release.

Pharmacology of scorpion (Lychas laevifrons Pock) venom with special reference to its neuromuscular activity.

L. laevifrons venom caused irreversible blockade of electrically induced twitch responses on phrenic nerve diaphragm and chick biventer cervicis preparation. The venom lowered cat blood pressure, caused a brief cardiac arrest and increased cutaneous capillary permeability. It contracted several smooth muscle preparations. The quick contraction produced on guinea pig ileum was partly antagonized by mepyramine and completely by methysergide. The residual slow contraction was antagonized by SC 19220, a prostaglandin blocker. Haemolysis was not produced by the venom on human RBC. LD50 of crude venom in mice was 13.8 mg/kg (iv).

Effect of tityustoxin and quabain on protein phosphorylation in a crude synaptosome fraction

1. The effect of titiyustoxin (TsTX) and ouabain on the incorporation of 32P into a protein of the same apparent molecular weight as synapsin I it described. 2. Tityustoxin-stimulated protein phosphorylation in a crude synaptosome fraction increased up to a concentration of 3.0 micron time of 15 s. 3. Trifluoperazizne (100 micronM) inhibited, while trifluoperazine sulphoxide (100 micronM) did not alter the effect on the protein phosphorylation induced by tityustoxin. 4. Unlike tityutixin, ouabain (100 micronM) hada no effect on protein phosphorylation even after incubation up to 20 min. 5. Ouabain at at 10 micronM, a concentration having no effect on rates of respiration and ATP hydrolysis in brain cortical slices, also had no effect on protein phosphorylation

Impact of scorpion Heterometrus fulvipes venom on cholinesterase rhythmicity in the tropical mouse Mus booduga.

Rhythmicities of acetylcholine (ACh) and acetylcholinesterase (AChE) were studied in the mouse, Mus booduga (Gray), following intramuscular injection of scorpion, Heterometrus fulvipes (C. Koch) venom. Envenomation inhibited the activity levels of ACh and AChE in all the tissues selected for experimentation. Control animals exhibited diel rhythmicity in ACh and AChE while envenomated animals showed fluctuation in the Phase (delta phi), amplitude (A), Acrophase (phi) and the extreme activity hours (X and X1).