RESUMO
Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Cav2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Cav2.2 in fluorimetric assays and rat Cav2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA (125I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC50 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Cav2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.
Assuntos
Analgésicos/química , Bloqueadores dos Canais de Cálcio/química , Evolução Molecular , ômega-Conotoxinas/química , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Gânglios Espinais/citologia , Humanos , Neuralgia/tratamento farmacológico , Neurônios Aferentes/efeitos dos fármacos , Ratos , Ratos Wistar , Caramujos , ômega-Conotoxinas/genética , ômega-Conotoxinas/farmacologia , ômega-Conotoxinas/uso terapêuticoRESUMO
OBJECTIVE: A hallmark of rheumatoid arthritis (RA) is the chronic pain that accompanies inflammation and joint deformation. Patients with RA rate pain relief as the highest priority; however, few studies have addressed the efficacy and safety of therapies directed specifically toward pain pathways. The ω-conotoxin MVIIA (ziconotide) is used in humans to alleviate persistent pain syndromes, because it specifically blocks the voltage-gated calcium 2.2 (CaV 2.2) channel, which mediates the release of neurotransmitters and proinflammatory mediators from peripheral nociceptor nerve terminals. The aims of this study were to investigate whether blockade of CaV 2.2 can suppress arthritis pain, and to examine the progression of induced arthritis during persistent CaV 2.2 blockade. METHODS: Transgenic mice expressing a membrane-tethered form of MVIIA under the control of a nociceptor-specific gene (MVIIA-transgenic mice) were used in the experiments. The mice were subjected to unilateral induction of joint inflammation using a combination of antigen and collagen. RESULTS: CaV 2.2 blockade mediated by tethered MVIIA effectively suppressed arthritis-induced pain; however, in contrast to their wild-type littermates, which ultimately regained use of their injured joint as inflammation subsided, MVIIA-transgenic mice showed continued inflammation, with up-regulation of the osteoclast activator RANKL and concomitant joint and bone destruction. CONCLUSION: Taken together, our results indicate that alleviation of peripheral pain by blockade of CaV 2.2- mediated calcium influx and signaling in nociceptor sensory neurons impairs recovery from induced arthritis and point to the potentially devastating effects of using CaV 2.2 channel blockers as analgesics during inflammation.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Canais de Cálcio Tipo N/metabolismo , Dor Nociceptiva/metabolismo , Nociceptores/metabolismo , Ligante RANK/metabolismo , Joelho de Quadrúpedes/metabolismo , ômega-Conotoxinas/genética , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo N/imunologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Dor Nociceptiva/tratamento farmacológico , Dor Nociceptiva/imunologia , Joelho de Quadrúpedes/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , ômega-Conotoxinas/uso terapêuticoRESUMO
ω-Conotoxins MVIIA (ω-CTX MVIIA) is a peptide with 25 amino acid residues. It is a selective and reversible N-type voltage-gated calcium channel blocker, which could be used as an analgesic for pain. To date, there are no monoclonal antibodies (MAb) for immunoassay against ω-conotoxin MVIIA. In this study, an MAb against ω-conotoxin MVIIA was prepared. The conotoxin-coding DNA sequence was chemically synthesized and cloned into expression vector pGEX-6p-1 and pET32a (+), respectively. The fusion protein GST-CTX was expressed and purified, and was used to immunize BALB/c mice for preparing the anti-CTX antibody. The spleen cells were fused with SP2/0 myeloma cells after the titer of antiserum was detected and qualified. After being screened by indirect ELISA and cloned by limiting dilution, a hybridoma named 4A12, which produces monoclonal antibody specifically against ω-CTX MVIIA, was successfully obtained. It was found that there are 102 chromosomes in the 4A12 cell, and the subclass for the MAb is IgM. The MAb affinity against ω-CTX MVIIA was 7.33×10(9) L/mol, and the cross-reaction test showed that the MAb specifically bound ω-CTX MVIIA. The MAb could be used as a specific antagonist for ω-CTX MVIIA in the physiological study on the CaV channels in the nervous system.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Proteínas Recombinantes de Fusão/imunologia , ômega-Conotoxinas/imunologia , Animais , Anticorpos Monoclonais Murinos/genética , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Hibridomas/imunologia , Imunoensaio/métodos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , ômega-Conotoxinas/genéticaRESUMO
Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.
Assuntos
Analgésicos/farmacologia , ômega-Conotoxinas/farmacologia , Sequência de Aminoácidos , Analgésicos/classificação , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Caramujo Conus/genética , Caramujo Conus/metabolismo , Avaliação de Medicamentos/tendências , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Proteínas Recombinantes/farmacologia , ômega-Conotoxinas/classificação , ômega-Conotoxinas/genéticaRESUMO
A number of omega-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. omega-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different omega-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200nM) and MVIIA (500nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200nM) reversibly reduced evoked EPSC amplitude an average of 34+/-8%, whereas MVIIC (200nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC amplitude and the "success rate" calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of omega-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Células do Corno Posterior , Medula Espinal/citologia , ômega-Conotoxinas/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Técnicas In Vitro , Técnicas de Patch-Clamp , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/fisiologia , Células do Corno Posterior/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Raízes Nervosas Espinhais/fisiologia , Raízes Nervosas Espinhais/efeitos da radiação , ômega-Conotoxinas/classificação , ômega-Conotoxinas/genéticaRESUMO
BACKGROUND: omega-Conotoxin (CTX) MVIIA is a specific antagonist of N-type voltage-sensitive calcium channels. A synthetic peptide version of CTX MVIIA (ziconotide) has been approved by the US FDA for severe and chronic pain. Given the high cost and complexity of the synthetic process of the disulfide-rich peptide, the genetic recombinant approach may simplify the development of this potent therapeutic agent. AIM: In this study, we report a new method for production of the recombinant CTX MVIIA. METHOD: A novel DNA fragment encoding CTX MVIIA was designed using Escherichia coli-preferred codons, and the fragment was cloned into the expression vector pGEX(2T). The fusion protein, CTX MVIIA and glutathione-S-transferase (GST) [GST-CTX MVIIA], was expressed in E. coli and purified by affinity chromatography on a glutathione-agarose column. After digestion with thrombin, the CTX MVIIA fragment was purified on a Sephacryl S-100 HR column and identified by mass spectrometry. The bioactivity of the peptide was evaluated by the hot tail-flick assay, in which the CTX MVIIA was intracerebroventricularly administered into Sprague-Dawley rats and its antinociceptive effect measured. RESULTS: The analgesic activity of the conotoxin was about 800 times stronger than that of morphine. CONCLUSION: The recombinant CTX MVIIA expressed in E. coli has shown marked analgesic activity, which may have potential in clinical application.
Assuntos
Analgésicos/farmacologia , DNA Recombinante/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , ômega-Conotoxinas/biossíntese , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/metabolismo , ômega-Conotoxinas/genética , ômega-Conotoxinas/isolamento & purificaçãoRESUMO
Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.
Assuntos
Anticorpos/imunologia , ômega-Conotoxinas/isolamento & purificação , Animais , Western Blotting , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Masculino , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/imunologia , ômega-Conotoxinas/genética , ômega-Conotoxinas/imunologiaAssuntos
Analgésicos não Narcóticos , Venenos de Moluscos/química , Animais , Doença Crônica , Ensaios Clínicos como Assunto , Custos de Medicamentos , Desenho de Fármacos , Humanos , Dor/tratamento farmacológico , Tecnologia Farmacêutica , ômega-Conotoxinas/administração & dosagem , ômega-Conotoxinas/efeitos adversos , ômega-Conotoxinas/genéticaRESUMO
Ziconotide (PRIALT) is a neuroactive peptide in the final stages of clinical development as a novel non-opioid treatment for severe chronic pain. It is the synthetic equivalent of omega-MVIIA, a component of the venom of the marine snail, Conus magus. The mechanism of action underlying ziconotide's therapeutic profile derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels (N-VSCCs). Direct blockade of N-VSCCs inhibits the activity of a subset of neurons, including pain-sensing primary nociceptors. This mechanism of action distinguishes ziconotide from all other analgesics, including opioid analgesics. In fact, ziconotide is potently anti-nociceptive in animal models of pain in which morphine exhibits poor anti-nociceptive activity. Moreover, in contrast to opiates, tolerance to ziconotide is not observed. Clinical studies of ziconotide in more than 2,000 patients reveal important correlations to ziconotide's non-clinical pharmacology. For example, ziconotide provides significant pain relief to severe chronic pain sufferers who have failed to obtain relief from opiate therapy and no evidence of tolerance to ziconotide is seen in these patients. Contingent on regulatory approval, ziconotide will be the first in a new class of neurological drugs: the N-type calcium channel blockers, or NCCBs. Its novel mechanism of action as a non-opioid analgesic suggests ziconotide has the potential to play a valuable role in treatment regimens for severe chronic pain. If approved for clinical use, ziconotide will further validate the neuroactive venom peptides as a source of new and useful medicines.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Dor/tratamento farmacológico , ômega-Conotoxinas/uso terapêutico , Sequência de Aminoácidos/genética , Animais , Bloqueadores dos Canais de Cálcio/química , Doença Crônica , Humanos , Dados de Sequência Molecular , ômega-Conotoxinas/química , ômega-Conotoxinas/genéticaRESUMO
omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.
Assuntos
Escherichia coli/metabolismo , Limiar da Dor/efeitos dos fármacos , Tiorredoxinas/biossíntese , Tiorredoxinas/farmacologia , ômega-Conotoxinas/biossíntese , ômega-Conotoxinas/farmacologia , Analgésicos , Animais , Comportamento Animal/efeitos dos fármacos , Escherichia coli/genética , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Tiorredoxinas/genética , ômega-Conotoxinas/genéticaRESUMO
A revised, high-resolution structure of psi-conotoxin Piiie (psi-Piiie), a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR), produced through the use of NMR and molecular modeling calculations is presented. The original structures of psi-Piiie had a relatively high degree of disorder, particularly in the conformation of the disulfide bridges. Our studies utilized (13)C-labeling of selected cysteine residues allowing the resolution of all problems of resonance overlap for the cysteine residues. The improved data were used to produce a new set of structures by a molecular modeling process incorporating relaxation matrix methods for the determination of interproton distance restraints and a combination of distance geometry and simulated annealing for structure generation. The structures produced are very well converged with the RMSD of backbone atom positions of the main body of the peptide improving from 0.73 to 0.13 A. Other indicators of correlation with the experimental data and quality of covalent geometry showed significant improvement in the new structures. The overall conformation of the peptide backbone is similar between the two determinations with the exception of the N-terminus. This difference leads to a significant effect on the predicted distribution of positive charge within psi-Piiie, a property likely to influence interpretation of future mutational studies.
Assuntos
ômega-Conotoxinas/química , Algoritmos , Cisteína/química , Dissulfetos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Modelos Teóricos , Mutação , Biossíntese Peptídica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estereoisomerismo , ômega-Conotoxinas/genéticaRESUMO
omega-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha(1B-d)) and peripheral (alpha(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta(3) in Xenopus oocytes. However, the potency of CVID and MVIIA increased when alpha(1B-d) and alpha(1B-b) were expressed in the absence of rat beta(3), an effect most pronounced for CVID at alpha(1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. (1)H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.
Assuntos
Canais de Cálcio/metabolismo , Neurônios/metabolismo , ômega-Conotoxinas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Ligação de Hidrogênio , Íons , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Biossíntese Peptídica , Peptídeos/química , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Caramujos , Fatores de Tempo , Ducto Deferente/metabolismo , Xenopus laevis , ômega-Conotoxinas/química , ômega-Conotoxinas/genética , ômega-Conotoxinas/farmacologiaRESUMO
Replacement of the N-terminal half of omega-conotoxin MVIIC, a peptide blocker of P/Q-type calcium channels, with that of omega-conotoxin MVIIA significantly increased the affinity for N-type calcium channels. To identify the residues essential for subtype selectivity, we examined single reverse mutations from MVIIA-type to MVIIC-type in this chimeric analog. A reverse mutation from Lys(7) to Pro(7) decreased the affinity for both P/Q- and N-type channels, whereas that from Leu(11) to Thr(11) increased the affinity for P/Q-type channels and decreased the affinity for N-type channels. The roles of these two residues were confirmed by synthesizing two MVIIC analogs in which Pro(7) and Thr(11) were replaced with Lys(7) and Leu(11), respectively.
Assuntos
Canais de Cálcio/metabolismo , ômega-Conotoxinas/genética , ômega-Conotoxinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/classificação , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Cerebelo/metabolismo , Dicroísmo Circular , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ômega-Conotoxinas/químicaRESUMO
Conopeptide content in Conus textile and Conus striatus venoms were examined by polymerase chain reaction amplification of alpha-conopeptide cDNA and rapid amplification of 3' cDNA ends of O-superfamily conopeptide cDNA. Two new alpha-conopeptide sequences and six new O-superfamily conopeptide sequences from C. textile, four new O-superfamily conopeptide sequences, and four previously biochemically characterized conopeptide sequences from C. striatus were identified. The results suggest that this cDNA method is rapid and requires less material for the study of conopeptides.