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1.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921462

RESUMO

Cardiovascular diseases (CVDs) are considered as a major cause of death worldwide. Therefore, identifying and developing therapeutic strategies to treat and reduce the prevalence of CVDs is a major medical challenge. Several drugs used for the treatment of CVDs, such as captopril, emerged from natural products, namely snake venoms. These venoms are complex mixtures of bioactive molecules, which, among other physiological networks, target the cardiovascular system, leading to them being considered in the development and design of new drugs. In this review, we describe some snake venom molecules targeting the cardiovascular system such as phospholipase A2 (PLA2), natriuretic peptides (NPs), bradykinin-potentiating peptides (BPPs), cysteine-rich secretory proteins (CRISPs), disintegrins, fibrinolytic enzymes, and three-finger toxins (3FTXs). In addition, their molecular targets, and mechanisms of action-vasorelaxation, inhibition of platelet aggregation, cardioprotective activities-are discussed. The dissection of their biological effects at the molecular scale give insights for the development of future snake venom-derived drugs.


Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Venenos de Serpentes/química , Venenos de Serpentes/uso terapêutico , Animais , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/patologia , Humanos , Modelos Biológicos , Venenos de Serpentes/farmacologia
2.
Nat Protoc ; 16(3): 1494-1510, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33504990

RESUMO

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.


Assuntos
Técnicas de Cultura de Células/métodos , Organoides/crescimento & desenvolvimento , Venenos de Serpentes/biossíntese , Animais , Antivenenos/genética , Hibridização in Situ Fluorescente/métodos , Venenos de Serpentes/química , Venenos de Serpentes/genética , Serpentes/genética
3.
Food Chem Toxicol ; 149: 112009, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33503469

RESUMO

SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. Studies have shown that smokers are less likely to be diagnosed with or be hospitalized for COVID-19 but, once hospitalized, have higher odds for an adverse outcome. We have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a "toxin-like" epitope on the Spike glycoprotein, with homology to a sequence of a snake venom toxin. This epitope coincides with the well-described cryptic epitope for the human anti-SARS-CoV antibody CR3022. In this study, we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike glycoproteins, at their open or closed conformations, with the model of the human α7 nAChR. We found that all studied protein complexes' interface involves a large part of the "toxin-like" sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human α7 nAChR. Our findings provide further support to the hypothesis about the protective role of nicotine and other cholinergic agonists. The potential therapeutic role of CR3022 and other similar monoclonal antibodies with increased affinity for SARS-CoV-2 Spike glycoprotein against the clinical effects originating from the dysregulated cholinergic pathway should be further explored.


Assuntos
COVID-19/virologia , Epitopos , Nicotina/farmacologia , Vírus da SARS/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Sítios de Ligação de Anticorpos , COVID-19/metabolismo , COVID-19/prevenção & controle , Humanos , Modelos Moleculares , Agonistas Nicotínicos/química , Agonistas Nicotínicos/uso terapêutico , Sistema Colinérgico não Neuronal , Pandemias , Fatores de Proteção , Conformação Proteica , Homologia de Sequência , Transdução de Sinais , Fumantes , Fumar , Venenos de Serpentes/química
4.
PLoS Negl Trop Dis ; 14(6): e0008366, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579606

RESUMO

Snakebite envenoming is a major neglected tropical disease that affects millions of people every year. The only effective treatment against snakebite envenoming consists of unspecified cocktails of polyclonal antibodies purified from the plasma of immunized production animals. Currently, little data exists on the molecular interactions between venom-toxin epitopes and antivenom-antibody paratopes. To address this issue, high-density peptide microarray (hdpm) technology has recently been adapted to the field of toxinology. However, analysis of such valuable datasets requires expert understanding and, thus, complicates its broad application within the field. In the present study, we developed a user-friendly, and high-throughput web application named "Snake Toxin and Antivenom Binding Profiles" (STAB Profiles), to allow straight-forward analysis of hdpm datasets. To test our tool and evaluate its performance with a large dataset, we conducted hdpm assays using all African snake toxin protein sequences available in the UniProt database at the time of study design, together with eight commercial antivenoms in clinical use in Africa, thus representing the largest venom-antivenom dataset to date. Furthermore, we introduced a novel method for evaluating raw signals from a peptide microarray experiment and a data normalization protocol enabling intra-microarray and even inter-microarray chip comparisons. Finally, these data, alongside all the data from previous similar studies by Engmark et al., were preprocessed according to our newly developed protocol and made publicly available for download through the STAB Profiles web application (http://tropicalpharmacology.com/tools/stab-profiles/). With these data and our tool, we were able to gain key insights into toxin-antivenom interactions and were able to differentiate the ability of different antivenoms to interact with certain toxins of interest. The data, as well as the web application, we present in this article should be of significant value to the venom-antivenom research community. Knowledge gained from our current and future analyses of this dataset carry the potential to guide the improvement and optimization of current antivenoms for maximum patient benefit, as well as aid the development of next-generation antivenoms.


Assuntos
Antivenenos/farmacologia , Reações Cruzadas , Gerenciamento de Dados , Peptídeos , Análise Serial de Proteínas/métodos , África , Animais , Sítios de Ligação , Epitopos/química , Humanos , Mordeduras de Serpentes/terapia , Venenos de Serpentes/química , Serpentes/classificação , Serpentes/metabolismo
5.
PLoS Negl Trop Dis ; 14(4): e0007802, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236099

RESUMO

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.


Assuntos
Anticoagulantes/análise , Fatores Biológicos/análise , Coagulantes/análise , Peptídeos/análise , Proteínas/análise , Venenos de Serpentes/química , Animais , Coagulação Sanguínea/efeitos dos fármacos , Fracionamento Químico , Cromatografia Líquida , Humanos , Plasma/efeitos dos fármacos , Proteômica , Espectrometria de Massas em Tandem
6.
Sci Rep ; 10(1): 4476, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161292

RESUMO

Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel.


Assuntos
Hemostasia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Serina Proteases/toxicidade , Venenos de Serpentes/toxicidade , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Catálise , Linhagem Celular , Desenho de Fármacos , Fenômenos Eletrofisiológicos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/química , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Bloqueadores dos Canais de Potássio/química , Canais de Potássio/química , Proteínas Recombinantes , Serina Proteases/química , Venenos de Serpentes/química , Relação Estrutura-Atividade
7.
Toxicon ; 178: 92-99, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32135198

RESUMO

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought.


Assuntos
Adaptação Fisiológica/fisiologia , Didelphis/fisiologia , Venenos de Serpentes/toxicidade , Fator de von Willebrand/metabolismo , Animais , Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Metaloproteases/metabolismo , Agregação Plaquetária , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , América do Sul
8.
PLoS One ; 15(2): e0229657, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106235

RESUMO

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.


Assuntos
Boidae/sangue , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Inibidores de Fosfolipase A2/sangue , Sequência de Aminoácidos , Animais , Bothrops/metabolismo , Brasil , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/química , Fosfolipases A2 do Grupo IV/química , Peso Molecular , Inibidores de Fosfolipase A2/química , Domínios e Motivos de Interação entre Proteínas , Venenos de Serpentes/antagonistas & inibidores , Venenos de Serpentes/química , Espectrometria de Massas em Tandem
9.
Annu Rev Anim Biosci ; 8: 91-116, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31702940

RESUMO

Snake venoms are primarily composed of proteins and peptides, and these toxins have developed high selectivity to their biological targets. This makes venoms interesting for exploration into protein evolution and structure-function relationships. A single venom protein superfamily can exhibit a variety of pharmacological effects; these variations in activity originate from differences in functional sites, domains, posttranslational modifications, and the formations of toxin complexes. In this review, we discuss examples of how the major venom protein superfamilies have diversified, as well as how newer technologies in the omics fields, such as genomics, transcriptomics, and proteomics, can be used to characterize both known and unknown toxins.Because toxins are bioactive molecules with a rich diversity of activities, they can be useful as therapeutic and diagnostic agents, and successful examples of toxin applications in these areas are also reviewed. With the current rapid pace of technology, snake venom research and its applications will only continue to expand.


Assuntos
Evolução Molecular , Venenos de Serpentes/química , Venenos de Serpentes/farmacologia , Animais , Genômica , Proteômica , Venenos de Serpentes/genética , Serpentes/genética , Transcriptoma
10.
Artigo em Inglês | MEDLINE | ID: mdl-31634575

RESUMO

Dispholidus typus and Thelotornis mossambicanus are closely related rear-fanged colubrid snakes that both possess strongly procoagulant venoms. However, despite similarities in overall venom biochemistry and resulting clinical manifestations, the underlying venom composition differs significantly between the two species. As a result, the only available antivenom-which is a monovalent antivenom for D. typus-has minimal cross reactivity with T. mossambicanus and is not a clinically viable option. It was hypothesised that this lack of cross reactivity is due to the additional large metalloprotease protein within T. mossambicanus venom, which may also be responsible for faster coagulation times. In this study, we found that T. mossambicanus venom is a more powerful activator of prothrombin than that of D. typus and that the SVMP transcripts from T. mossambicanus form a clade with those from D. typus. The sequences from D. typus and T. mossambicanus were highly similar in length, with the calculated molecular weights of the T. mossambicanus transcripts being significantly less than the molecular weights of some isoforms on the 1D SDS-PAGE gels. Analyses utilising degylcosylating enzymes revealed that T. mossambicanus SVMPs are glycosylated during post-translational modification, but that this does not lead to the different molecular weight bands observed in 1D SDS-PAGE gels. However, differences in glycosylation patterns may still explain some of the difference between the enzymatic activities and neutralization by antivenom that have been observed in these venoms. The results of this study provide new information regarding the treatment options for patients envenomated by T. mossambicanus as well as the evolution of these dangerous snakes.


Assuntos
Colubridae/fisiologia , Metaloproteases/metabolismo , Protrombina/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Animais , Colubridae/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicosilação , Metaloproteases/genética , Filogenia , Protrombina/química , Protrombina/farmacologia , Transcriptoma
11.
PLoS Negl Trop Dis ; 13(12): e0007899, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31805055

RESUMO

BACKGROUND: Snakebite in India causes the highest annual rates of death (46,000) and disability (140,000) than any other country. Antivenom is the mainstay treatment of snakebite, whose manufacturing protocols, in essence, have remained unchanged for over a century. In India, a polyvalent antivenom is produced for the treatment of envenomations from the so called 'big four' snakes: the spectacled cobra (Naja naja), common krait (Bungarus caeruleus), Russell's viper (Daboia russelii), and saw-scaled viper (Echis carinatus). In addition to the 'big four', India is abode to many other species of venomous snakes that have the potential to inflict severe clinical or, even, lethal envenomations in their human bite victims. Unfortunately, specific antivenoms are not produced against these species and, instead, the 'big four' antivenom is routinely used for the treatment. METHODS: We characterized the venom compositions, biochemical and pharmacological activities and toxicity profiles (mouse model) of the major neglected yet medically important Indian snakes (E. c. sochureki, B. sindanus, B. fasciatus, and two populations of N. kaouthia) and their closest 'big four' congeners. By performing WHO recommended in vitro and in vivo preclinical assays, we evaluated the efficiencies of the commercially marketed Indian antivenoms in recognizing venoms and neutralizing envenomations by these neglected species. FINDINGS: As a consequence of dissimilar ecologies and diet, the medically important snakes investigated exhibited dramatic inter- and intraspecific differences in their venom profiles. Currently marketed antivenoms were found to exhibit poor dose efficacy and venom recognition potential against the 'neglected many'. Premium Serums antivenom failed to neutralise bites from many of the neglected species and one of the 'big four' snakes (North Indian population of B. caeruleus). CONCLUSIONS: This study unravels disturbing deficiencies in dose efficacy and neutralisation capabilities of the currently marketed Indian antivenoms, and emphasises the pressing need to develop region-specific snakebite therapy for the 'neglected many'.


Assuntos
Antitoxinas/farmacologia , Antivenenos/uso terapêutico , Mordeduras de Serpentes/terapia , Venenos de Serpentes/química , Venenos de Serpentes/toxicidade , Animais , Modelos Animais de Doenças , Índia , Masculino , Camundongos , Análise de Sobrevida , Resultado do Tratamento
12.
Molecules ; 24(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744153

RESUMO

We carried out surveys on the use of Cordia nodosa Lam. in the jungles of Bobonaza (Ecuador). We documented this knowledge to prevent its loss under the Framework of the Convention on Biological Diversity and the Nagoya Protocol. We conducted bibliographic research and identified quercetrin as a significant bioactive molecule. We studied its in silico biological activity. The selected methodology was virtual docking experiments with the proteins responsible for the venomous action of snakes. The molecular structures of quercetrin and 21 selected toxins underwent corresponding tests with SwissDock and Chimera software. The results point to support its antiophidic use. They show reasonable geometries and a binding free energy of -7 to -10.03 kcal/mol. The most favorable values were obtained for the venom of the Asian snake Naja atra (5Z2G, -10.03 kcal/mol). Good results were also obtained from the venom of the Latin American Bothrops pirajai (3CYL, -9.71 kcal/mol) and that of Ecuadorian Bothrops asper snakes (5TFV, -9.47 kcal/mol) and Bothrops atrox (5TS5, -9.49 kcal/mol). In the 5Z2G and 5TS5 L-amino acid oxidases, quercetrin binds in a pocket adjacent to the FAD cofactor, while in the myotoxic homologues of PLA2, 3CYL and 5TFV, it joins in the hydrophobic channel formed when oligomerizing, in the first one similar to α-tocopherol. This study presents a case demonstration of the potential of bioinformatic tools in the validation process of ethnobotanical phytopharmaceuticals and how in silico methods are becoming increasingly useful for sustainable drug discovery.


Assuntos
Antídotos/química , Antídotos/farmacologia , Cordia/química , Modelos Moleculares , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Sítios de Ligação , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Venenos de Serpentes/antagonistas & inibidores , Venenos de Serpentes/química , Relação Estrutura-Atividade , Toxinas Biológicas/antagonistas & inibidores , Toxinas Biológicas/química , Árvores
13.
Dokl Biochem Biophys ; 487(1): 251-255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31559591

RESUMO

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Neurotoxinas/química , Antagonistas Nicotínicos/química , Polissacarídeos/química , Receptores Nicotínicos/metabolismo , Sulfatos/química , Cápsulas , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Tamanho da Partícula , Venenos de Serpentes/química
14.
PLoS One ; 14(9): e0222206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31513632

RESUMO

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution.


Assuntos
Bothrops/metabolismo , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Animais , Bothrops/genética , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Masculino , Espectrometria de Massas , Camundongos , Plasma , Venenos de Serpentes/genética
15.
Anal Chem ; 91(16): 10458-10466, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31373797

RESUMO

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.


Assuntos
Antifibrinolíticos/isolamento & purificação , Bioensaio , Cromatografia Líquida/métodos , Fibrinolíticos/isolamento & purificação , Nanotecnologia/métodos , Peptídeo Hidrolases/isolamento & purificação , Animais , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Cromatografia Líquida/instrumentação , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Nanotecnologia/instrumentação , Peptídeo Hidrolases/análise , Venenos de Serpentes/química , Serpentes/metabolismo
16.
Blood Coagul Fibrinolysis ; 30(8): 379-384, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31415248

RESUMO

BACKGROUND: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. METHODS: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. RESULTS: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. CONCLUSION: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.


Assuntos
Anticoagulantes , Monóxido de Carbono/farmacologia , Venenos de Serpentes/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Coleta de Amostras Sanguíneas , Bungarotoxinas/antagonistas & inibidores , Bungarotoxinas/química , Bungarotoxinas/farmacologia , Bungarus , Cobras Corais , Venenos Elapídicos/antagonistas & inibidores , Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Elapidae , Humanos , Neurotoxinas/antagonistas & inibidores , Proteoma/análise , Venenos de Serpentes/antagonistas & inibidores , Venenos de Serpentes/química , Tromboelastografia
17.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370142

RESUMO

Hypertension is considered a major public health issue due to its high prevalence and subsequent risk of cardiovascular and kidney diseases. Thus, the search for new antihypertensive compounds remains of great interest. Snake venoms provide an abundant source of lead molecules that affect the cardiovascular system, which makes them prominent from a pharmaceutical perspective. Such snake venom components include bradykinin potentiating peptides (proline-rich oligopeptides), natriuretic peptides, phospholipases A2, serine-proteases and vascular endothelial growth factors. Some heparin binding hypotensive factors, three-finger toxins and 5' nucleotidases can also exert blood pressure lowering activity. Great advances have been made during the last decade regarding the understanding of the mechanism of action of these hypotensive proteins. Bradykinin potentiating peptides exert their action primarily by inhibiting the angiotensin-converting enzyme and increasing the effect of endogenous bradykinin. Snake venom phospholipases A2 are capable of reducing blood pressure through the production of arachidonic acid, a precursor of cyclooxygenase metabolites (prostaglandins or prostacyclin). Other snake venom proteins mimic the effects of endogenous kallikrein, natriuretic peptides or vascular endothelial growth factors. The aim of this work was to review the current state of knowledge regarding snake venom components with potential antihypertensive activity and their mechanisms of action.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Hipotensão/tratamento farmacológico , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Anti-Hipertensivos/química , Bradicinina/química , Bradicinina/uso terapêutico , Humanos , Peptídeos/química , Peptídeos/uso terapêutico , Venenos de Serpentes/química
18.
Curr Top Med Chem ; 19(22): 1962-1980, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31345151

RESUMO

Among the ophidians that inhabit the Northeast of Argentina, the genus Bothrops such as B. alternatus and B. diporus species (also known as yararás) and Crotalus durisus terrificus (named cascabel), represent the most studied snake venom for more than thirty years. These two genera of venomous snakes account for the majority of poisonous snake envenomations and therefore, constitute a medical emergency in this region. This review presents a broad description of the compiled knowledge about venomous snakebite: its pathophysiological action, protein composition, isolated toxins, toxin synergism, toxin-antitoxin cross-reaction assays. Properties of some isolated toxins support a potential pharmacological application.


Assuntos
Venenos de Serpentes/farmacologia , Toxinas Biológicas/farmacologia , Animais , Argentina , Bothrops , Crotalus , Humanos , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Toxinas Biológicas/química , Toxinas Biológicas/isolamento & purificação
19.
J Proteome Res ; 18(9): 3419-3428, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337208

RESUMO

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.


Assuntos
Metaloproteases/genética , Proteoma/genética , Proteômica , Venenos de Serpentes/genética , Sequência de Aminoácidos/genética , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/isolamento & purificação , Bothrops/genética , Humanos , Marcação por Isótopo , Metaloproteases/química , Metaloproteases/isolamento & purificação , Camundongos , Biblioteca de Peptídeos , Proteoma/química , Venenos de Serpentes/química , Especificidade por Substrato/genética , Espectrometria de Massas em Tandem
20.
Curr Top Med Chem ; 19(22): 2041-2048, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31340737

RESUMO

BACKGROUND: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. METHODS: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. RESULTS: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 µg/mL against Leishmania infantum promastigotes and of 13.8 µg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 µg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 µg/mL. CONCLUSION: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.


Assuntos
Antiparasitários/farmacologia , Leishmania infantum/efeitos dos fármacos , Fosfolipases A2/farmacologia , Venenos de Serpentes/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiparasitários/química , Antiparasitários/isolamento & purificação , Bothrops , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Camundongos , Paraguai , Testes de Sensibilidade Parasitária , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Relação Estrutura-Atividade
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