RESUMEN
ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35 residue polypeptide cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role of these disulfides in the structure and channel-blocking activity of ShK toxin, a series of analogues was synthesized by selective replacement of each pair of half-cystines with two alpha-amino-butyrate (Abu) residues. The remaining two disulfide pairs were formed unambiguously using an orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the appropriate position. The peptides were tested in vitro for their ability to block Kv1.1 and Kv1.3 potassium channels and their ability to displace [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The monocyclic peptides showed no activity in these assays. Of the dicyclic peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates disulfide connectivities) had weak activity on Kv1.3 and Kv1.1. [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)) against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native toxin of 18 in the displacement assay, whereas [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had ratios of 69 and 390, respectively. Thus, the disulfide bond linking the N- and C-terminal regions is less important for activity than the internal disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues indicated that they had little residual structure, consistent with their significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution structure, with a mean pairwise root-mean-square deviation of 1.33 A over the backbone heavy atoms. This structure nevertheless showed significant differences from that of native ShK toxin. The possible interactions of this analogue with the channel and the distinction between native secondary and tertiary structure on one hand and global topology imposed by the disulfide bridges on the other are discussed.
Asunto(s)
Venenos de Cnidarios/química , Venenos de Cnidarios/toxicidad , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Venenos de Cnidarios/genética , Disulfuros/química , Femenino , Técnicas In Vitro , Cinética , Canal de Potasio Kv.1.1 , Canal de Potasio Kv1.3 , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Anémonas de Mar , Relación Estructura-Actividad , XenopusRESUMEN
The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.
Asunto(s)
Inmunosupresores/metabolismo , Péptidos/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Canal de Potasio Kv1.3 , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Abnormal sensory inhibition is a measurable indicator of a sensory processing deficit which is observed in schizophrenia, and other disorders, and which may be heritable. This deficit has also been observed in certain inbred mouse strains where the intensity of the deficit has been correlated with reduction in the number of hippocampal alpha-bungarotoxin-sensitive nicotinic receptors. Nicotine and certain nicotinic agonists produce brief periods of normal sensory inhibition in these mice. Similarly, nicotine also transiently normalizes sensory inhibition in schizophrenics. The present study assessed the effects of a novel nicotinic partial agonist (GTS-21), selective for the alpha-bungarotoxin site, on sensory inhibition in DBA mice, a strain with no sensory inhibition under routine experimental conditions. GTS-21 produced a dose-dependent normalization of sensory inhibition which was blocked by alpha-bungarotoxin but not mecamylamine. In contrast to other nicotinic agonists, normalization of sensory inhibition by GTS-21 and two related anabaseine compounds, DMAB-anabaseine and DMAC-anabaseine, was observed when administered a second time to the animal, after a 40-min delay. Our results indicated that the anabaseine compounds increase sensory inhibition through alpha7 nicotinic receptors, and that their ability to act repeatedly on these receptors may be less affected by desensitization.
Asunto(s)
Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Reflejo de Sobresalto/efectos de los fármacos , Anabasina/análogos & derivados , Anabasina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Psicotrópicos/farmacología , Piridinas/farmacología , Receptores Nicotínicos/fisiología , Reflejo de Sobresalto/fisiología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
DMXBA (3-(2,4-dimethoxybenzylidene)-anabaseine, also known as GTS-21) is currently being tested as a possible pharmacological treatment of cognitive dysfunction in Alzheimer's disease. In this study, plasma and brain pharmacokinetics as well as urinary excretion of this compound have been evaluated in adult rats. DMXBA concentrations were determined by HPLC. Following a 5 mg kg-1 iv dose, DMXBA plasma concentration declined bi-exponentially with mean (+/- SE) absorption and elimination half-lives of 0.71 +/- 0.28 and 3.71 +/- 1.12 h, respectively. The apparent steady state volume of distribution was 2150 +/- 433 mL kg-1, total body clearance was 1480 +/- 273 mL h-1 kg-1, and AUC0-infinity was 3790 +/- 630 ng h mL-1. Orally administered DMXBA was rapidly absorbed. After oral administration of 10 mg kg-1, a peak plasma concentration of 1010 +/- 212 ng mL-1 was observed at 10 min after dosing. Elimination half-life was 1.740 +/- 0.34 h, and AUC0-infinity was 1440 +/- 358 ng h mL-1. DMXBA peak brain concentration after oral administration was 664 +/- 103 ng g-1 tissue, with an essentially constant brain-plasma concentration ratio of 2.61 +/- 0.34, which indicates that the drug readily passes across the blood-brain barrier. Serum protein binding was 80.3 +/- 1.1%. Apparent oral bioavailability was 19%. Renal clearance (21.8 mL h-1 kg-1) was less than 2% of the total clearance (1480 +/- 273 mL h-1 kg-1); urinary excretion of unchanged DMXBA over a 96 h period accounted for only 0.28 +/- 0.03% of the total orally administered dose. Our data indicates that DMXBA oral bioavailability is primarily limited by hepatic metabolism.
Asunto(s)
Compuestos de Bencilideno/farmacocinética , Compuestos de Bencilideno/orina , Agonistas Nicotínicos/farmacocinética , Agonistas Nicotínicos/orina , Piridinas/farmacocinética , Piridinas/orina , Animales , Compuestos de Bencilideno/sangre , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Trastornos del Conocimiento/tratamiento farmacológico , Masculino , Agonistas Nicotínicos/sangre , Piridinas/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
We assessed the pharmacological activity of anabaseine, a toxin found in certain animal venoms, relative to nicotine and anabasine on a variety of vertebrate nicotinic receptors, using cultured cells, the Xenopus oocyte expression system, contractility assays with skeletal and smooth muscle strips containing nicotinic receptors and in vivo rat prostration assay involving direct injection into the lateral ventricle of the brain. Anabaseine stimulated every subtype of nicotinic receptor that was tested. It was the most potent frog skeletal muscle nicotinic receptor agonist. At higher concentrations it also blocked the BC3H1 (adult mouse) muscle type receptor ion channel. The affinities of the three nicotinoid compounds for rat brain membrane alpha-bungarotoxin binding sites and their potencies for stimulating Xenopus oocyte homomeric alpha7 receptors, expressed in terms of their active monocation concentrations, displayed the same rank order, anabaseine>anabasine> nicotine. Although the maximum currents generated by anabaseine and anabasine at alpha7 receptors were equivalent to that of acetylcholine, the maximum response to nicotine was only about 65% of the acetylcholine response. At alpha4-beta2 receptors the affinities and apparent efficacies of anabaseine and anabasine were much less than that of nicotine. Anabaseine, nicotine and anabasine were nearly equipotent on sympathetic (PC12) receptors, although parasympathetic (myenteric plexus) receptors were much more sensitive to anabaseine and nicotine but less sensitive to anabasine. These differences suggest that there may be different subunit combinations in these two autonomic nicotinic receptors. The preferential interactions of anabaseine, anabasine and nicotine with different receptor subtypes provides molecular clues that should be helpful in the design of selective nicotinic agonists.
Asunto(s)
Anabasina/análogos & derivados , Encéfalo/efectos de los fármacos , Bungarotoxinas/farmacología , Músculos/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Anabasina/administración & dosificación , Anabasina/farmacología , Animales , Inyecciones Intraventriculares , Ratones , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Ensayo de Unión Radioligante , Rana pipiens , Ratas , Receptores Nicotínicos/análisis , Relación Estructura-Actividad , Torpedo , XenopusRESUMEN
An "Ala scan" analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.
Asunto(s)
Encéfalo/metabolismo , Venenos de Cnidarios/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Venenos de Cnidarios/síntesis química , Venenos de Cnidarios/química , Técnicas In Vitro , Membranas/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Ratas , TermodinámicaRESUMEN
Eighteen synthetic analogs of ShK toxin, a thirty-five residue K channel blocker derived from the sea anemone Stichodactyla helianthus, were prepared in order to identify functionally important residues. CD spectra of sixteen of the analogs were virtually identical with the spectrum of wild-type toxin, indicating that the conformations were not affected by the substitutions. A conserved residue, Lys22, is essential for ShK binding to rat brain K channels which are primarily of the Kv1.2 type. However, a cationic side chain at position 22 is not essential for binding to the human Jurkat T-lymphocyte Kv1.3 channel. While decreasing bulkiness at this position affected toxin affinity for the brain K channels, increasing bulkiness decreased toxin affinity for both brain and lymphocyte K channels. In contrast to the rat brain channels, ShK binding to Kv1.3 was sensitive to substitution at Lys9 and Arg11.
Asunto(s)
Encéfalo/metabolismo , Venenos de Cnidarios/química , Venenos de Cnidarios/farmacología , Bloqueadores de los Canales de Potasio , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Venenos de Cnidarios/síntesis química , Humanos , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Canales de Potasio/fisiología , Ratas , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
ShK-toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]-dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]-charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 pM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels.
Asunto(s)
Venenos de Cnidarios/síntesis química , Bloqueadores de los Canales de Potasio , Anémonas de Mar , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Membrana Celular/metabolismo , Caribdotoxina/metabolismo , Cromatografía Líquida de Alta Presión , Venenos de Cnidarios/farmacología , Disulfuros/química , Venenos Elapídicos/metabolismo , Masculino , Datos de Secuencia Molecular , Canales de Potasio/metabolismo , Pliegue de Proteína , Ratas , Ratas Sprague-Dawley , Homología de SecuenciaRESUMEN
1. Twenty-two sea anemone samples from seven species were collected in Aleutian and Comandorskiye Islands from sub-littoral region (> 50m depth). 2. Water-ethanol extracts of sea anemones were tested using various test-systems after ethanol evaporation. 3. All sea anemones extracts inhibited DNA and most of them inhibited RNA synthesis in Ehrlich carcinoma tumor cells. 4. Extracts of most sea anemones species showed high hemolytic activity. 5. The extracts proved to be nontoxic or display low toxicity being i.p. injected into mice. 6. Some extracts precipitated virus of aleutian disease of mink. 7. None of the extracts showed activity toward Gram +ve, Gram -ve bacteria or yeast.
Asunto(s)
Anémonas de Mar/metabolismo , Extractos de Tejidos/farmacología , Virus de la Enfermedad Aleutiana del Visón/aislamiento & purificación , Animales , Antibacterianos , Antiinfecciosos/farmacología , Antifúngicos/farmacología , Antineoplásicos/farmacología , Carcinoma de Ehrlich/tratamiento farmacológico , ADN de Neoplasias/biosíntesis , Hemólisis/efectos de los fármacos , Técnicas In Vitro , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , ARN Neoplásico/biosíntesis , Extractos de Tejidos/análisis , Extractos de Tejidos/toxicidadRESUMEN
A very potent non-protein toxin was isolated from the sea anemone Radianthus macrodactylus with the use of chromatography on polytetrafluoroethylene, CM-Sephadex C-25 and by cation and anion exchange HPLC. The toxin was identified as palytoxin by u.v.-, i.r.- and 500 MHz 1H NMR spectroscopy. Its LD50 was 0.74 +/- 0.29 micrograms/kg by i.v. injection into mice. So far, palytoxin has been associated with zoanthids only. The toxin caused the loss of haemoglobin from erythrocytes but only in about 2 hr after the beginning of incubation, which is characteristic for palytoxin from zoanthids. Sea anemone palytoxin was divided into major and minor components by HPLC. The latter proved to be a product of degradation of palytoxin.
Asunto(s)
Acrilamidas , Venenos de Cnidarios/análisis , Anémonas de Mar/química , Animales , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Venenos de Cnidarios/aislamiento & purificación , Venenos de Cnidarios/toxicidad , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Dosificación Letal Mediana , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Espectrofotometría Infrarroja , Espectrofotometría UltravioletaRESUMEN
The effect of modification of amino groups on RTX-III induced lethality in mice has been studied. The toxicity was not affected by guanidination of one or two lysine residues with O-methylisourea, but guanidination of three or four lysine residues decreased lethality two-fold. Acetylation of the N-terminal amino group with [3H]acetic anhydride caused a 12-fold decrease of lethality. The toxin containing acetylated Lys-4 or one of three C-terminal lysine residues had half the lethal potency of the native RTX-III. Diacetylated derivatives were 30- to 35-fold less toxic than the native toxin. By circular dichroism, it was shown that modification of one or two amino groups did not affect the secondary structure of the toxin. We conclude that protonated amino groups are essential for neurotoxicity.
Asunto(s)
Venenos de Cnidarios/toxicidad , Anémonas de Mar , Anhídridos Acéticos/farmacología , Acetilación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Dicroismo Circular , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Masculino , Compuestos de Metilurea/farmacología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mapeo Peptídico , Conformación Proteica , Relación Estructura-Actividad , Tripsina/metabolismoRESUMEN
The toxin was treated with [14C]trimethyloxonium tetrafluoroborate or [3H]glycine methyl ester in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide. Esterification of separate carboxyl groups with [14C]trimethyloxonium tetrafluoroborate decreased the toxicity no more than two-fold. Blocking of any single carboxyl group with [3H]glycine methyl ester did not cause more than a two-fold decrease of toxicity, and modification of two carboxyl groups caused no more than a six-fold decrease. Partial localization of modified residues in the amino acid sequence was performed. By circular dichroism, it was shown that the decrease of toxicity was not associated with alteration of secondary or tertiary structure. It is concluded that free carboxyl groups are not absolutely essential for toxicity, however they are necessary for expression of the maximum RTX-III toxicity.
Asunto(s)
Venenos de Cnidarios/toxicidad , Neurotoxinas/toxicidad , Secuencia de Aminoácidos , Animales , Boratos/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Ésteres , Glicina/análogos & derivados , Glicina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neurotoxinas/metabolismo , Mapeo Peptídico , Conformación Proteica , Anémonas de MarRESUMEN
Chemical modifications of the polypeptide neurotoxin RTX-III have allowed us to study the functional role of Arg residues. The effect of chemical modification has been estimated by measuring toxicity in mice. 2,4-Pentanedione did not react with Arg residues of RTX-III even after 100 hr incubation. Malonic aldehyde reacted readily with RTX-III, yielding an unusual derivative; a Schiff's base obtained by condensation of one of two aldehyde groups of malonic aldehyde with the guanidine group. The derivative was one-fourth as toxic as the native toxin. Modification of the guanidine side chain of Arg-13 with both 1,2-cyclohexanedione and phenylglyoxal decreased the toxicity of RTX-III by a factor of five. We conclude that Arg-13 is not fully responsible for toxicity. The toxin-receptor attachment might be multipoint, involving several structural elements of the protein molecule, with Arg-13, probably being one of them. The guanidine side chain of Arg-45 is buried in the sequence and apparently functionally nonessential.