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1.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35131940

RESUMO

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.


Assuntos
Venenos de Formiga/química , Formigas/fisiologia , Hipersensibilidade a Drogas , Fator de Crescimento Epidérmico/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Mordeduras e Picadas de Insetos , Camundongos , Mimetismo Molecular
2.
Circulation ; 144(12): 947-960, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34264749

RESUMO

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.


Assuntos
Canais Iônicos Sensíveis a Ácido/biossíntese , Canais Iônicos Sensíveis a Ácido/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Preparação de Coração Isolado/métodos , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/terapia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Venenos de Aranha/farmacologia
3.
Bioinformatics ; 34(6): 1074-1076, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29069336

RESUMO

Summary: ArachnoServer is a manually curated database that consolidates information on the sequence, structure, function and pharmacology of spider-venom toxins. Although spider venoms are complex chemical arsenals, the primary constituents are small disulfide-bridged peptides that target neuronal ion channels and receptors. Due to their high potency and selectivity, these peptides have been developed as pharmacological tools, bioinsecticides and drug leads. A new version of ArachnoServer (v3.0) has been developed that includes a bioinformatics pipeline for automated detection and analysis of peptide toxin transcripts in assembled venom-gland transcriptomes. ArachnoServer v3.0 was updated with the latest sequence, structure and functional data, the search-by-mass feature has been enhanced, and toxin cards provide additional information about each mature toxin. Availability and implementation: http://arachnoserver.org. Contact: support@arachnoserver.org. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Venenos de Aranha/química , Animais , Automação Laboratorial , Dissulfetos/química , Proteínas de Insetos/química , Peptídeos/química , Venenos de Aranha/análise
5.
Microb Cell Fact ; 16(1): 6, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28095880

RESUMO

BACKGROUND: Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.


Assuntos
Escherichia coli/genética , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Peptídeos/genética , Proteínas Recombinantes/isolamento & purificação , Peçonhas/genética , Animais , Dissulfetos/química , Descoberta de Drogas/métodos , Endopeptidases/metabolismo , Proteínas de Escherichia coli/genética , Oxirredução , Peptídeos/isolamento & purificação , Peptídeos/uso terapêutico , Periplasma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Peçonhas/química
6.
Microb Cell Fact ; 16(1): 4, 2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28093085

RESUMO

BACKGROUND: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. RESULTS: The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. CONCLUSIONS: This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.


Assuntos
Endopeptidases/metabolismo , Escherichia coli/genética , Biossíntese Peptídica , Peptídeos/genética , Peçonhas/biossíntese , Peçonhas/genética , Animais , Biotecnologia/métodos , Clonagem Molecular , Dissulfetos/química , Endopeptidases/química , Vetores Genéticos , Ensaios de Triagem em Larga Escala , Oxirredução , Peptídeos/química , Peptídeos/isolamento & purificação , Periplasma/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Peçonhas/química , Peçonhas/metabolismo
7.
Proc Natl Acad Sci U S A ; 108(3): 1040-5, 2011 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-21193638

RESUMO

Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.


Assuntos
Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Munc18/metabolismo , Conformação Proteica , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Sintaxina 1/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Cristalografia , Dados de Sequência Molecular , Proteínas Munc18/química , Ligação Proteica , Alinhamento de Sequência
8.
ACS Pharmacol Transl Sci ; 7(4): 1043-1054, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38638162

RESUMO

Activation of acid-sensing ion channel 1a (ASIC1a) plays a major role in mediating acidosis-induced neuronal injury following a stroke. Therefore, the inhibition of ASIC1a is a potential therapeutic avenue for the treatment of stroke. Venom-peptide Hi1a, a selective and highly potent ASIC1a inhibitor, reduces the infarct size and functional deficits when injected into the brain after stroke in rodents. However, its efficacy when administered using a clinically relevant route of administration remains to be established. Therefore, the current investigation aims to examine the efficacy of systemically administered Hi1a, using two different models of stroke in different species. Mice were subjected to the filament model of middle cerebral artery occlusion (MCAO) and treated with Hi1a systemically using either a single- or multiple-dosing regimen. 24 h poststroke, mice underwent functional testing, and the brain infarct size was assessed. Rats were subjected to endothelin-1 (ET-1)-induced MCAO and treated with Hi1a intravenously 2 h poststroke. Rats underwent functional tests prior to and for 3 days poststroke, when infarct volume was assessed. Mice receiving Hi1a did not show any improvements in functional outcomes, despite a trend toward reduced infarct size. This trend for reduced infarct size in mice was consistent regardless of the dosing regimen. There was also a trend toward lower infarct size in rats treated with Hi1a. More specifically, Hi1a reduced the amount of damage occurring within the somatosensory cortex, which was associated with an improved sensorimotor function in Hi1a-treated rats. Thus, this study suggests that Hi1a or more brain-permeable ASIC1a inhibitors are a potential stroke treatment.

9.
Microb Cell Fact ; 12: 37, 2013 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-23607455

RESUMO

BACKGROUND: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. RESULTS: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. CONCLUSIONS: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.


Assuntos
Escherichia coli/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Citoplasma/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
10.
Org Lett ; 25(24): 4439-4444, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37306339

RESUMO

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.


Assuntos
Canais Iônicos Sensíveis a Ácido , Peptídeos , Ligadura , Peptídeos/química , Venenos de Aranha/metabolismo , Venenos de Aranha/farmacologia , AVC Isquêmico/fisiopatologia , Infarto do Miocárdio/fisiopatologia
11.
J Neurotrauma ; 2023 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-36924276

RESUMO

Acid-sensing ion channel 1a (ASIC1a) is a proton-activated channel that is expressed ubiquitously throughout the central nervous system and in various types of immune cells. Its role in spinal cord injury (SCI) is controversial; inhibition of ASIC1a has been reported to improve SCI pathology in vivo, but conversely, gene ablation increased kainite-mediated excitotoxic cell death in vitro. Here, we re-examined the role of ASIC1a in a mouse model of SCI. First, we observed functional outcomes up to 42 days post-operation (DPO) in SCI mice with a selective genetic ablation of ASIC1a. Mice lacking ASIC1a had significantly worsened locomotor ability and increased lesion size compared with mice possessing the ASIC1a gene. Next, we explored pharmacological antagonism of this ion channel by administering the potent ASIC1a inhibitor, Hi1a. Consistent with a role for ASIC1a to attenuate excitotoxicity, accelerated neuronal cell loss was found at the lesion site in SCI mice treated with Hi1a, but there were no differences in locomotor recovery. Moreover, ASIC1a inhibition did not cause significant alterations to neutrophil migration, microglial density, or blood-spinal cord barrier integrity.

12.
Mol Pharmacol ; 80(5): 796-808, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21825095

RESUMO

Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a-ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a ß-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.


Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Venenos de Aranha/farmacologia , Canais Iônicos Sensíveis a Ácido , Sequência de Aminoácidos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos , Mutação Puntual , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Canais de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Aranha/química , Venenos de Aranha/genética
13.
Front Bioeng Biotechnol ; 9: 811905, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35127675

RESUMO

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

14.
Org Lett ; 23(21): 8375-8379, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34632783

RESUMO

Hi1a is a venom peptide from the Australian funnel-web spider Hadronyche infensa with a complex tertiary structure. Hi1a has neuroprotective and cardioprotective properties due to its potent inhibition of acid-sensing ion channel 1a (ASIC1a) and is currently being pursued as a novel therapy for acute ischemic events. Herein, we describe the total synthesis of Hi1a using native chemical ligation. The synthetic peptide was successfully folded and exhibited similar inhibitory activity on ASIC1a to recombinant Hi1a.


Assuntos
Venenos de Aranha
15.
J Med Chem ; 63(15): 8250-8264, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32602722

RESUMO

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.


Assuntos
Proteômica/métodos , Receptores de Melanocortina/agonistas , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Receptores de Melanocortina/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/metabolismo
16.
Toxicon ; 158: 109-126, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30543821

RESUMO

Spiders have been evolving complex and diverse repertoires of peptides in their venoms with vast pharmacological activities for more than 300 million years. Spiders use their venoms for prey capture and defense, hence they contain peptides that target both prey (mainly arthropods) and predators (other arthropods or vertebrates). This includes peptides that potently and selectively modulate a range of targets such as ion channels, receptors and signaling pathways involved in physiological processes. The contribution of these targets in particular disease pathophysiologies makes spider venoms a valuable source of peptides with potential therapeutic use. In addition, peptides with insecticidal activities, used for prey capture, can be exploited for the development of novel bioinsecticides for agricultural use. Although we have already reviewed potential applications of spider venom peptides as therapeutics (in 2010) and as bioinsecticides (in 2012), a considerable number of research articles on both topics have been published since, warranting an updated review. Here we explore the most recent research on the use of spider venom peptides for both medical and agricultural applications.


Assuntos
Inseticidas/farmacologia , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Animais , Descoberta de Drogas , Inseticidas/química , Peptídeos/química , Venenos de Aranha/química , Venenos de Aranha/toxicidade , Aranhas/química
17.
Vet Parasitol ; 270: 40-46, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213240

RESUMO

Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC50 4.9 µM and 16.1 µM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC50 of 22.9 ± 1.9 µM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target.


Assuntos
Anti-Helmínticos/farmacologia , Diminazena/farmacologia , Haemonchus/efeitos dos fármacos , Pentamidina/farmacologia , Animais , Antiprotozoários/farmacologia , Técnicas In Vitro , Concentração Inibidora 50
18.
Biochem Pharmacol ; 163: 381-390, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30849303

RESUMO

Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the spider venom peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core peptide pharmacophore residues that bind to the thumb domain, while peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Peptídeos/metabolismo , Venenos de Aranha/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Animais , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Humanos , Modelos Moleculares , Mutação , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Subunidades Proteicas , Ratos , Especificidade da Espécie , Venenos de Aranha/química , Xenopus laevis
19.
Methods Mol Biol ; 2025: 165-190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267452

RESUMO

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.


Assuntos
Escherichia coli/metabolismo , Animais , Dissulfetos/metabolismo , Escherichia coli/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Peçonhas/metabolismo
20.
Br J Pharmacol ; 175(12): 2204-2218, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29134638

RESUMO

BACKGROUND AND PURPOSE: Acid-sensing ion channels (ASICs) are primary acid sensors in mammals, with the ASIC1b and ASIC3 subtypes being involved in peripheral nociception. The antiprotozoal drug diminazene is a moderately potent ASIC inhibitor, but its analgesic activity has not been assessed. EXPERIMENTAL APPROACH: We determined the ASIC subtype selectivity of diminazene and the mechanism by which it inhibits ASICs using voltage-clamp electrophysiology of Xenopus oocytes expressing ASICs 1-3. Its peripheral analgesic activity was then assessed relative to APETx2, an ASIC3 inhibitor, and morphine, in a Freund's complete adjuvant (FCA)-induced rat model of inflammatory pain. KEY RESULTS: Diminazene inhibited homomeric rat ASICs with IC50 values of ~200-800 nM, via an open channel and subtype-dependent mechanism. In rats with FCA-induced inflammatory pain in one hindpaw, diminazene and APETx2 evoked more potent peripheral antihyperalgesia than morphine, but the effect was partial for APETx2. APETx2 potentiated rat ASIC1b at concentrations 30-fold to 100-fold higher than the concentration inhibiting ASIC3, which may have implications for its use in in vivo experiments. CONCLUSIONS AND IMPLICATIONS: Diminazene and APETx2 are moderately potent ASIC inhibitors, both inducing peripheral antihyperalgesia in a rat model of chronic inflammatory pain. APETx2 has a more complex ASIC pharmacology, which must be considered when it is used as a supposedly selective ASIC3 inhibitor in vivo. Our use of outbred rats revealed responders and non-responders when ASIC inhibition was used to alleviate inflammatory pain, which is aligned with the concept of number-needed-to-treat in human clinical studies. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Dor Crônica/tratamento farmacológico , Venenos de Cnidários/metabolismo , Diminazena/farmacologia , Hiperalgesia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Inflamação/tratamento farmacológico , Bloqueadores do Canal Iônico Sensível a Ácido/química , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Animais , Dor Crônica/metabolismo , Diminazena/química , Modelos Animais de Doenças , Hiperalgesia/metabolismo , Hipoglicemiantes/química , Inflamação/metabolismo , Masculino , Medição da Dor , Ratos , Ratos Sprague-Dawley , Xenopus laevis
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