RESUMEN
Several therapeutic options are currently available to treat excessive daytime sleepiness (EDS) in patients suffering from narcolepsy or obstructive sleep apnea. However, there are no comparisons between the various wake-promoting agents in terms of mechanism of action, efficacy, or safety. The goal of this study was to compare amphetamine, modafinil, solriamfetol, and pitolisant at their known primary pharmacological targets, histamine H3 receptors (H3R), dopamine, norepinephrine, and serotonin transporters, and in various in vivo preclinical models in relation to neurochemistry, locomotion, behavioral sensitization, and food intake. Results confirmed that the primary pharmacological effect of amphetamine, modafinil, and solriamfetol was to increase central dopamine neurotransmission, in part by inhibiting its transporter. Furthermore, solriamfetol increased levels of extracellular dopamine in the nucleus accumbens, and decreased the 3,4-dihydroxyphenyl acetic acid (DOPAC)/DA ratio in the striatum, as reported for modafinil and amphetamine. All these compounds produced hyperlocomotion, behavioral sensitization, and hypophagia, which are common features of psychostimulants and of compounds with abuse potential. In contrast, pitolisant, a selective and potent H3R antagonist/inverse agonist that promotes wakefulness, had no effect on striatal dopamine, locomotion, or food intake. In addition, pitolisant, devoid of behavioral sensitization by itself, attenuated the hyperlocomotion induced by either modafinil or solriamfetol. Therefore, pitolisant presents biochemical, neurochemical, and behavioral profiles different from those of amphetamine and other psychostimulants such as modafinil or solriamfetol. In conclusion, pitolisant is a differentiated therapeutic option, when compared with psychostimulants, for the treatment of EDS, as this agent does not show any amphetamine-like properties within in vivo preclinical models.
Asunto(s)
Anfetamina/farmacología , Carbamatos/farmacología , Cuerpo Estriado/efectos de los fármacos , Trastornos de Somnolencia Excesiva/tratamiento farmacológico , Conducta Alimentaria/efectos de los fármacos , Locomoción/efectos de los fármacos , Modafinilo/farmacología , Fenilalanina/análogos & derivados , Piperidinas/farmacología , Promotores de la Vigilia/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Inhibidores de Captación Adrenérgica/farmacología , Animales , Cuerpo Estriado/metabolismo , Trastornos de Somnolencia Excesiva/etiología , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Evaluación Preclínica de Medicamentos , Agonismo Inverso de Drogas , Antagonistas de los Receptores Histamínicos/farmacología , Ratones , Narcolepsia/tratamiento farmacológico , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Fenilalanina/farmacología , Receptores Histamínicos H3 , Apnea Obstructiva del Sueño/complicacionesRESUMEN
Since the discovery and early characterization of the histamine H3 receptor (H3R) in the 1980's, predominantly imidazole-based agonists were presented to the scientific community such as Nα-methylhistamine (Nα-MeHA) or (R)-α-methylhistamine ((R)α-MeHA). Whereas therapeutic applications have been prompted for H3R agonists such as treatment of pain, asthma and obesity, several drawbacks associated with imidazole-containing ligands makes the search for new agonists for this receptor demanding. Accordingly, high interest arose after publication of several pyrrolidindione-based, highly affine H3R agonists within this journal that avoid the imidazole moiety and thus, presenting a novel type of potential pharmacophores (Ghoshal, Anirban et al., 2018). In our present study performed in two independent laboratories, we further evaluated the exposed lead-compound (EC50 = 0.1 nM) of the previous research project with regards to pharmacological behavior at H3R. Thereby, no binding affinity was observed in neither [3H]Nα-MeHA nor bodilisant displacement assays that contradicts the previously published activity. Additional functional exploration employing GTPγ[35S], cAMP-accumulation assay and cAMP response element (CRE)-driven reporter gene assays exhibited slight partial agonist properties of such pyrrolidindiones but acting apart from the reported concentration range. We conclude, that the previously reported actions of such pyrrolidindiones result from an overestimation based on the method of measurement and thus, we cast doubt on the new pharmacophores with H3R agonist activity.
Asunto(s)
Pirrolidinonas/farmacología , Receptores Histamínicos H3/metabolismo , Relación Dosis-Respuesta a Droga , Polarización de Fluorescencia , Células HEK293 , Humanos , Estructura Molecular , Unión Proteica/efectos de los fármacos , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Relación Estructura-ActividadRESUMEN
The involvement of histamine H4 receptor (H4R) in immune cells chemotaxis and mediator release makes it an attractive target for the treatment of inflammation disorders. A decade of medicinal chemistry efforts has led to several promising ligands, although the chemical structures described so far possesses a singular limited diversity. We report here the discovery of novel structures, belonging to completely different scaffolds. The virtual screening was planed as a two-steps process. First, using a "scout screening" methodology, we have experimentally probed the H4R ligand binding site using a small size chemical library with very diverse structures, and identified a hit that further assist us in refining a raw 3D homology model. Second, the refined 3D model was used to conduct a widened virtual screening. This two-steps strategy proved to be very successful, both in terms of structural diversity and hit rate (23%). Moreover, the hits have high affinity for the H4R, with most potent ligands in the nanomolar range.
Asunto(s)
Descubrimiento de Drogas , Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Receptores Histamínicos/química , Humanos , Ligandos , Modelos Moleculares , Receptores Histamínicos H4 , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Synthesis and biological evaluation of a new class of histamine H4 receptor ligands, distinct from the previously reported chemotypes, are described. A virtual screening of our corporate compound collection identified a hit with an undesired dual H3R/H4R activity. Chemical exploration led to the discovery of a more potent and selective 2-benzothiazolylphenylmethyl ether lead compound.
Asunto(s)
Benzotiazoles/síntesis química , Antagonistas de los Receptores Histamínicos/síntesis química , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Benzotiazoles/química , Benzotiazoles/farmacología , Línea Celular , Evaluación Preclínica de Medicamentos , Humanos , Receptores Histamínicos , Receptores Histamínicos H4RESUMEN
Fibrosis of lung tissue is a disease where a chronic inflammatory process determines a pathological remodelling of lung parenchyma. The animal model obtained by intra-tracheal administration of bleomycin in C57BL/6 mice is one of the most validated murine model. Bleomycin stimulates oxidative stress and the production of pro-inflammatory mediators. Histamine H4R have recently been implicated in inflammation and immune diseases. This study was focused to investigate the effects of H4R ligands in the modulation of inflammation and in the reduction of lung fibrosis in C57BL/6 mice treated with bleomycin. C57BL/6 mice were treated with vehicle, JNJ7777120 (JNJ, selective H4R antagonist) or ST-1006 (partial H4R agonist), ST-994 (H4R neutral antagonist) and ST-1012 (inverse H4R agonist) at equimolar doses, released by micro-osmotic pumps for 21days. Airway resistance to inflation was assayed and lung samples were processed to measure malondialdehyde (TBARS); 8-hydroxy-2'-deoxyguanosine (8OHdG); myeloperoxidase (MPO); COX-2 expression and activity as markers of oxidative stress and inflammation. Fibrosis and airway remodelling were evaluated throughout transforming growth factor-ß (TGF-ß), percentage of positive Goblet cells, smooth muscle layer thickness determination. Our results indicated that JNJ, ST-994 and ST-1012 decreased inflammation and oxidative stress markers, i.e. the number of infiltrating leukocytes evaluated as lung tissue MPO, COX-2 expression and activity, TBARS and 8OHdG production. They also reduced the level of TGF-ß, a pro-fibrotic cytokine, collagen deposition, thickness of smooth muscle layer, Goblet cells hyperplasia; resulting in a decrease of airway functional impairment. The results here reported clearly demonstrated that H4R ligands have a beneficial effect in a model of lung fibrosis in the mouse, thus indicating that H4R antagonists or inverse agonists could be a novel therapeutic strategy for lung inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Bleomicina , Antagonistas de los Receptores Histamínicos/farmacología , Indoles/farmacología , Pulmón/efectos de los fármacos , Piperazinas/farmacología , Fibrosis Pulmonar/prevención & control , Pirimidinas/farmacología , Receptores Histamínicos H4/antagonistas & inhibidores , Animales , Biomarcadores/metabolismo , Colágeno/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Agonismo Parcial de Drogas , Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patología , Hiperplasia , Mediadores de Inflamación/metabolismo , Ligandos , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/prevención & control , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptores Histamínicos H4/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Due to its involvement in major CNS functions, the histamine H3 receptor (H3R) is the subject of intensive medicinal chemistry investigation, supported by the range of modern drug discovery tools, such as receptor modeling and ligand docking. Although the receptor models described to date share a majority of common traits, they display discrete alternatives in amino-acid conformation, rendering ligand binding modes quite different. Such variations impede structure-based drug design in the H3R field. In the present study, we used a combination of medicinal chemistry, receptor-guided and ligand-based methods to elucidate the binding mode of antagonists. The approaches converged towards a ligand orientation perpendicular to the membrane plane, bridging Glu206 of the transmembrane helix 5 to acidic amino acids of the extracellular loops. This consensus will help future structure-based drug design for H3R ligands.
Asunto(s)
Aminoácidos/metabolismo , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos H3/química , Secuencia de Aminoácidos , Diseño de Fármacos , Descubrimiento de Drogas , Ligandos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Receptores Histamínicos H3/metabolismoRESUMEN
Structure-based design methods commonly used in medicinal chemistry rely on a three-dimensional representation of the receptor. However, few crystal structures are solved in comparison with the huge number of pharmaceutical targets. This often renders homology models the only information available. It is particularly true for G protein-coupled receptors (GPCRs), one of the most important targets for approved medicines and current drug discovery projects. However, very few studies have tested their validity in comparison with corresponding crystal structures, especially in a lead optimization perspective. The recent solving of dopamine D3 receptor crystal structure allowed us to assess our historical homology model. We performed a statistical analysis, by docking our in-house lead optimization library of 1500 molecules. We demonstrate here that the refined homology model suits at least as well as the X-ray structure. It is concluded that when the crystal structure of a given GPCR is not available, homology modeling can be an excellent surrogate to support drug discovery efforts.
RESUMEN
Conjugated linoleic acids (CLA) are a class of positional, geometric, conjugated dienoic isomers of linoleic acid (LA). Dietary CLA supplementation results in a dramatic decrease in body fat mass in mice, but also causes considerable liver steatosis. However, little is known of the molecular mechanisms leading to hepatomegaly. Although c9,t11- and t10,c12-CLA isomers are found in similar proportions in commercial preparations, the respective roles of these two molecules in liver enlargement has not been studied. We show here that mice fed a diet enriched in t10,c12-CLA (0.4% w/w) for 4 weeks developed lipoatrophy, hyperinsulinemia, and fatty liver, whereas diets enriched in c9,t11-CLA and LA had no significant effect. In the liver, dietary t10,c12-CLA triggered the ectopic production of peroxisome proliferator-activated receptor gamma (PPARgamma), adipocyte lipid-binding protein and fatty acid transporter mRNAs and induced expression of the sterol responsive element-binding protein-1a and fatty acid synthase genes. In vitro transactivation assays demonstrated that t10,c12- and c9,t11-CLA were equally efficient at activating PPARalpha, beta/delta, and gamma and inhibiting liver-X-receptor. Thus, the specific effect of t10,c12-CLA is unlikely to result from direct interaction with these nuclear receptors. Instead, t10,c12-CLA-induced hyperinsulinemia may trigger liver steatosis, by inducing both fatty acid uptake and lipogenesis.
Asunto(s)
Grasas de la Dieta/farmacología , Hígado Graso/inducido químicamente , Hiperinsulinismo/inducido químicamente , Ácido Linoleico/administración & dosificación , Ácido Linoleico/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Proteínas de Unión al ADN , Metabolismo Energético/efectos de los fármacos , Hígado Graso/sangre , Hígado Graso/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hiperinsulinismo/sangre , Hiperinsulinismo/genética , Insulina/sangre , Isomerismo , Ácido Linoleico/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Receptores X del Hígado , Ratones , Receptores Nucleares Huérfanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The presence of a neuropeptide AF and FF receptor (NPFF-R2) mRNA in human adipose tissue (Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965-25971) suggested these peptides, principally recognized for their pain modulating effects, may also impact on adipocyte metabolism, an aspect that has not been explored previously. Our aim was thus to obtain more insights into the actions of these peptides on adipocytes, an approach initially undertaken with a functional genomic assay. First we showed that 3T3-L1 adipocytes express both NPFF-R1 and NPFF-R2 transcripts, and that NPAF binds adipocyte membranes with a nanomolar affinity as assessed by surface plasmon resonance technology. Then, and following a 24-h treatment with NPFF or NPAF (1 microm), we have measured using real-time quantitative reverse transcriptase-PCR the mRNA steady state levels of already well characterized genes involved in key pathways of adipose metabolism. Among the 45 genes tested, few were modulated by NPFF ( approximately 10%) and a larger number by NPAF ( approximately 27%). Interestingly, NPAF increased the mRNA levels of beta2- and beta3-adrenergic receptors (AR), and to a lesser extent those of beta1-ARs. These variations in catecholamine receptor mRNAs correlated with a clear induction in the density of beta2- and beta3-AR proteins, and in the potency of beta-AR subtype-selective agonists to stimulate adenylyl cyclase activity. Altogether, these data show that NPFF-R1 and NPFF-R2 are functionally present in adipocytes and suggest that besides their well described pain modulation effects, NPAF and to a lesser extent NPFF, may have a global impact on body energy storage and utilization.
Asunto(s)
Adipocitos/metabolismo , Oligopéptidos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Células 3T3 , Adenilil Ciclasas/metabolismo , Tejido Adiposo/metabolismo , Animales , Técnicas Biosensibles , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Ratones , Neuropéptidos/metabolismo , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
We evaluated the hypothesis of sterol-regulatory element-binding protein (SREBP)-1c being a general mediator of the transcriptional effects of insulin, with a focus on adipocytes, in which insulin profoundly influences specific gene expression. Using real time quantitative reverse transcriptase-PCR to monitor changes in the expression of about 50 genes that cover a wide range of adipocyte functions, we have compared the impact of insulin treatment with that of adenoviral overexpression of either dominant positive or dominant negative SREBP-1c mutants in 3T3-L1 adipocytes. As expected, insulin up-regulated, dominant positive stimulated, and dominant negative decreased previously characterized direct SREBP targets (FAS, SCD-1, and low density lipoprotein receptor). We also identified three novel SREBP-1c transcriptional targets in adipocytes, which were confirmed by run-on assays: plasminogen activator inhibitor 1, CCAAT/enhancer-binding protein delta (C/EBPdelta), and C/EBPbeta. Because most insulin-regulated genes were also modulated by SREBP-1c mutants, our data establish that 1) SREBP-1c is an important mediator of insulin transcriptional effects in adipocytes, and 2) C/EBPbeta is under the direct control of SREBP-1c, as demonstrated by the ability of SREBP-1c to activate the transcription from C/EBPbeta promoter through canonical SREBP binding sites. Thus, some of the effects of insulin and/or SREBP-1c in mature fat cells might require C/EBPbeta or C/EBPdelta as transcriptional relays.
Asunto(s)
Adipocitos/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Insulina/fisiología , Factores de Transcripción , Células 3T3 , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Ratones , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Transcripción Genética/fisiologíaRESUMEN
Ileal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acid (BA) specifically. In the ileum, it is thought to be implied in their enterohepatic circulation. Because the fecal excretion of BA represents the main physiological way of elimination for cholesterol (CS), the I-BABP gene could have a major function in CS homeostasis. Therefore, the I-BABP gene expression might be controlled by CS. I-BABP mRNA levels were significatively increased when the human enterocyte-like CaCo-2 cells were CS-deprived and repressed when CS were added to the medium. A highly conserved sterol regularory element-like sequence (SRE) and a putative GC box were found in human I-BABP gene promoter. Different constructs of human I-BABP promoter, cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene, have been used in transfections studies. CAT activity of the wild type promoter was increased in presence of CS-deprived medium, and conversely, decreased by a CS-supplemented medium. The inductive effect of CS depletion was fully abolished when the putative SRE sequence and/or GC box were mutated or deleted. Co-transfections experiments with the mature isoforms of human sterol responsive element-binding proteins (SREBPs) and Sp1 demonstrate that the CS-mediated regulation of I-BABP gene was dependent of these transcriptional factors. Paradoxically, mice subjected to a standard chow supplemented with 2% CS for 14 days exhibited a significant rise in both I-BABP and SREBP1c mRNA levels. We show that in vivo, this up-regulation could be explained by a recently described regulatory pathway involving a positive regulation of SREBP1c by liver-X-receptor following a high CS diet.