RESUMEN
Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling baker's yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1-mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP-glutamate dehydrogenase activity of Gdh1 negatively regulates NCR-sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation.
Asunto(s)
Ácido Glutámico/metabolismo , Glutamina/metabolismo , Nitrógeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The Rhesus blood-group antigens are defined by a complex association of membrane polypeptides that includes the non-glycosylated Rh proteins (RhD and RhCE) and the RHag glycoprotein, which is strictly required for cell surface expression of these antigens. RhAG and the Rh polypeptides are erythroid-specific transmembrane proteins belonging to the same family (36% identity). Despite their importance in transfusion medicine, the function of RhAG and Rh proteins remains unknown, except that their absence in Rh(null) individuals leads to morphological and functional abnormalities of erythrocytes, known as the Rh-deficiency syndrome. We recently found significant sequence similarity between the Rh family proteins, especially RhAG, and Mep/Amt ammonium transporters. We show here that RhAG and also RhGK, a new human homologue expressed in kidney cells only, function as ammonium transport proteins when expressed in yeast. Both specifically complement the growth defect of a yeast mutant deficient in ammonium uptake. Moreover, ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export. Our results provide the first experimental evidence for a direct role of RhAG and RhGK in ammonium transport. These findings are of high interest, because no specific ammonium transport system has been characterized so far in human.
Asunto(s)
Proteínas Sanguíneas , Proteínas de Transporte de Catión , Riñón/metabolismo , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Animales , Western Blotting , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Proteínas del Helminto/metabolismo , Humanos , Proteínas de Insectos/metabolismo , Transporte Iónico , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Sistema del Grupo Sanguíneo Rh-Hr/química , Sistema del Grupo Sanguíneo Rh-Hr/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
Astrocytes have many neuronal characteristics, such as neurotransmitter receptors, ion channels, and neurotransmitter uptake systems. Cultured astrocytes were shown to express certain neuropeptide genes, with specificity for both the gene expressed and the brain region from which the cells were prepared. Somatostatin messenger RNA and peptides were detected only in cerebellar astrocytes, whereas proenkephalin messenger RNA and enkephalin peptides were present in astrocytes of cortex, cerebellum, and striatum. Cholecystokinin was not expressed in any of the cells. These results support the hypothesis that peptides synthesized in astrocytes may play a role in the development of the central nervous system.
Asunto(s)
Astrocitos/metabolismo , Regulación de la Expresión Génica , Neuropéptidos/genética , Animales , Animales Recién Nacidos , Northern Blotting , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Encefalina Metionina/biosíntesis , Encefalina Metionina/genética , Encefalinas/biosíntesis , Encefalinas/genética , Neuropéptidos/biosíntesis , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , ARN Mensajero/análisis , Radioinmunoensayo , Ratas , Somatostatina/biosíntesis , Somatostatina/genéticaRESUMEN
This study reports the synthesis of a number of 1- and 2-phenyl derivatives of the 1,4-dihydrobenzothiopyrano[4,3-c]pyrazole nucleus, which were obtained by the reaction of the versatile 7-substituted 2,3-dihydro-3-hydroxymethylene-4H-1-benzothiopyran-4-ones with hydrazine and substituted phenylhydrazines. The antiproliferative activity of the synthesized compounds was evaluated by an in vitro assay on human tumor cell lines (HL-60 and HeLa) and showed a significant capacity of the 7-methoxy-substituted benzothiopyrano[4,3-c]pyrazoles 3b-d, carrying the pendant phenyl group in the 1-position, to inhibit cell growth. Investigation of the mechanism of action indicated the induction of the mitochondrial permeability transition (MPT) as the molecular event responsible for the inhibition of cell growth. This phenomenon is related to the ability of the test compounds to cause a rapid Ca2+-dependent and cyclosporin A-sensitive collapse of the transmembrane potential (DeltaPsi) and matrix swelling. All this leads to the release of caspase activators, such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF), which trigger the pro-apoptotic pathway leading to DNA fragmentation.
Asunto(s)
Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Pirazoles/síntesis química , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Derivados del Benceno , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Potenciales de la Membrana , Membranas Mitocondriales , Permeabilidad , Pirazoles/farmacologíaRESUMEN
Ammonium is a nitrogen source supporting growth of yeast cells at an optimal rate. We recently reported the first characterization of an NH4+ transport protein (Mep1p) in Saccharomyces cerevisiae. Here we describe the characterization of two additional NH4+ transporters, Mep2p and Mep3p, both of which are highly similar to Mep1p. The Mep2 protein displays the highest affinity for NH4+ (Km, 1 to 2 microM), followed closely by Mep1p (Km, 5 to 10 microM) and finally by Mep3p, whose affinity is much lower (Km, approximately 1.4 to 2.1 mM). A strain lacking all three MEP genes cannot grow on media containing less than 5 mM NH4+ as the sole nitrogen source, while the presence of individual NH4+ transporters enables growth on these media. Yet, the three Mep proteins are not essential for growth on NH4+ at high concentrations (>20 mM). Feeding experiments further indicate that the Mep transporters are also required to retain NH4+ inside cells during growth on at least some nitrogen sources other than NH4+. The MEP genes are subject to nitrogen control. In the presence of a good nitrogen source, all three MEP genes are repressed. On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression. High-level MEP2 transcription requires at least one of the two GATA family factors Gln3p and Nil1p, which are involved in transcriptional activation of many other nitrogen-regulated genes. In contrast, expression of either MEP1 or MEP3 requires only Gln3p and is unexpectedly down-regulated in a Nil1p-dependent manner. Analysis of databases suggests that families of NH4+ transporters exist in other organisms as well.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Compuestos de Amonio Cuaternario/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/genética , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Factores de Transcripción GATA , Regulación Fúngica de la Expresión Génica/fisiología , Genes Fúngicos/genética , Cinética , Datos de Secuencia Molecular , Mutación , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
OBJECTIVE: Hair loss generates severe psychosocial implications. To date, exploring the prognostic factors of possible clinical benefit of autologous blood concentrate platelet rich plasma (PRP) was failed. The aim of our pilot study was to explore the correlation between the individual inflammation genetic profile and PRP efficacy in the treatment of hair follicle regeneration. PATIENTS AND METHODS: 41 volunteers (25 men, 16 women) took part in this retrospective study. All the patients were scheduled for 4 sessions of PRP application with intervals of 40-60 days. All the patients were checked up at 6 weekly intervals for 6 months and, then, at the end of the first year. A panel of 5 polymorphisms on 4 genes (IL-1a, IL-1b, IL-6, and IL-10) implicated in the individual genetic inflammation profile were performed. RESULTS: A significant increase rate in hair density was noticed after the third month of treatment in 32/41 (78%) of the subjects. We found an interesting association between the pro-inflammatory cytokine IL-1α polymorphism C>A (rs17561) and responders to PRP treatment. The cases carrying C/C genotype (coding for Ser114) were 21 (66%) in responders and only 2 (22%) in non-responders (p<0.05). In addition, about IL-1a, the frequency of G/G genotype in responder patients was over two times lower in responder (31%) than in non-responder patients (78%). CONCLUSIONS: Our pilot study demonstrated a correlation between the individual genetic inflammatory profile and the efficacy of the PRP treatment in males. On the contrary, in females, it showed a negative correlation. IL-1a could be used as a prognostic value for PRP efficacy. Also, these results provide preliminary evidence that may encourage the design of controlled clinical trials to properly test this modus operandi on a large number of subjects.
Asunto(s)
Folículo Piloso/efectos de los fármacos , Inflamación/genética , Interleucina-1alfa/genética , Plasma Rico en Plaquetas , Adolescente , Adulto , Anciano , Femenino , Genotipo , Cabello/crecimiento & desarrollo , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Polimorfismo de Nucleótido Simple , Pronóstico , Regeneración/efectos de los fármacos , Estudios Retrospectivos , Caracteres Sexuales , Adulto JovenRESUMEN
BACKGROUND: Small-cell lung cancer (SCLC) is a common malignancy that is usually fatal, since it metastasizes and recurs even after aggressive chemotherapy. While the cellular origin of this cancer is not well established, the cells of certain tumors exhibit neuroendocrine markers, including L-dopa decarboxylase. PURPOSE: We designed in vitro and in vivo studies to investigate whether the neuroendocrine features in classic SCLC cell lines were sufficient to make them sensitive to 1-methyl-4-phenylpyridinium (MPP+), a known neurotoxin that destroys nigrostriatal dopaminergic neurons. METHODS: Both classic SCLC cell lines (NCI-H345, NCI-H510, NCI-H187, and NCI-H146) and variant SCLC cell lines (NCI-H417, NCI-H82, NCI-H446, and NCI-H524) were exposed to MPP+ (0-512 microM) for 3 days. Inhibition of DNA synthesis was determined by [3H]thymidine incorporation assays. In a related experiment, MPP+ was removed from the classic cell line culture, and the incorporation of [3H]thymidine was determined. In the in vivo study, male athymic nude mice received subcutaneous injections of 0.5 mL tumor cells with matrigel for 10 days to enhance tumor growth, followed by MPP+ at doses of 100-400 micrograms/d given intraperitoneally for 2 days. RESULTS: All four classic SCLC cell lines showed great sensitivity to MPP+, with detachment from laminin substrates and inhibition of DNA synthesis. MPP+ interfered with [3H]thymidine incorporation and, thus, with DNA synthesis in classic SCLC cell lines at low doses (median +/- SD, 12 +/- 4 microM), whereas much higher doses (median, > 512 microM) were required to inhibit [3H]thymidine incorporation in the variant lines. Treated cells excluded trypan blue dye, showing that inhibition of DNA synthesis was not due to cytotoxicity, and the cells incorporated [3H]thymidine when MPP+ was removed from the culture medium, demonstrating that the inhibition was reversible. MPP+ inhibited the growth of the classic NCI-H187 and variant NCI-H417 cell lines implanted in nude mice. CONCLUSIONS: These results suggest that MPP+ differentially interferes with DNA synthesis in SCLC cell lines in vitro; the selective inhibitory effect on classic cell lines suggests that the neuroendocrine properties expressed by classic SCLC cells may be responsible for the differential effect. IMPLICATIONS: MPP+ exerts a cytostatic effect on these cell lines, and the differential sensitivity observed in vitro is maintained in vivo, suggesting that MPP+ or other pyridinium compounds may be of therapeutic value in SCLC.
Asunto(s)
1-Metil-4-fenilpiridinio/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , ADN de Neoplasias/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Timidina/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Saccharomyces cerevisiae possesses three ammonium transporters from the Mep/Amt family involved in ammonium acquisition and retention. We have shown that Rh proteins are structurally related to Mep/Amt proteins and that human RhAG and RhCG perform bi-directional ammonium transport upon heterologous expression in yeast. Using yeast as an expression tool, we have started a structure-function analysis of distinct members from the Mep/Amt/Rh super-family.
Asunto(s)
Proteínas Sanguíneas/fisiología , Proteínas de Transporte de Catión/fisiología , Glicoproteínas de Membrana/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Animales , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/aislamiento & purificación , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas de Escherichia coli/fisiología , Humanos , Invertebrados/metabolismo , Transporte Iónico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Ammonium excretion into urine is a major process essential to the regulation of acid-base homeostasis. We have shown that Rh-type proteins, including renal RhCG, belong to the Mep/Amt family of ammonium transporters and promote bi-directional ammonium transport upon heterologous expression in yeast. To study the physiological role of RhCG and to test a potential function in ammonium excretion, we have generated mice bearing an invalidation of the corresponding gene.
Asunto(s)
Proteínas de Transporte de Catión/fisiología , Glicoproteínas de Membrana/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Acidosis/metabolismo , Animales , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Glicoproteínas/metabolismo , Humanos , Transporte Iónico , Riñón/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Noqueados , Familia de Multigenes , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus. External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed. The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae. Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells. The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Delta cells during pseudohyphal differentiation. Northern blot analysis in H. cylindrosporum showed that the HcAMTs were up-regulated upon nitrogen deprivation and down-regulated by ammonium.
Asunto(s)
Amoníaco/metabolismo , Basidiomycota/metabolismo , Proteínas Portadoras/metabolismo , Isoformas de Proteínas/metabolismo , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/genética , Clonación Molecular , Cartilla de ADN , Regulación Fúngica de la Expresión Génica , Cinética , Isoformas de Proteínas/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Valproic acid (VPA) consistently and reproducibly elevates arterial ammonia in rats injected with an amino acid load. The extent of the hyperammonemia is dependent on the dose of VPA injected or VPA plasma concentration and the amino acid dose. Bilaterally nephrectomized rats injected with VPA and an amino acid load also develop hyperammonemia, which overall approximates 75% of that achieved in non-nephrectomized animals. In non-nephrectomized animals injected with an amino acid load, valproic acid produces a marked reduction in baseline and activated hepatic mitochondrial carbamyl phosphate synthetase I activity. Our results suggest that VPA-induced hyperammonemia after an amino acid load results from inhibition of hepatic intramitochondrial citrullinogenesis with only a limited contribution from the kidneys.
Asunto(s)
Amoníaco/sangre , Riñón/fisiología , Hígado/fisiología , Ácido Valproico/farmacología , Aminoácidos/farmacología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Inyecciones Intraperitoneales , Hígado/enzimología , Masculino , Nefrectomía , Concentración Osmolar , Ratas , Ratas EndogámicasRESUMEN
Infarction in the anterior cerebral artery (ACA) territory is an uncommon cause of stroke. The clinical findings of ACA infarctions are not fully characterized but include contralateral hemiparesis, urinary incontinence, transcortical aphasia, agraphia, apraxia, and executive dysfunction. We report a patient with a large right ACA infarction, who in addition to previously reported findings also had a complete hemiplegia, profound sensory neglect, and micrographia.
Asunto(s)
Arterias Cerebrales/patología , Infarto Cerebral/diagnóstico , Anciano , Femenino , Lateralidad Funcional/fisiología , Humanos , Imagen por Resonancia Magnética , Desempeño Psicomotor/fisiologíaRESUMEN
Three adults on unrestricted protein diets receiving valproic aci (VPA) developed gastrointestinal symptoms with encephalopathy and/or deteriorating seizure control associated with arterial hyperammonemia and normal liver function tests. The signs and symptoms did not directly correlate with VPA dosage and the arterial ammonium levels did not correlate with serum VPA concentrations. Two of the three patients had phenobarbital concentrations above the therapeutic range, but remission of neurologic or gastrointestinal symptoms was dependent on a reduction of the VPA concentration. In one case, rechallenge with VPA reproduced hyperammonemia.
Asunto(s)
Amoníaco/sangre , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Ácido Valproico/efectos adversos , Adulto , Encefalopatías/inducido químicamente , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Electroencefalografía , Potenciales Evocados/efectos de los fármacos , Femenino , Humanos , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Ácido Valproico/uso terapéuticoRESUMEN
Several optically active N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. IC50 values were measured and revealed that the D form of the amino acid moiety of the compounds was more potent than both the L form and racemic form, suggesting a key role of the amino acid stereochemistry on the affinity to the benzodiazepine receptors. GABA ratio and proconvulsant/convulsant data reported for the most active compounds reveal they behave as inverse agonists at the benzodiazepine receptor.
Asunto(s)
Aminoácidos/farmacología , Indoles/farmacología , Receptores de GABA-A/efectos de los fármacos , Aminoácidos/síntesis química , Animales , Bovinos , Corteza Cerebral/metabolismo , Fenómenos Químicos , Química , Indoles/síntesis química , Conformación Molecular , Receptores de GABA-A/metabolismo , EstereoisomerismoRESUMEN
Several N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. From these compounds, the N-[(5-chloro-, 5-bromo-, or 5-nitroindol-3-yl)glyoxylyl]glycine or -alanine esters were clearly the most potent, while the 5-methoxy analogues were considerably less active. Moreover, esters were more active than the corresponding acids. It is concluded that the affinity of these derivatives for the benzodiazepine receptor is profoundly dependent on amino acid molecular size, as well as the hydrophobic and electronic properties of the compounds.
Asunto(s)
Aminoácidos/farmacología , Indoles/farmacología , Receptores de GABA-A/efectos de los fármacos , Alanina/análogos & derivados , Alanina/síntesis química , Alanina/farmacología , Aminoácidos/síntesis química , Animales , Encéfalo/metabolismo , Bovinos , Flunitrazepam/metabolismo , Glicina/análogos & derivados , Glicina/síntesis química , Glicina/farmacología , Técnicas In Vitro , Indoles/síntesis química , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
A series of N'-phenylindol-3-ylglyoxylohydrazides, isosters of the N-benzylindol-3-ylglyoxylamide derivatives previously described by us, were synthesized and tested for their ability to displace [3H]Ro 15-1788 from bovine brain membranes. These compounds were designed with the aim of obtaining products which could exert an in vivo activity, thanks to a higher hydrosolubility and consequently a better bioavailability. Affinity was restricted to the derivatives unsubstituted in the 5 position of the indole nucleus (1, 6, 9, 12, 15, 18, 23, and 26), with Ki values ranging from 510 to 11 nM. The most active compounds (6, 9, 23, and 29) proved to be effective in antagonizing pentylenetetrazole-induced seizures. Molecular modeling studies were performed to rationalize the lack of affinity of hydrazides with a chloro or a nitro group in the 5 position of the indole nucleus. It was hypothesized that the conformational preference of the hydrazide side chain, characterized by a gauche disposition of lone pairs and substituents about the N-N bond, prevents all hydrazides from binding to the receptor similarly to other classes of indole analogues previously investigated. The potency of 5-H hydrazides was attributed to a binding mode which is not feasible for 5-Cl and 5-NO2 counterparts. This theoretical model of ligand-receptor interaction permitted a more stringent interpretation of structure-affinity relationships of hydrazides and of recently described benzylamide derivatives (Da Settimo et al. J. Med. Chem. 1996, 39, 5083-5091).
Asunto(s)
Encéfalo/metabolismo , Glioxilatos , Hidrazinas , Indoles , Modelos Moleculares , Receptores de GABA-A/efectos de los fármacos , Animales , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Anticonvulsivantes/farmacología , Unión Competitiva , Bovinos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Convulsivantes/síntesis química , Convulsivantes/química , Convulsivantes/metabolismo , Convulsivantes/farmacología , Diazepam/farmacología , Flumazenil/metabolismo , Moduladores del GABA/metabolismo , Glioxilatos/síntesis química , Glioxilatos/química , Glioxilatos/metabolismo , Glioxilatos/farmacología , Hidrazinas/síntesis química , Hidrazinas/química , Hidrazinas/metabolismo , Hidrazinas/farmacología , Técnicas In Vitro , Indoles/síntesis química , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Ligandos , Ratones , Conformación Molecular , Receptores de GABA-A/metabolismo , Convulsiones/inducido químicamente , Relación Estructura-ActividadRESUMEN
A series of N-(arylalkyl)indol-3-ylglyoxylylamides (4-8) was synthesized as ligands of the benzodiazepine receptor (BzR) and tested for their ability to displace [(3)H]flumazenil from bovine brain membranes. The new compounds, bearing a branched (4) or a geometrically constrained benzyl/phenylethyl amide side chain (5-8), represent the continuation of our research on N-benzylindol-3-ylglyoxylylamides 1 (Da Settimo et al., 1996), N'-phenylindol-3-ylglyoxylohydrazides 2 (Da Settimo et al., 1998), and N-(indol-3-ylglyoxylyl)alanine derivatives 3 (Primofiore et al., 1989). A few indoles belonging to the previously investigated benzylamides 1 and phenylhydrazides 2 were synthesized and tested to enrich the SARs in these two series. The affinities and the GABA ratios of selected compounds for clonal mammalian alpha(1)beta(2)gamma(2), alpha(3)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzR subtypes were also determined. It was hypothesized that the reduced flexibility of indoles 4-8 would both facilitate the mapping of the BzR binding cleft and increase the chances of conferring selectivity for the considered receptor subtypes. In the series of indoles 4, the introduction of a methyl group on the benzylic carbon with the R configuration improved affinity of the 5-substituted (5-Cl and 5-NO(2)) derivatives, whereas it was detrimental for their 5-unsubtituted (5-H) counterparts. All S enantiomers were less potent than the R ones. Replacement of the methyl with hydrophilic substituents on the benzylic carbon lowered affinity. The isoindolinylamide side chain was tolerated if the 5-position was unsubstituted (K(i) of 5a = 123 nM), otherwise affinity was abolished (5b, c). All the 2-indanylamides 6 and (S)-1-indanylamides 8 were devoid of any appreciable affinity. The 5-Cl and 5-NO(2) (R)-1-indanylamides 7b (K(i) 80 nM) and 7c (K(i) 28 nM) were the most potent among the indoles 5-8 geometrically constrained about the side chain. The 5-H (R)-1-indanylamide 7a displayed a lower affinity (K(i) 675 nM). The SARs developed from the new compounds, together with those collected from our previous studies, confirmed the hypothesis of different binding modes for 5-substituted and 5-unsubstituted indoles, suggesting that the shape of the lipophilic pocket L(1) (notation in accordance with Cook's BzR topological model) is asymmetric and highlighted the stereoelectronic and conformational properties of the amide side chain required for high potency. Several of the new indoles showed selectivity for the alpha(1)beta(2)gamma(2) subtype compared with the alpha(3)beta(2)gamma(2) and alpha(5)beta(3)gamma(2) subtypes (e.g.: 4t and 7c bind to these three BzR isoforms with K(i) values of 14 nM, 283 nM, 239 nM, and 9 nM, 1960 nM, 95 nM, respectively). The GABA ratios close to unity exhibited by all the tested compounds on each BzR subtype were predictive of an efficacy profile typical of antagonists.
Asunto(s)
Glioxilatos/síntesis química , Indoles/síntesis química , Receptores de GABA-A/metabolismo , Amidas/síntesis química , Amidas/química , Amidas/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Glioxilatos/química , Glioxilatos/metabolismo , Técnicas In Vitro , Indoles/química , Indoles/metabolismo , Ligandos , Modelos Moleculares , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us as ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96-102), behaved as antagonists at the A1 adenosine receptor (A1AR). Alkylation of the nitrogen at position 10 of the triazinobenzimidazole nucleus conferred selectivity for the A1AR vs the BzR. The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 12) displayed a Ki value of 63 nM at the A1AR without binding appreciably to the adenosine A2A and A3 nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A1AR antagonists suggested that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) and three lipophilic pockets (L1, L2, and L3) might be available to antagonists within the A1AR binding cleft. According to the proposed pharmacophore scheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replacement of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (23) was characterized by a Ki value of 18 nM which represents a 3.5-fold gain of A1AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A1AR was built to highlight the binding mode of 23 and two well-known A1AR antagonists (III and VII) and to guide future lead optimization projects. In our docking simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills the L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.
Asunto(s)
Bencimidazoles/síntesis química , Antagonistas de Receptores Purinérgicos P1 , Secuencia de Aminoácidos , Animales , Bencimidazoles/química , Bencimidazoles/metabolismo , Encéfalo/metabolismo , Bovinos , Técnicas In Vitro , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Ensayo de Unión Radioligante , Receptores Purinérgicos P1/metabolismoRESUMEN
A number of N-(indol-3-ylglyoxylyl)benzylamine derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. Some of these derivatives (9, 12, 14, 15, 17, 27, 34, 35, 38, 41, and 45) exhibited high affinity for the benzodiazepine receptor (BzR) with Ki values ranging from 67 to 11 nM. The GABA ratio and [35S]-tert-butylbicyclophosphorothionate binding data, determined for the most active compounds, showed that they elicit an efficacy profile at the BzR which depends on the kind of substituent present on the phenyl ring of the benzylamine moiety. Moreover, lengthening (propylamine derivatives 1-3) and shortening (aniline derivatives 46-54) of the distance between the phenyl ring and the amide group of the side chain gave compounds with a drastically lower binding potency. The biological results are discussed in the light of a recently proposed pharmacophore model and compared, by molecular modeling studies, with those obtained from effective BzR ligands.
Asunto(s)
Indoles/síntesis química , Receptores de GABA-A/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bovinos , Indoles/química , Indoles/farmacología , Ratones , Modelos Moleculares , Ensayo de Unión Radioligante , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues of the previously described N-benzylindolylglyoxylylamide derivatives IV. The title compounds V showed an affinity which depended directly on the presence of the N(10)-H group and an aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to that of the indolylglyoxylylamide derivatives IV (R = H). The GABA ratio and [(35)S]-tert-butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/antagonists for compounds 1c,e,f,j,k, and of a partial agonist for 2c. This last compound proved to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made to interpret the structure-affinity relationships of compounds V in the light of possible tautomeric equilibria involving the ligands.