RESUMEN
Partial agonists of peripheral cannabinoid receptors (CBRs) have potential therapeutic applications in several medical conditions. However, (-)-trans-Δ9-tetrahydrocannabinol (THC), the principal active component of marijuana, which is a partial agonist of CB1 and CB2 penetrates the central nervous system (CNS) and produces adverse effects. Peripherally restricted partial agonists of CBRs, particularly of CB1, can be used to treat illnesses safely and effectively with a better therapeutic index. Here, we report on our efforts to synthesize pyrazole partial CBR agonists with peripheral selectivity, resulting in lead compound 40. This compound is a potent partial agonist of CB1 with â¼ 5-fold higher plasma biodistribution over brain and represents an early lead for optimization.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Dronabinol , Agonistas de Receptores de Cannabinoides/farmacología , Distribución Tisular , Pirazoles/farmacología , Receptores de Cannabinoides , Receptor Cannabinoide CB1 , Receptor Cannabinoide CB2RESUMEN
Selective modulation of peripheral cannabinoid receptors (CBRs) has potential therapeutic applications in medical conditions, including obesity, diabetes, liver diseases, GI disorders and pain. While there have been considerable efforts to produce selective antagonists or full agonists of CBRs, there has been limited reports on the development of partial agonists. Partial agonists targeting peripheral CBRs may have desirable pharmacological profiles while not producing centrally mediated dissociative effects. Bayer reported that BAY 59-3074 is a CNS penetrant partial agonist of both CB1 and CB2 receptors with efficacy in rat models of neuropathic and inflammatory pain. In this report, we demonstrate our efforts to synthesize analogs that would favor peripheral selectivity, while maintaining partial agonism of CB1. Our efforts led to the identification of a novel compound, which is a partial agonist of the human CB1 (hCB1) receptor with vastly diminished brain exposure compared to BAY 59-3074.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Dolor , Alcanosulfonatos , Animales , Agonistas de Receptores de Cannabinoides/farmacología , Humanos , Nitrilos , Dolor/tratamiento farmacológico , Ratas , Receptor Cannabinoide CB1 , Receptor Cannabinoide CB2 , Receptores de Cannabinoides , Relación Estructura-ActividadRESUMEN
Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Inmunomodulación/genética , Factores de Transcripción/deficiencia , Alelos , Animales , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Edición Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Masculino , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.
Asunto(s)
ADN/genética , Regulación de la Expresión Génica/genética , MicroARNs/genética , Algoritmos , Composición de Base/genética , Secuencia de Bases , Sitios de Unión , Biología Computacional , ADN/química , Humanos , Leucemia/genéticaRESUMEN
The physiological impact of cannabinoid receptor agonists is of great public health interest due to their increased use in recreational and therapeutic contexts. However, the body of literature on cannabinoid receptor agonists includes multiple confounding variables that complicate comparisons across studies, including route of administration, timeline across which phenotypes are observed, agonist dose, and sex of the study cohort. In this study, we characterized the impact of sex and route of administration on Δ9-tetrahydrocannabinol (THC)-induced changes in cardiopulmonary phenotypes in mice. Using noninvasive plethysmography and telemetry, we monitored heart rate and respiration in the same cohort of animals across aerosol, oral gavage, subcutaneous, and intraperitoneal administrations of THC (0-30 mg/kg THC for oral gavage, subcutaneous, and intraperitoneal, and 0-300 mg/ml THC for aerosol). All routes of THC administration altered respiratory minute volume and heart rate, with the direction of effects typically being consistent across dependent measures. THC primarily decreased respiration and heart rate, but females given oral gavage THC showed increased heart rate. Intraperitoneal and subcutaneous THC produced the longest-lasting effects, including THC-induced alterations in physiological parameters for up to 10 h, whereas effects of aerosolized THC were short lived. The fastest onset of effects of THC occurred for aerosolized and intraperitoneal THC. Altogether, the work herein establishes the impact of dosing route on THC-induced heart rate and respiratory alteration in male and female mice. This study highlights important differences in the timeline of cardiopulmonary response to THC following the most common preclinical routes of administration.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Dronabinol , Humanos , Ratones , Masculino , Femenino , Animales , Dronabinol/toxicidad , Agonistas de Receptores de Cannabinoides/toxicidad , Frecuencia Cardíaca , Aerosoles , RespiraciónRESUMEN
Introduction: Excessive alcohol consumption can result in alcoholic liver disease (ALD). There is no FDA-approved drug to specifically treat ALD and current management approaches have limited efficacy. Past studies indicate that monoacylglycerol lipase (MAGL) inhibition can have a positive impact on nonalcoholic fatty liver disease. However, the effect of MAGL inhibition in ALD has not been reported. Materials and Methods: We tested the highly selective and clinically evaluated MAGL inhibitor ABX-1431 in the Lieber-DeCarli liquid alcohol diet-induced model of ALD in C57BL/6 mice. Results: ABX-1431 failed to reduce ALD-associated steatosis and elevated levels of liver enzymes associated with hepatic injury. Furthermore, survival rate declined with increasing doses of ABX-1431 when compared with mice administered vehicle only. Conclusion: These data suggest that MAGL inhibition does not improve ALD and is unlikely to be a good strategy for this condition.
RESUMEN
Metabolic syndrome (MetS) is a complex disorder that stems from the additive effects of multiple underlying causes such as obesity, insulin resistance, and chronic low-grade inflammation. The endocannabinoid system plays a central role in appetite regulation, energy balance, lipid metabolism, insulin sensitivity, and ß-cell function. The type 1 cannabinoid receptor (CB1R) antagonist SR141716A (rimonabant) showed promising antiobesity effects, but its use was discontinued due to adverse psychiatric events in some users. These adverse effects are due to antagonism of CB1R in the central nervous system (CNS). As such, CNS-sparing CB1R antagonists are presently being developed for various indications. In this study, we report that a recently described compound, 3-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[6-(difluoromethoxy)pyridin-3-yl]urea (RTI1092769), a pyrazole based weak inverse agonist/antagonist of CB1 with very limited brain exposure, improves MetS related complications. Treatment with RTI1092769 inhibited weight gain and improved glucose utilization in obese mice maintained on a high fat diet. Hepatic triglyceride content and steatosis significantly improved with treatment. These phenotypes were supported by improvement in several biomarkers associated with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). These results reinforce the idea that CB1 antagonists with limited brain exposure should be pursued for MetS and other important indications.
RESUMEN
The orphan receptor GPR88 has been implicated in a number of striatal-associated disorders, yet its endogenous ligand has not been discovered. We have previously reported that the amine functionality in the 2-AMPP-derived GPR88 agonists can be replaced with an amide (e.g., 4) without losing activity. Later, we have found that the amide can be replaced with a bioisosteric 1,3,4-oxadiazole with improved potency. Here, we report a further study of amide bioisosteric replacement with a variety of azoles containing three heteroatoms, followed by a focused structure-activity relationship study, leading to the discovery of a series of novel 1,4-disubstituted 1H-1,2,3-triazoles as GPR88 agonists. Collectively, our medicinal chemistry efforts have resulted in a potent, efficacious, and brain-penetrant GPR88 agonist 53 (cAMP EC50 = 14 nM), which is a suitable probe to study GPR88 functions in the brain.
Asunto(s)
Bencenoacetamidas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Triazoles/farmacología , Animales , Bencenoacetamidas/síntesis química , Bencenoacetamidas/farmacocinética , Barrera Hematoencefálica/metabolismo , Cuerpo Estriado/metabolismo , Diseño de Fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Receptores Acoplados a Proteínas G/deficiencia , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacocinéticaRESUMEN
The G-protein-coupled receptor APLNR and its ligands apelin and ELABELA/TODDLER/apela comprise the apelinergic system, a signaling pathway that is critical during development and physiological homeostasis. Targeted regulation of the receptor has been proposed to treat several important diseases including heart failure, pulmonary arterial hypertension and metabolic syndrome. The apelinergic system is widely expressed within the central nervous system (CNS). However, the role of this system in the CNS has not been completely elucidated. Utilizing an Aplnr knockout mouse model, we report here results from tests of sensory ability, locomotion, reward preference, social preference, learning and memory, and anxiety. We find that knockout of Aplnr leads to significant effects on acoustic startle response and sex-specific effects on conditioned fear responses without significant changes in baseline anxiety. In particular, male Aplnr knockout mice display enhanced context- and cue-dependent fear responses. Our results complement previous reports that exogenous Apelin administration reduced conditioned fear and freezing responses in rodent models, and future studies will explore the therapeutic benefit of APLNR-targeted drugs in rodent models of PTSD.
Asunto(s)
Receptores de Apelina/fisiología , Conducta Animal/fisiología , Condicionamiento Clásico/fisiología , Miedo/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores Sexuales , Trastornos por Estrés Postraumático/fisiopatologíaRESUMEN
Apelin receptor agonism improves symptoms of metabolic syndrome. However, endogenous apelin peptides have short half-lives, making their utility as potential drugs limited. Previously, we had identified a novel pyrazole-based agonist scaffold. Systematic modification of this scaffold was performed to produce compounds with improved ADME properties. Compound 13 with favorable agonist potency (cAMPi EC50 = 162 nM), human liver microsome stability (T1/2 = 62 min), and pharmacokinetic profile in rodents was identified. The compound was tested in a mouse model of diet-induced obesity (DIO) and metabolic syndrome for efficacy. Treatment with 13 led to significant weight loss, hypophagia, improved glucose utilization, reduced liver steatosis, and improvement of disease-associated biomarkers. In conclusion, a small-molecule agonist of the apelin receptor has been identified that is suitable for in vivo investigation of the apelinergic system in DIO and perhaps other diseases where this receptor has been implicated to play a role.
Asunto(s)
Receptores de Apelina/agonistas , Síndrome Metabólico/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Pirazoles/uso terapéutico , Animales , Receptores de Apelina/metabolismo , Humanos , Masculino , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Pirazoles/química , Pirazoles/farmacocinética , Pirazoles/farmacología , Pérdida de Peso/efectos de los fármacosRESUMEN
Cannabinoid subtype 1 receptor (CB1R) antagonists were originally developed as anti-obesity agents. Unfortunately, SR1417116A (rimonabant), the first marketed inverse agonist of CB1R, produced CNS-related adverse effects including depression and suicidal ideation, and thus it was withdrawn from the market. These effects of rimonabant became evident in patients following chronic dosing. Standard preclinical toxicity studies failed to detect these adverse effects. The goal of these studies was to perform an integrated battery of behavioral assays to better understand the behavioral effects of rimonabant following both acute and chronic administration. In the present study, acute dosing with rimonabant in rats significantly decreased food consumption; decreased measures of locomotor activity; increased scratching, grooming and wet-dog shakes; and increased defecation. Subsequently, animals were tested using a chronic dosing regimen but prior to drug administration for that day. The highest dose of rimonabant tested significantly decreased marble burying behavior, presumably anxiolysis. There were also significant effects in social interaction after chronic dosing. Our results did not reveal significant rimonabant-induced anxiogenic behaviors. Future studies to characterize behavioral screens for anxiogenic effects of CB1 antagonists in rodents should further explore social interaction paradigms and potential comorbid factors of rimonabant dosing such as sex, age, and obesity.
Asunto(s)
Conducta Animal/efectos de los fármacos , Antagonistas de Receptores de Cannabinoides/farmacología , Locomoción/efectos de los fármacos , Receptor Cannabinoide CB1/antagonistas & inhibidores , Rimonabant/farmacología , Conducta Social , Animales , Antagonistas de Receptores de Cannabinoides/administración & dosificación , Antagonistas de Receptores de Cannabinoides/efectos adversos , Masculino , Ratas , Ratas Sprague-Dawley , Rimonabant/administración & dosificación , Rimonabant/efectos adversosRESUMEN
Increasing evidence implicates the orphan G protein-coupled receptor 88 (GPR88) in a number of striatal-associated disorders. In this study, we report the design and synthesis of a series of novel (4-alkoxyphenyl)glycinamides (e.g., 31) and the corresponding 1,3,4-oxadiazole bioisosteres derived from the 2-AMPP scaffold (1) as GPR88 agonists. The 5-amino-1,3,4-oxadiazole derivatives (84, 88-90) had significantly improved potency and lower lipophilicity compared to 2-AMPP. Compound 84 had an EC50 of 59 nM in the GPR88 overexpressing cell-based cAMP assay. In addition, 84 had an EC50 of 942 nM in the [35S]GTPγS binding assay using mouse striatal membranes but was inactive in membranes from GPR88 knockout mice, even at a concentration of 100 µM. In vivo pharmacokinetic testing of 90 in rats revealed that the 5-amino-1,3,4-oxadiazole analogues may have limited brain permeability. Taken together, these results provide the basis for further optimization to develop a suitable agonist to probe GPR88 functions in the brain.
Asunto(s)
Glicina/análogos & derivados , Glicina/farmacología , Oxadiazoles/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Diseño de Fármacos , Glicina/farmacocinética , Masculino , Ratones Noqueados , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/farmacocinética , Ratas Long-Evans , Solubilidad , Relación Estructura-ActividadRESUMEN
Neutrophil chemotaxis to the airways is a key aspect of host response to microbes and a feature of multiple pulmonary diseases including asthma. Tight regulation of this recruitment is critical to prevent unwanted host tissue damage and inflammation. Using a mouse (Mus musculus) model of asthma applied to the Collaborative Cross population, we previously identified a lung gene expression quantitative trait locus (eQTL) for Zinc finger protein 30 (Zfp30) that was also a QTL for neutrophil recruitment and the hallmark neutrophil chemokine CXCL1. The Zfp30 eQTL is defined by three functionally distinct haplotypes. In this study, we searched for causal genetic variants that underlie the Zfp30 eQTL to gain a better understanding of this candidate repressor's regulation. First, we identified a putative regulatory region spanning 500 bp upstream of Zfp30, which contains 10 SNPs that form five haplotypes. In reporter gene assays in vitro, these haplotypes recapitulated the three previously identified in vivo expression patterns. Second, using site-directed mutagenesis followed by reporter gene assays, we identified a single variant, rs51434084, which explained the majority of variation in expression between two out of three haplotype groups. Finally, using a combination of in silico predictions and electrophoretic mobility shift assays, we identified ZFP148 as a transcription factor that differentially binds to the Zfp30 promoter region harboring rs51434084. In conclusion, we provide evidence in support of rs51434084 being a causal variant for the Zfp30 eQTL, and have identified a mechanism by which this variant alters Zfp30 expression, namely differential binding of ZFP148.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Polimorfismo de Nucleótido Simple , Mucosa Respiratoria/metabolismo , Factores de Transcripción/genética , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Haplotipos , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos , Regiones Promotoras Genéticas/genética , Unión Proteica , Sitios de Carácter Cuantitativo , Factores de Transcripción/metabolismoRESUMEN
Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.