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1.
Biomed Pharmacother ; 150: 113094, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35658242

RESUMO

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.


Assuntos
Dendroaspis , Toxinas Biológicas , Animais , Artérias/metabolismo , Colinérgicos , Dendroaspis/metabolismo , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacologia , Humanos , Peptídeos/farmacologia , Ratos , Receptores Muscarínicos/metabolismo
2.
Am J Trop Med Hyg ; 106(1): 338-341, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34724630

RESUMO

The black mamba is known for its notorious potent neurotoxic venom. For this reason, their bites are often erroneously treated in the field with the application of a tourniquet in the hope of delaying systemic spread of the venom. Observational studies have shown that inappropriate tourniquet application is a common, harmful practice. An arterial tourniquet is not a recommended first aid measure because of the risk of limb ischemia and gangrene. When inappropriately applied, the rapid removal of the tourniquet in the emergency department may precipitate a life-threatening venom and metabolic toxin rush, leading to respiratory arrest. We present two cases of black mamba bites in Gauteng, South Africa, where gradual tourniquet removal was used to avoid a venom rush and rapid respiratory paralysis. Venom and metabolic toxin rush with potentially fatal respiratory muscle paralysis may be averted by gradual, cautious removal of field-applied tourniquets with concomitant antivenom administration.


Assuntos
Dendroaspis , Mordeduras de Serpentes/terapia , Torniquetes , Animais , Gasometria , Humanos , Masculino , Pessoa de Meia-Idade , Mordeduras de Serpentes/complicações , Adulto Jovem
3.
Clin Toxicol (Phila) ; 59(10): 860-868, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34219550

RESUMO

CONTEXT: The black mamba (Dendroaspis polylepis) is, due to its extremely toxic venom, one of the most dangerous snake species in Sub-Saharan Africa. A D. polylepis bite is a medical emergency and requires adequate action to prevent severe complications. However, there are no comprehensive reviews available based on clinical cases, and no readily accessible guidelines for standardized treatment. Therefore, we aim to provide an overview regarding the currently available clinical literature on D. polylepis envenomations; in order to promote knowledge on symptomatology and treatment options. METHODS: We searched for cases reporting humans bitten by D. polylepis in PubMed, Embase, Scopus, and Sabinet. We searched the reference lists of all eligible articles for additional articles. After quality assessment, 29 cases were included in this review. We used descriptive analysis to create an overview of the collected parameters. DISCUSSION: Among the included case reports and case series, D. polylepis envenomations most frequently resulted in decreased respiratory function, sweating and paralysis. The onset of symptoms usually occurred within 60 minutes. Neurological symptoms occurred more often than symptoms of autonomic dysfunction. In the reported cases most patients (26/29) received antivenom and most survived (25/29). We recommend the reporting of additional structured case reports to improve future analyses on the clinical course of envenomations, in order to improve public health response to D. polylepis envenomations.


Assuntos
Antivenenos/uso terapêutico , Dendroaspis , Venenos Elapídicos/antagonistas & inibidores , Mordeduras de Serpentes/tratamento farmacológico , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Dendroaspis/metabolismo , Venenos Elapídicos/metabolismo , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mordeduras de Serpentes/diagnóstico , Mordeduras de Serpentes/metabolismo , Mordeduras de Serpentes/mortalidade , Resultado do Tratamento , Adulto Jovem
4.
Acad Radiol ; 28(2): 149-150, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33272827
5.
Biochem J ; 477(20): 3951-3962, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33000863

RESUMO

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three ß-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.


Assuntos
Dendroaspis/metabolismo , Venenos Elapídicos/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Cristalografia por Raios X , Dimerização , Dissulfetos/química , Venenos Elapídicos/isolamento & purificação , Elapidae/metabolismo , Evolução Molecular , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas em Tandem
7.
Sci Rep ; 10(1): 5096, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198446

RESUMO

Historical data can clarify ecological attributes of fauna in sites that have subsequently been altered by anthropogenic activities. We used the 1960s notebooks of pioneering East African naturalist C.J.P. Ionides to extract quantitative information on captures of 484 snakes of five species (rhinoceros vipers Bitis nasicornis, black mambas Dendroaspis polylepis, Jameson's mambas D. jamesoni, water cobras Naja annulata, and eastern forest cobras N. subfulva). High capture rates suggest high abundances of all species. The relative numbers of each species collected changed over the years and differed seasonally, reflecting targeting by Ionides. Sex ratios and age-class distributions differed among species and were affected by factors such as month of collection and time of day. Habitat use was affected by species, sex and body size: for example, arboreality became less common with increasing body size in the rhinoceros viper and black mamba, and males were found in arboreal sites more often than were females. In both D. jamesoni and D. polylepis, adult males and females were recorded together in September-October, suggesting reproductive activity at this time of year. Although fragmentary, the data from Ionides' notebooks provide a unique glimpse into ecological patterns of snakes within an African landscape half a century ago.


Assuntos
Demografia/história , Dendroaspis/classificação , Naja/classificação , Viperidae/classificação , África Oriental , Animais , Conservação dos Recursos Naturais , Ecossistema , Feminino , História do Século XX , Masculino , Razão de Masculinidade
8.
Int J Mol Sci ; 21(6)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197368

RESUMO

Using thrombelastography to gain mechanistic insights, recent investigations have identified enzymes and compounds in Naja and Crotalus species' neurotoxic venoms that are anticoagulant in nature. The neurotoxic venoms of the four extant species of Dendroaspis (the Black and green mambas) were noted to be anticoagulant in nature in human blood, but the mechanisms underlying these observations have never been explored. The venom proteomes of these venoms are unique, primarily composed of three finger toxins (3-FTx), Kunitz-type serine protease inhibitors (Kunitz-type SPI) and <7% metalloproteinases. The anticoagulant potency of the four mamba venoms available were determined in human plasma via thrombelastography; vulnerability to inhibition of anticoagulant activity to ethylenediaminetetraacetic acid (EDTA) was assessed, and inhibition of anticoagulant activity after exposure to a ruthenium (Ru)-based carbon monoxide releasing molecule (CORM-2) was quantified. Black mamba venom was the least potent by more than two orders of magnitude compared to the green mamba venoms tested; further, Black Mamba venom anticoagulant activity was not inhibited by either EDTA or CORM-2. In contrast, the anticoagulant activities of the green mamba venoms were all inhibited by EDTA to a greater or lesser extent, and all had anticoagulation inhibited with CORM-2. Critically, CORM-2-mediated inhibition was independent of carbon monoxide release, but was dependent on a putative Ru-based species formed from CORM-2. In conclusion, there was great species-specific variation in potency and mechanism(s) responsible for the anticoagulant activity of Dendroaspis venom, with perhaps all three protein classes-3-FTx, Kunitz-type SPI and metalloproteinases-playing a role in the venoms characterized.


Assuntos
Anticoagulantes/química , Coagulação Sanguínea , Dendroaspis , Venenos Elapídicos/química , Neurotoxinas/química , Proteoma/química , Animais , Tromboelastografia
9.
Am J Forensic Med Pathol ; 40(4): 356-360, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31318708

RESUMO

We present the case of a male adult who was admitted to an emergency department after having sustained envenomation from a black mamba (Dendroaspis polylepis). According to the available history, a single fang hooked his right index finger, post venom extraction. After administering antivenom in the accident and emergency department, further vials were transfused in the intensive care unit. An urticarial rash was noted, which was thought to be related to the antivenom. The victim remained in a coma for 3 days, after which he was declared dead. A medicolegal postmortem examination was performed 4 days after death because of logistical reasons. The complexities of differentiating acute envenomation from black mamba versus early acute reactions to polyvalent antivenom administration are highlighted in this case study.


Assuntos
Antivenenos/administração & dosagem , Antivenenos/efeitos adversos , Dendroaspis , Mordeduras de Serpentes/complicações , Venenos de Serpentes/imunologia , Adulto , Anafilaxia/diagnóstico , Animais , Diagnóstico Diferencial , Coagulação Intravascular Disseminada/etiologia , Oxigenação por Membrana Extracorpórea , Humanos , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , África do Sul
10.
Toxicon ; 168: 76-82, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254601

RESUMO

The African elapid snake genus Dendroaspis comprises four species, with D. polylepsis the most dangerous of them. D. viridis is believed to cause stronger neurotoxic symptoms than the potentially least toxic of the genus, D. angusticeps, but seems less toxic compared to either of the D. jamesoni species (D. j. jamesoni(TRAILL 1843) and D. j. kaimosae (Loveridge 1936)). We present three episodes of bites byD. viridis in the same patient, sustained on three different occasions, caused by three different and unrelated snakes. While the first bite remained oligosymptomatic with a slight tightness of the throat and speedy resolution of symptoms without specific therapy, episodes two and three resulted in the patient developing massive local swelling. However, the patient showed only minimal neurologic and systemic symptoms such as tightness of the throat and a tingling sensation of the body. Episode two resolved with fasciotomy after compartment syndrome was diagnosed with a measured intracompartmental pressure of 52 mmHg. In episode three, antivenom was administered with good resolution of symptoms. The clinical courses in this patient were remarkable as he displayed mainly local symptoms after three individual bites by a supposedly neurotoxic snake.


Assuntos
Síndromes Compartimentais/induzido quimicamente , Dendroaspis , Mordeduras de Serpentes/tratamento farmacológico , Mordeduras de Serpentes/cirurgia , Adulto , Animais , Antivenenos/uso terapêutico , Síndromes Compartimentais/tratamento farmacológico , Síndromes Compartimentais/cirurgia , Venenos Elapídicos , Fasciotomia , Humanos , Masculino , Pessoa de Meia-Idade , Suíça
12.
Nat Commun ; 9(1): 3928, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279409

RESUMO

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.


Assuntos
Anticorpos Monoclonais Humanizados/química , Antivenenos/química , Dendroaspis , Venenos Elapídicos/imunologia , Fatores Imunológicos/química , Mordeduras de Serpentes/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivenenos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Venenos Elapídicos/antagonistas & inibidores , Fatores Imunológicos/uso terapêutico , Camundongos , Testes de Neutralização
13.
Dev Comp Immunol ; 81: 141-151, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29154857

RESUMO

Astakine1 was isolated as a hematopoietic cytokine in the freshwater crayfish Pacifastacus leniusculus. In this study we detect and compare 79 sequences in GenBank, which we consider to be possible astakine orthologs, among which eleven are crustacean, sixteen are chelicerate and 52 are from insect species. Available arthropod genomes are searched for astakines, and in conclusion all astakine sequences in the current study have a similar exon containing CCXX(X), thus potentially indicating that they are homologous genes with the structure of this exon highly conserved. Two motifs, RYS and YP(N), are also conserved among the arthropod astakines. A phylogenetic analysis reveals that astakine1 and astakine2 from P. leniusculus and Procambarus clarkii are distantly related, and may have been derived from a gene duplication occurring early in crustacean evolution. Moreover, a structural comparison using the Mamba intestinal toxin (MIT1) from Dendroaspis polylepis as template indicates that the overall folds are similar in all crustacean astakines investigated.


Assuntos
Artrópodes/genética , Astacoidea/genética , Dendroaspis/fisiologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Animais , Evolução Biológica , Bases de Dados de Ácidos Nucleicos , Especiação Genética , Hematopoese/genética , Peptídeos e Proteínas de Sinalização Intercelular , Estrutura Molecular , Peptídeos/genética , Filogenia , Domínios Proteicos/genética , Especificidade da Espécie , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo
14.
J Proteomics ; 172: 173-189, 2018 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28843532

RESUMO

Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation. BIOLOGICAL SIGNIFICANCE: The mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation.


Assuntos
Antivenenos/imunologia , Dendroaspis , Venenos Elapídicos/química , África ao Sul do Saara , Animais , Dieta , Venenos Elapídicos/imunologia , Venenos Elapídicos/toxicidade , Elapidae , Humanos , Filogenia , Especificidade da Espécie , Transcriptoma
15.
Int J Mol Sci ; 18(11)2017 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-29156586

RESUMO

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.


Assuntos
Dendroaspis/metabolismo , Glicopeptídeos/análise , Glicopeptídeos/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Dendroaspis/genética , Venenos Elapídicos/análise , Venenos Elapídicos/química , Venenos Elapídicos/genética , Glicopeptídeos/genética , Estrutura Molecular , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
16.
Toxicon ; 138: 151-158, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28867663

RESUMO

Polyvalent snakebite antivenoms derive their therapeutic success from the ability of their antibodies to neutralize venom toxins across multiple snake species. This ability results from a production process involving immunization of large mammals with a broad suite of toxins present in venoms. As a result of immunization with this wide range of toxins, many polyvalent antivenoms have a high degree of cross-reactivity to similar toxins in other snake venoms - a cross-reactivity which cannot easily be deconvoluted. As a proof of concept, we aimed at exploring the opposite scenario by performing a high-throughput evaluation of the extent of cross-reactivity of a polyclonal mixture of antibodies that was raised against only a single snake venom fraction. For this purpose, a venom fraction containing short neurotoxin 1 (SN-1; Uniprot accession number P01416, three-finger toxin (3FTx) family), which is the medically most important toxin from the notorious black mamba (Dendroaspis polylepis), was employed. Following immunization of a rabbit, a specific polyclonal antibody response was confirmed by ELISA and immunodiffusion. Subsequently, these antibodies were investigated by high-density peptide microarray to reveal linear elements of recognized epitopes across 742 3FTxs and 10 dendrotoxins. This exploratory study demonstrates in a single immunized animal that cross-reactivity between toxins of high similarity may be difficult to obtain when immunizing with a single 3FTx containing venom fraction. Additionally, this study explored the influence of employing different lengths of peptides in high-density peptide microarray experiments for identification of toxin epitopes. Using 8-mer, 12-mer, and 15-mer peptides, a single linear epitope element was identified in SN-1 with high precision.


Assuntos
Formação de Anticorpos/imunologia , Reações Cruzadas , Dendroaspis , Venenos Elapídicos/imunologia , Animais , Imunização , Peptídeos/imunologia , Análise Serial de Proteínas , Coelhos
17.
Sci Rep ; 7(1): 2701, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28578406

RESUMO

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.


Assuntos
Dendroaspis/metabolismo , Engenharia de Proteínas , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Sequência de Aminoácidos , Animais , Dendroaspis/genética , Evolução Molecular , Modelos Moleculares , Filogenia , Conformação Proteica , Venenos de Serpentes/genética , Venenos de Serpentes/farmacologia
18.
Proc Natl Acad Sci U S A ; 114(27): 7154-7159, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630289

RESUMO

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.


Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Dendroaspis , Peptídeos Natriuréticos/farmacologia , Peptídeos/farmacologia , Doenças Renais Policísticas/tratamento farmacológico , Receptores de Vasopressinas/genética , Venenos de Serpentes/farmacologia , Animais , Benzazepinas/farmacologia , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tolvaptan , Tripsina/química
19.
Sci Rep ; 6: 36629, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27824133

RESUMO

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.


Assuntos
Antivenenos/química , Dendroaspis , Venenos Elapídicos/química , Epitopos/química , Biblioteca de Peptídeos , Análise Serial de Proteínas/métodos , Animais
20.
J Proteomics ; 146: 148-64, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27318176

RESUMO

We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution. SIGNIFICANCE PARAGRAPH: We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution.


Assuntos
Dendroaspis , Venenos Elapídicos/química , Proteoma/análise , Animais , Cardiotoxinas/análise , Venenos Elapídicos/análise , Venenos Elapídicos/toxicidade , Lisina/metabolismo , Neurotoxinas/análise , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Especificidade da Espécie , Espectrometria de Massas em Tandem
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