RESUMO
Centipedes are a group of venomous arthropods widely distributed around the world, and due to their well-adapted nature to urban areas, they often cause accidents in humans. Despite their limited medical relevance, centipede envenomation can lead to a range of undesirable symptoms such as burning pain, paresthesia, swelling, and superficial necrosis at the bite site, occasionally progressing to a severe condition in rare cases. A clinical study involving patients treated at the Vital Brazil Hospital of the Butantan Institute showed that most centipede accidents were caused by the Cryptops and Otostigmus genera, accounting for about 90% of cases. Literature reports indicate that centipede venom contains several bioactive compounds, some with potential therapeutic interest. However, despite its significant pharmacological importance, very little is known about the active components of these animals' venom. Therefore, centipede venom could be an excellent source of as-yet-unknown toxins with unexplored biotechnological potential. Cryptops is one of the genera most associated with accidents in humans, and so far, there is no literature study on the toxins of its venom. Through a transcriptomic analysis of the venom gland of Cryotops iheringi conducted by our group, a significant proportion of acetylcholinesterase sequences was observed. As cholinesterases are involved in cholinergic synapses present in the central and peripheral nervous systems, this enzyme becomes an attractive target for the development of new drugs. This project was already underway in our group, where the enzyme was cloned into a bacterial vector. Consequently, this work aims to carry out the expression tests and purification of this molecule.
As centopeias são um grupo de artrópodes venenosos amplamente distribuídos pelo mundo, e por serem animais bem adaptados a áreas urbanas, frequentemente provocam acidentes em humanos. Apesar da pouca relevância médica, o envenenamento por lacraias pode causar uma série de sintomas indesejáveis, como dor ardente, parestesia, edema e necrose superficial no local da picada, podendo, em casos raros, evoluir para um quadro grave. Um estudo clínico com pacientes atendidos no Hospital Vital Brazil do Instituto Butantan, mostrou que a maioria dos acidentes com lacraias foram causados pelos gêneros Cryptops e Otostigmus, representando cerca de 90% dos casos. Têm sido relatados na literatura que o veneno das lacraias contém vários compostos bioativos, alguns com potencial interesse terapêutico, no entanto, apesar da importância farmacológica significante, muito pouco se sabe sobre os componentes ativos do veneno destes animais. Portanto, o veneno de lacraias pode ser uma excelente fonte de toxinas ainda desconhecidas e com potencial biotecnológico inexplorado. Cryptops é um dos gêneros mais associados a acidentes em humanos e, que até o presente momento, não há na literatura nenhum estudo sobre as toxinas de seu veneno. Através de uma análise transcriptômica da glândula de veneno de Cryotops iheringi realizado pelo nosso grupo, foi observada uma grande proporção de sequências de acetilcolinesterase. Uma vez que as colinesterases estão envolvidas em sinapses colinérgicas presentes no sistema nervoso central e periférico, esta enzima torna-se um alvo atraente para o desenvolvimento de novas drogas. Este projeto já estava em desenvolvimento pelo nosso grupo, onde foi realizada a clonagem da enzima em vetor bacteriano. Por conseguinte, este trabalho tem como objetivo realizar os testes de expressão e purificação desta molécula.
RESUMO
Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme's substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.
Assuntos
Fosfolipase D , Venenos de Aranha , Aranhas , Animais , Esfingomielinas/metabolismo , Diester Fosfórico Hidrolases/química , Fosfolipase D/metabolismo , Venenos de Aranha/química , Fosfolipídeos/metabolismo , Lisofosfatidilcolinas , Aranhas/metabolismoRESUMO
Alpha-latrotoxin (ÉLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits. ÉLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ÉLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans. The hyperimmune sera obtained using the complete ÉLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 µL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.
Assuntos
Viúva Negra , Venenos de Aranha , Animais , Anquirinas , Antivenenos/farmacologia , Antivenenos/uso terapêutico , Cavalos , Camundongos , CoelhosRESUMO
This study investigated the effects of intravenous (iv) administration of recombinant Phα1ß toxin, pregabalin, and diclofenac by the intrathecal route using an animal model fibromyalgia (FM). The reserpine administration (0.25 mg/kg s. c) once daily for three consecutive days significantly induced hyperalgesia, immobility time, and sucrose consumption in mice on the 4th day. Reserpine caused hyperalgesia on the mechanical and thermal hyperalgesia on the 4th day was reverted by recombinant Phα1ß (0.2 mg/kg iv) and pregabalin (1.25 µmol/site i. t) treatments. In contrast, diclofenac (215 nmol/site i. t) was ineffective. Recombinant Phα1ß toxin, pregabalin, and diclofenac did not affect the depressive-like behavioural effect induced by reserpine on mice during the forced swim and sucrose consumption tests. The data confirmed the analgesic effect of the recombinant Phα1ß toxin administered intravenously in a fibromyalgia mouse model.
Assuntos
Fibromialgia , Venenos de Aranha/toxicidade , Administração Intravenosa , Analgésicos/uso terapêutico , Animais , Modelos Animais de Doenças , Fibromialgia/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Camundongos , Reserpina/uso terapêutico , Venenos de Aranha/administração & dosagemRESUMO
Snakes, scorpions, and spiders are venomous animals that pose a threat to human health, and severe envenomings from the bites or stings of these animals must be treated with antivenom. Current antivenoms are based on plasma-derived immunoglobulins or immunoglobulin fragments from hyper-immunized animals. Although these medicines have been life-saving for more than 120 years, opportunities to improve envenoming therapy exist. In the later decades, new biotechnological tools have been applied with the aim of improving the efficacy, safety, and affordability of antivenoms. Within the avenues explored, novel immunization strategies using synthetic peptide epitopes, recombinant toxins (or toxoids), or DNA strings as immunogens have demonstrated potential for generating antivenoms with high therapeutic antibody titers and broad neutralizing capacity. Furthermore, these approaches circumvent the need for venom in the production process of antivenoms, thereby limiting some of the complications associated with animal captivity and venom collection. Finally, an important benefit of innovative immunization approaches is that they are often compatible with existing antivenom manufacturing setups. In this review, we compile all reported studies examining venom-independent innovative immunization strategies for antivenom development. In addition, a brief description of toxin families of medical relevance found in snake, scorpion, and spider venoms is presented, as well as how biochemical, bioinformatic, and omics tools could aid the development of next-generation antivenoms.
Assuntos
Antivenenos/administração & dosagem , Antivenenos/biossíntese , Mordeduras de Serpentes/tratamento farmacológico , Picada de Aranha/tratamento farmacológico , Animais , Antivenenos/imunologia , Humanos , Venenos de Serpentes/imunologia , Venenos de Aranha/imunologiaRESUMO
Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Artrópodes/antagonistas & inibidores , Aranha Marrom Reclusa/enzimologia , Desenho de Fármacos , Fosfolipase D/antagonistas & inibidores , Venenos de Aranha/antagonistas & inibidores , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Benzimidazóis/farmacologia , Aranha Marrom Reclusa/genética , Aranha Marrom Reclusa/patogenicidade , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Necrose , Fosfolipase D/química , Fosfolipase D/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Piperidinas/farmacologia , Coelhos , Proteínas Recombinantes/genética , Pele/efeitos dos fármacos , Pele/patologia , Picada de Aranha/tratamento farmacológico , Picada de Aranha/enzimologia , Venenos de Aranha/química , Venenos de Aranha/genética , Suramina/farmacologiaRESUMO
The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P. nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50 = 212.5 nM), and at 1 µM caused an increase on current peak amplitude of 105.2 ± 3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43 ± 8.04%, IC50 = 1.5 µM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.