RESUMEN

In mammals, nine genes encode trans-membrane adenylyl cyclase (tmAC) isoforms that synthesize the intracellular messenger compound cAMP from ATP. As cAMP is produced in virtually all types of cell, isoform-selective modulators of tmAC would have major research and therapeutic potential. This study investigated the effects of fungicide imidazoles previously shown to suppress cAMP production in various tissues on the activities of tmAC isoforms AC1, 2, or 9 stably expressed in human embryonic kidney 293 cells. Intact cells, as well as crude membranes, were exposed to various imidazoles or known stimulators of tmAC and the ensuing changes in the production of cAMP analyzed. In crude membranes, the activity of AC9 in the presence of GDP-ß-S was enhanced by miconazole with an EC50 of ~ 8 µM, while AC1 and AC2 were inhibited with an IC50 of ~ 20 µM. Clotrimazole (10-100 µM) was an inhibitor of all the ACs tested. Substrate saturation analysis indicated that miconazole increased the Vmax of AC9 by 3-fold while having no effect on the Km. In intact cells, the effect of miconazole on cAMP production through AC9 was additive with that of isoproterenol. The stimulation of cAMP production by miconazole was inhibited by Ca2+, and this could be prevented by the calcineurin blocker FK506. In sum, activation of AC9 by miconazole is through a mechanism distinct from that of forskolin, activated G proteins, or the COOH-terminal mediated autoinhibition. However, it is subject to the AC9 isoform-specific inhibition by Ca2+/calcineurin. Differential modulation of mammalian tmAC paralogs appears to be achievable by an imidazole with phenylated side chains. Optimization of the lead compound and exploration of the underlying mechanism(s) of action in more detail could exploit this further.

Asunto(s)

Adenilil Ciclasas/metabolismo , Fungicidas Industriales/farmacología , Imidazoles/farmacología , Adenilil Ciclasas/genética , AMP Cíclico/metabolismo , Células HEK293 , Humanos

RESUMEN

Trans-membrane adenylyl cyclase (tmAC) isoforms show markedly distinct regulatory properties that have not been fully explored. AC9 is highly expressed in vital organs such as the heart and the brain. Here, we report that the isoform-specific carboxyl-terminal domain (C2b) of AC9 inhibits the activation of the enzyme by Gs-coupled receptors (GsCR). In human embryonic kidney cells (HEK293) stably overexpressing AC9, cAMP production by AC9 induced upon the activation of endogenous ß-adrenergic and prostanoid GsCRs was barely discernible. Cells expressing AC9 lacking the C2b domain showed a markedly enhanced cAMP response to GsCR. Subsequent studies of the response of AC9 mutants to the activation of GsCR revealed that residues 1268-1276 in the C2b domain were critical for auto-inhibition. Two main species of AC9 of 130 K and ≥ 170 K apparent molecular weight were observed on immunoblots of rodent and human myocardial membranes with NH2-terminally directed anti-AC9 antibodies. The lower molecular weight AC9 band did not react with antibodies directed against the C2b domain. It was the predominant species of AC9 in rodent heart tissue and some of the human samples. There is a single gene for AC9 in vertebrates, moreover, amino acids 957-1353 of the COOH-terminus are encoded by a single exon with no apparent signs of mRNA splicing or editing making it highly unlikely that COOH-terminally truncated AC9 could arise through the processing or editing of mRNA. Thus, deductive reasoning leads to the suggestion that proteolytic cleavage of the C2b auto-inhibitory domain may govern the activation of AC9 by GsCR.

Asunto(s)

Adenilil Ciclasas/metabolismo , Membrana Celular/metabolismo , Giro del Cíngulo/metabolismo , Hipocampo/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Adenilil Ciclasas/genética , Animales , AMP Cíclico/metabolismo , Células HEK293 , Ventrículos Cardíacos/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Dominios Proteicos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal

RESUMEN

Gamma-amino butyric acid (GABA) is an abundant neurotransmitter in the CNS. GABAergic interneurons orchestrate pyramidal neurons in the cerebral cortex, and thus control learning and memory. Ionotropic receptors for GABA (GABAAR) are heteropentameric complexes of α, ß and γ integral membrane-protein subunits forming Cl- -channels operated by GABA, which are vital for brain function and are important drug targets. However, knowledge on how GABAAR bind GABA is controversial. Structural biology versus functional modelling combined with site-directed mutagenesis suggest markedly different roles for loop F of the extracellular domain of the α-subunit when complexed with GABA. Here, we report that contrary to the results of structural studies, loop F of the α-subunit controls the potency of GABA on GABAAR. We examined the effect of replacing a short, variable segment of loop F of the GABAA α5-subunit with the corresponding segment of the α2-subunit (GABAA5_LF2) and vice versa (GABAA2-LF5). When compared with their respective wild-type counterparts, GABAA5_LF2 receptors displayed enhanced sensitivity towards GABA, whilst in GABAA2-LF5 sensitivity was diminished. Mice homozygous for the genetic knock-in of the GABAA5_LF2 subunit showed a marked deficit in long- but not short-term object recognition memory. Working memory in place learning, spontaneous alternation and the rewarded T-maze were all normal. The deficit in long-term recognition memory was reversed by an α5-GABAA negative allosteric modulator compound. The data show that loop F governs GABA potency in a receptor isoform-specific manner in vitro. Moreover, this mechanism of ligand recognition appears to be operative in vivo and impacts cognitive performance.

Asunto(s)

Subunidades de Proteína/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Unión Competitiva , Conducta Exploratoria/fisiología , Células HEK293 , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Mutación/genética , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Receptores de GABA-A/genética , Reconocimiento en Psicología/fisiología , Relación Estructura-Actividad , Factores de Tiempo , Transfección , Ácido gamma-Aminobutírico/farmacología

RESUMEN

In the mammalian central nervous system (CNS) GABAA receptors (GABAARs) mediate neuronal inhibition and are important therapeutic targets. GABAARs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20-30 GABAAR subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABAARs (α5-GABAARs). Current α5-GABAAR inhibitors bind to the "benzodiazepine site". However, in HEK293 cells expressing recombinant α5-GABAARs, S44819 had no effect on 3H-flumazenil binding, but displaced the GABAAR agonist 3H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABAARs, but had no effect on synaptic GABAARs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABAARs, or on synaptic (α1ß2γ2) GABAARs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABAARs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.

Asunto(s)

Benzodiazepinas/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Nootrópicos/farmacología , Oxazoles/farmacología , Receptores de GABA-A/metabolismo , Animales , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Femenino , Flumazenil/farmacología , Agonistas de Receptores de GABA-A/farmacología , Células HEK293 , Humanos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones Endogámicos C57BL , Muscimol/farmacología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Ratas Sprague-Dawley , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/farmacología

RESUMEN

The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABAA) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABAA antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABAA receptors of αxß2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and ß-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α-subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo. The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABAA receptors.

Asunto(s)

Benzodiazepinas/química , Benzodiazepinas/farmacología , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacología , Subunidades de Proteína/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Benzodiazepinas/metabolismo , Unión Competitiva , Simulación por Computador , Antagonistas de Receptores de GABA-A/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Oxazoles/química , Conformación Proteica , Subunidades de Proteína/química , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

Asunto(s)

Anticonvulsivantes/farmacología , Benzodiazepinas/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Nootrópicos/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Anticonvulsivantes/síntesis química , Anticonvulsivantes/metabolismo , Anticonvulsivantes/toxicidad , Conducta Animal/efectos de los fármacos , Benzodiazepinas/síntesis química , Benzodiazepinas/metabolismo , Benzodiazepinas/toxicidad , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Antagonistas de Receptores de GABA-A/síntesis química , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/toxicidad , Células HEK293 , Humanos , Masculino , Ratones , Estructura Molecular , Actividad Motora/efectos de los fármacos , Nootrópicos/síntesis química , Nootrópicos/metabolismo , Nootrópicos/toxicidad , Pentilenotetrazol , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Relación Estructura-Actividad , Xenopus laevis

RESUMEN

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.

Asunto(s)

Bacteriófagos/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Genes Virales/fisiología , Sinorhizobium meliloti/virología , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismo , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Mutación

RESUMEN

Rhizobial surface polysaccharides, including capsular polysaccharides (KPS), are involved in symbiotic infection. The rkp-3 locus of Sinorhizobium meliloti 41 is responsible for the production of pseudaminic acid, one of the components of the KR5 antigen, a strain-specific KPS. We have extended the sequence determination and genetic dissection of the rkp-3 region to clarify the structure and function of the rkpY gene and to identify additional rkp genes. Except for rkpY, no other genes were found where mutation affected the KPS structure and symbiosis. These mutants show a unique phenotype producing a low molecular weight polysaccharide (LMW PS). Creating double mutants, we have shown that biosynthesis genes of the KR5 antigen except rkpZ are not necessary for the production of this LMW PS. Polysaccharide analysis of genetically modified strains suggests that rkpY has pleiotropic effects on polysaccharide production. It directs KPS synthesis to the KR5 antigen and influences lipo-oligo 3-deoxy-d-manno-2 octulosonic acid (Kdo) production in S. meliloti 41. In addition, rkpY suppresses the lipo-oligoKdo production when it is introduced into S. meliloti 1021.

Asunto(s)

Antígenos Bacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Polisacáridos Bacterianos/biosíntesis , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Datos de Secuencia Molecular

RESUMEN

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.

Asunto(s)

Proteínas Bacterianas/metabolismo , Bacteriófagos/fisiología , Sinorhizobium meliloti/virología , Proteínas de la Cola de los Virus/metabolismo , Adsorción , Proteínas Bacterianas/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Datos de Secuencia Molecular , Mutación Missense , Análisis de Secuencia de ADN , Sinorhizobium meliloti/genética , Proteínas de la Cola de los Virus/genética