RESUMO
The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.
Assuntos
Imunossupressores/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio , Linfócitos T/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Humanos , Imunossupressores/química , Ativação do Canal Iônico , Canal de Potássio Kv1.3 , Estrutura Molecular , Suínos , Porco Miniatura , Linfócitos T/imunologia , Triterpenos/químicaRESUMO
The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.
Assuntos
Quinolinas/metabolismo , Canais de Sódio/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Ativação do Canal Iônico , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Técnicas de Patch-Clamp , Quinolinas/farmacocinética , Quinolinas/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Bloqueadores dos Canais de Sódio , Suínos , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismo , Distribuição TecidualRESUMO
Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.
Assuntos
Resistência a Medicamentos/genética , Imunossupressores/farmacologia , Linfoma de Células T/patologia , Mutação/genética , Polienos/farmacologia , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Divisão Celular/efeitos dos fármacos , Fusão Celular/fisiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Camundongos , Proteínas Serina-Treonina Quinases/análise , RNA Mensageiro/biossíntese , Proteínas Quinases S6 Ribossômicas , Sirolimo , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo , Células Tumorais CultivadasRESUMO
Spacer arms 2.1-3.7 nm (21-37 A) long were prepared, and coupled with the methyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine benzyl ester, to give blocked 6-acylates. Deprotection was effected with palladium chloride and triethyl-silane. Chemical conjugates of MDP-meningococcal group C polysaccharide were then synthesized, in attempts to enhance the immunogenicity of the polysaccharide antigen.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Metilglicosídeos/síntese química , Polissacarídeos Bacterianos/imunologia , Indicadores e Reagentes , Relação Estrutura-AtividadeRESUMO
The 6-aminohexyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine and its spacer-arm-linked analog (3.8 nm) were synthesized from 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2- oxazoline, and coupled with meningococcal group C polysaccharide in attempts to enhance the immunogenicity of the polysaccharide antigen.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Aminoglicosídeos/síntese química , Polissacarídeos Bacterianos/imunologia , Indicadores e Reagentes , Relação Estrutura-AtividadeRESUMO
2',3'-O-[1-(2-Carboxyethyl) ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.
Assuntos
Proteínas de Transporte/isolamento & purificação , Nucleotídeos de Guanina , Guanosina Difosfato/análogos & derivados , Proteínas de Transporte/genética , Cromatografia de Afinidade/métodos , Guanosina Difosfato/síntese química , Células HeLa/metabolismo , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Peso Molecular , Biossíntese de Proteínas , Proteínas de Ligação ao Cap de RNA , Sindbis virus/genética , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.