RESUMEN
EDP-938 is a novel non-fusion replication inhibitor of respiratory syncytial virus (RSV). It is highly active against all RSV-A and B laboratory strains and clinical isolates tested in vitro in various cell lines and assays, with half-maximal effective concentrations (EC50s) of 21, 23 and 64 nM against Long (A), M37 (A) and VR-955 (B) strains, respectively, in the primary human bronchial epithelial cells (HBECs). EDP-938 inhibits RSV at a post-entry replication step of the viral life cycle as confirmed by time-of-addition study, and the activity appears to be mediated by viral nucleoprotein (N). In vitro resistance studies suggest that EDP-938 presents a higher barrier to resistance compared to viral fusion or non-nucleoside L polymerase inhibitors with no cross-resistance observed. Combinations of EDP-938 with other classes of RSV inhibitors lead to synergistic antiviral activity in vitro. Finally, EDP-938 has also been shown to be efficacious in vivo in a non-human primate model of RSV infection.
Asunto(s)
Antivirales/farmacología , Infecciones por Virus Sincitial Respiratorio , Animales , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Humanos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/virología , Virus Sincitial Respiratorio Humano/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: EDP-305 is an oral farnesoid X receptor (FXR) agonist under development for the treatment of non-alcoholic steatohepatitis (NASH). Herein, we aimed to assess the efficacy, safety and tolerability of EDP-305 in patients with fibrotic NASH. METHODS: In this double-blind phase II study, patients with fibrotic NASH (without cirrhosis), diagnosed by historical biopsy or phenotypically, were randomized to EDP-305 1 mg, EDP-305 2.5 mg, or placebo, for 12 weeks. The primary endpoint was mean change in alanine aminotransferase (ALT) from baseline to Week 12, and the key secondary endpoint was mean change in liver fat content from baseline to Week 12. RESULTS: Between January 2018 and July 2019, 134 patients were randomized and 132 were evaluated. At Week 12, the least squares mean reductions from baseline in ALT for patients receiving 2.5 mg EDP-305 and 1 mg EDP-305 were -27.9 U/L (95% CI 0.03 to 24.9; p = 0.049) and -21.7 U/L (-5.8 to 18.3: p = 0.304), respectively, compared to -15.4 U/L for those receiving placebo. Absolute liver fat reduction was -7.1% (2.0-7.5; p = 0.0009) with 2.5 mg EDP-305, -3.3% with EDP-305 1 mg, and -2.4% with placebo. The most common (≥5%) adverse events were pruritus, nausea, vomiting, diarrhea, headache, and dizziness. Pruritus occurred in 50.9%, 9.1%, and 4.2% of patients in the 2.5 mg, 1 mg, and placebo groups, respectively, and led to study drug discontinuation in 20.8% of patients in the 2.5 mg group and 1.8% in the 1 mg group. CONCLUSIONS: EDP-305 reduced ALT levels and liver fat content, providing support for a longer-term trial assessing histological endpoints in patients with NASH. CLINICALTRIALS. GOV NUMBER: NCT03421431 LAY SUMMARY: Non-alcoholic fatty liver disease is a chronic hepatic disease that can progress to non-alcoholic steatohepatitis (NASH), which is associated with an increased risk of cirrhosis and liver cancer. Results from this phase II study support continued development of EDP-305, an oral farnesoid X receptor agonist, for the treatment of patients with NASH.
Asunto(s)
Relación Dosis-Respuesta a Droga , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Esteroides/administración & dosificación , Administración Oral , Adulto , Análisis de Varianza , Canadá , Método Doble Ciego , Femenino , Francia , Alemania , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Nueva Zelanda , Placebos , Receptores Citoplasmáticos y Nucleares/administración & dosificación , Receptores Citoplasmáticos y Nucleares/uso terapéutico , Esteroides/uso terapéutico , Resultado del Tratamiento , Reino Unido , Estados UnidosRESUMEN
Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Ácidos y Sales Biliares/metabolismo , Grasas de la Dieta/farmacología , Enterocitos/enzimología , Metabolismo de los Lípidos/fisiología , Triglicéridos/farmacología , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Animales , Ratones , Ratones Noqueados , Fosfolípidos/genética , Fosfolípidos/metabolismoRESUMEN
OBJECTIVE: Very low-density lipoprotein assembly and secretion are regulated by the availability of triacylglycerol. Although compelling evidence indicates that the majority of triacylglycerol in very low-density lipoprotein is derived from re-esterification of lipolytic products released by endoplasmic reticulum-associated lipases, little is known about roles of acyl-CoA:diacylglycerol acyltransferases (DGATs) in this process. We aimed to investigate the contribution of DGAT1 and DGAT2 in lipid metabolism and lipoprotein secretion in primary mouse and human hepatocytes. APPROACH AND RESULTS: We used highly selective small-molecule inhibitors of DGAT1 and DGAT2, and we tracked storage and secretion of lipids synthesized de novo from [(3)H]acetic acid and from exogenously supplied [(3)H]oleic acid. Inactivation of individual DGAT activity did not affect incorporation of either radiolabeled precursor into intracellular triacylglycerol, whereas combined inactivation of both DGATs severely attenuated triacylglycerol synthesis. However, inhibition of DGAT2 augmented fatty acid oxidation, whereas inhibition of DGAT1 increased triacylglycerol secretion, suggesting preferential channeling of separate DGAT-derived triacylglycerol pools to distinct metabolic pathways. Inactivation of DGAT2 impaired cytosolic lipid droplet expansion, whereas DGAT1 inactivation promoted large lipid droplet formation. Moreover, inactivation of DGAT2 attenuated expression of lipogenic genes. Finally, triacylglycerol secretion was significantly reduced on DGAT2 inhibition without altering extracellular apolipoprotein B levels. CONCLUSIONS: Our data suggest that DGAT1 and DGAT2 can compensate for each other to synthesize triacylglycerol, but triacylglycerol synthesized by DGAT1 is preferentially channeled to oxidation, whereas DGAT2 synthesizes triacylglycerol destined for very low-density lipoprotein assembly.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Hepatocitos/enzimología , Triglicéridos/biosíntesis , Acilcoenzima A/metabolismo , Animales , Células Cultivadas , Diacilglicerol O-Acetiltransferasa/genética , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/fisiología , Lipogénesis/fisiología , Ratones , Rol , Sensibilidad y EspecificidadRESUMEN
Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4. To investigate the in vivo functions of GPAT3, we generated Gpat3-deficient (Gpat3(-/-)) mice. Total GPAT activity in white adipose tissue of Gpat3(-/-) mice was reduced by 80%, suggesting that GPAT3 is the predominant GPAT in this tissue. In liver, GPAT3 deletion had no impact on total GPAT activity but resulted in a 30% reduction in N-ethylmaleimide-sensitive GPAT activity. The Gpat3(-/-) mice were viable and fertile and exhibited no obvious metabolic abnormalities on standard laboratory chow. However, when fed a high-fat diet, female Gpat3(-/-) mice showed decreased body weight gain and adiposity and increased energy expenditure. Increased energy expenditure was also observed in male Gpat3(-/-) mice, although it was not accompanied by a significant change in body weight. GPAT3 deficiency lowered fed, but not fasted, glucose levels and tended to improve glucose tolerance in diet-induced obese male and female mice. On a high-fat diet, Gpat3(-/-) mice had enlarged livers and displayed a dysregulation in cholesterol metabolism. These data establish GPAT3 as the primary GPAT in white adipose tissue and reveal an important role of the enzyme in regulating energy, glucose, and lipid homeostasis.
Asunto(s)
Tejido Adiposo Blanco/enzimología , Colesterol/metabolismo , Metabolismo Energético/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Obesidad/enzimología , Animales , Dieta/efectos adversos , Femenino , Glicerol-3-Fosfato O-Aciltransferasa/genética , Homeostasis/genética , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genéticaRESUMEN
The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.
Asunto(s)
Antivirales , COVID-19 , Proteasas 3C de Coronavirus , SARS-CoV-2 , Replicación Viral , Animales , Cricetinae , Femenino , Humanos , Masculino , Antivirales/farmacología , Chlorocebus aethiops , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , COVID-19/virología , COVID-19/transmisión , Tratamiento Farmacológico de COVID-19 , Modelos Animales de Enfermedad , Hurones , Lactamas , Leucina , Pulmón/virología , Pulmón/efectos de los fármacos , Pulmón/patología , Mesocricetus , Nitrilos , Compuestos Orgánicos , Pandemias/prevención & control , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Neumonía Viral/transmisión , Neumonía Viral/prevención & control , Prolina , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
RESUMEN
Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.
Asunto(s)
Aciltransferasas/genética , Aciltransferasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Adulto , Anciano , Diacilglicerol O-Acetiltransferasa/metabolismo , Hígado Graso/enzimología , Hígado Graso/patología , Femenino , Células Hep G2 , Humanos , Resistencia a la Insulina , Hígado/citología , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico , Obesidad/enzimología , Obesidad/patología , Adulto JovenRESUMEN
Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.
Asunto(s)
Diacilglicerol O-Acetiltransferasa , Enfermedad del Hígado Graso no Alcohólico , Humanos , Diseño de Fármacos , Cirrosis Hepática , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológicoRESUMEN
The liver and intestine play crucial roles in maintaining bile acid homeostasis. Here, we demonstrate that fibroblast growth factor 15 (FGF15) signals from intestine to liver to repress the gene encoding cholesterol 7alpha-hydroxylase (CYP7A1), which catalyzes the first and rate-limiting step in the classical bile acid synthetic pathway. FGF15 expression is stimulated in the small intestine by the nuclear bile acid receptor FXR and represses Cyp7a1 in liver through a mechanism that involves FGF receptor 4 (FGFR4) and the orphan nuclear receptor SHP. Mice lacking FGF15 have increased hepatic CYP7A1 mRNA and protein levels and corresponding increases in CYP7A1 enzyme activity and fecal bile acid excretion. These studies define FGF15 and FGFR4 as components of a gut-liver signaling pathway that synergizes with SHP to regulate bile acid synthesis.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Circulación Enterohepática/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Homeostasis , Transducción de Señal , Animales , Células CACO-2 , Colesterol 7-alfa-Hidroxilasa/biosíntesis , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Circulación Enterohepática/efectos de los fármacos , Epitelio/metabolismo , Factores de Crecimiento de Fibroblastos/deficiencia , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Perfilación de la Expresión Génica , Homeostasis/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citología , Hígado/enzimología , Masculino , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
The orphan nuclear receptor liver receptor homolog 1 (LRH-1) has been reported to play an important role in bile acid biosynthesis and reverse cholesterol transport. Here, we show that LRH-1 is a key player in the control of the hepatic acute-phase response. Ectopic expression of LRH-1 with adenovirus resulted in strong inhibition of both interleukin-6 (IL-6)- and IL-1beta-stimulated haptoglobin, serum amyloid A, and fibrinogen beta gene expression in hepatocytes. Furthermore, induction of the hepatic inflammatory response was significantly exacerbated in HepG2 cells expressing short hairpin RNA targeting LRH-1 expression. Moreover, transient-transfection experiments and electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that LRH-1 regulates this cytokine-elicited inflammatory response by, at least in part, antagonizing the CCAAT/enhancer binding protein beta signaling pathway. Finally, we show, by using LRH-1 heterozygous mice, that LRH-1 is involved in the control of the inflammatory response at the hepatic level in vivo. Taken together, our results outline an unexpected role for LRH-1 in the modulation of the hepatic acute-phase response.
Asunto(s)
Reacción de Fase Aguda/inmunología , Hígado/inmunología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Expresión Génica , Haptoglobinas/genética , Heterocigoto , Humanos , Interleucina-1/farmacología , Interleucina-6/farmacología , Lipopolisacáridos/inmunología , Hígado/efectos de los fármacos , Macaca fascicularis , Ratones , Unión Proteica/efectos de los fármacos , Interferencia de ARNRESUMEN
Liver receptor homolog 1 (LRH-1), an orphan nuclear receptor, is highly expressed in liver and intestine, where it is implicated in the regulation of cholesterol, bile acid, and steroid hormone homeostasis. Among the proposed LRH-1 target genes in liver are those encoding cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12alpha-hydroxylase (CYP8B1), which catalyze key steps in bile acid synthesis. In vitro studies suggest that LRH-1 may be involved both in stimulating basal CYP7A1 and CYP8B1 transcription and in repressing their expression as part of the nuclear bile acid receptor [farnesoid X receptor (FXR)]-small heterodimer partner signaling cascade, which culminates in small heterodimer partner binding to LRH-1 to repress gene transcription. However, in vivo analysis of LRH-1 actions has been hampered by the embryonic lethality of Lrh-1 knockout mice. To overcome this obstacle, mice were generated in which Lrh-1 was selectively disrupted in either hepatocytes or intestinal epithelium. LRH-1 deficiency in either tissue changed mRNA levels of genes involved in cholesterol and bile acid homeostasis. Surprisingly, LRH-1 deficiency in hepatocytes had no significant effect on basal Cyp7a1 expression or its repression by FXR. Whereas Cyp8b1 repression by FXR was also intact in mice deficient for LRH-1 in hepatocytes, basal CYP8B1 mRNA levels were significantly decreased, and there were corresponding changes in the composition of the bile acid pool. Taken together, these data reveal a broad role for LRH-1 in regulating bile acid homeostasis but demonstrate that LRH-1 is either not involved in the feedback regulation of bile acid synthesis or is compensated for by other factors.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Retroalimentación Fisiológica/genética , Homeostasis/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Retroalimentación Fisiológica/fisiología , Hepatocitos/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.
Asunto(s)
Cumestrol/farmacología , Fitoestrógenos/farmacología , Receptores de Esteroides/antagonistas & inhibidores , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Línea Celular , Células Cultivadas , Receptor de Androstano Constitutivo , Cumestrol/química , Cumestrol/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Inmunohistoquímica , Espectrometría de Masas , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Coactivador 1 de Receptor Nuclear , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Fitoestrógenos/química , Fitoestrógenos/metabolismo , Receptor X de Pregnano , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Nonalcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatocyte triglycerides, the synthesis of which is catalyzed by diacylglycerol acyltransferases (DGATs). Here, we investigate DGAT2 as a potential therapeutic target using an orally administered, selective DGAT2 inhibitor, PF-06427878. Treatment with PF-06427878 resulted in the reduction of hepatic and circulating plasma triglyceride concentrations and decreased lipogenic gene expression in rats maintained on a Western-type diet. In a mouse model of NASH, histological improvements in steatosis, ballooning, and fibrosis were evident in the livers of animals receiving PF-06427878 compared with mice treated with vehicle alone. We extended these nonclinical studies to two phase 1 studies in humans [NCT02855177 (n = 24) and NCT02391623 (n = 39; n = 38 completed)] and observed that PF-06427878 was well tolerated and influenced markers of liver function (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin) in healthy adults, with statistically significant reductions from baseline at day 14 in participants treated with PF-06427878 1500 milligrams per day (P < 0.05). Moreover, magnetic resonance imaging using proton density fat fraction showed that PF-06427878 1500 milligrams per day reduced hepatic steatosis in healthy adult participants. Our findings highlight DGAT2 inhibition by a small, potent, selective compound as a potential therapeutic approach for the treatment of NASH.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/enzimología , Administración Oral , Adulto , Animales , Diacilglicerol O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lípidos , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Pruebas de Función Hepática , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Piridinas/efectos adversos , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas Sprague-DawleyRESUMEN
The nuclear pregnane X receptor (PXR; NR1I2) is an important component of the body's adaptive defense mechanism against toxic substances including foreign chemicals (xenobiotics). PXR is activated by a large number of endogenous and exogenous chemicals including steroids, antibiotics, antimycotics, bile acids, and the herbal antidepressant St. John's wort. Elucidation of the three-dimensional structure of the PXR ligand binding domain revealed that it has a large, spherical ligand binding cavity that allows it to interact with a wide range of hydrophobic chemicals. Thus, unlike other nuclear receptors that interact selectively with their physiological ligands, PXR serves as a generalized sensor of hydrophobic toxins. PXR binds as a heterodimer with the 9-cis retinoic acid receptor (NR2B) to DNA response elements in the regulatory regions of cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and elimination of xenobiotics from the body. Although PXR evolved to protect the body, its activation by a variety of prescription drugs represents the molecular basis for an important class of harmful drug-drug interactions. Thus, assays that detect PXR activity will be useful in developing safer prescription drugs.
Asunto(s)
Núcleo Celular/química , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Esteroides/fisiología , Xenobióticos/metabolismo , Secuencia de Aminoácidos , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Clonación Molecular , Citocromo P-450 CYP3A , ADN/metabolismo , Dimerización , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Oxidorreductasas N-Desmetilantes/genética , Polimorfismo Genético , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Elementos de Respuesta , Receptores X Retinoide , Factores de Transcripción/metabolismo , Xenobióticos/farmacologíaRESUMEN
Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/metabolismo , Animales , Blastocisto/química , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Embrión de Mamíferos/citología , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Silenciador del Gen , Genes Letales , Ratones , Factor 3 de Transcripción de Unión a Octámeros , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Elementos de Respuesta/genética , Células Madre , Factores de Transcripción/análisis , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Fibroblast growth factor 21 (FGF21) is a hormone secreted by the liver in response to metabolic stress. In addition to its well-characterized effects on energy homeostasis, FGF21 has been shown to increase water intake in animals. In this study, we sought to further explore the effects of FGF21 on fluid homeostasis in rats. A single dose of a long-acting FGF21 analog, PF-05231023, significantly increased water consumption, which was accompanied by an elevation in urine output that appeared prior to a significant change in water intake. We observed that FGF21 rapidly and significantly increased heart rate and blood pressure in telemeter-implanted rats, before changes in urine output and water intake were observed. Our data suggest that sympathetic activation may contribute to the pathogenesis by which FGF21 increases blood pressure as the baroreceptor unloading induced reflex tachycardia was significantly elevated in FGF21-treated animals. However, FGF21 was still capable of causing hypertension in animals in which approximately 40% of the sympathetic post-ganglionic neurons were ablated. Our data suggest that FGF21-induced water intake is in fact secondary to diuresis, which we propose to be a compensatory mechanism engaged to alleviate the acute hypertension caused by FGF21.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Presión Sanguínea/efectos de los fármacos , Fármacos Cardiovasculares/farmacología , Diuresis/efectos de los fármacos , Diuréticos/farmacología , Ingestión de Líquidos/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Animales , Barorreflejo/efectos de los fármacos , Barorreflejo/fisiología , Presión Sanguínea/fisiología , Preparaciones de Acción Retardada , Diuresis/fisiología , Ingestión de Líquidos/fisiología , Agua Potable , Electrólitos/sangre , Electrólitos/orina , Factores de Crecimiento de Fibroblastos/metabolismo , Guanetidina/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratas WistarRESUMEN
Farnesoid X receptor (FXR) is a bile acid-activated transcription factor that is a member of the nuclear hormone receptor superfamily. Fxr-null mice exhibit a phenotype similar to Byler disease, an inherited cholestatic liver disorder. In the liver, activation of FXR induces transcription of transporter genes involved in promoting bile acid clearance and represses genes involved in bile acid biosynthesis. We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct-ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease.
Asunto(s)
Colestasis/tratamiento farmacológico , Citoprotección , Proteínas de Unión al ADN/fisiología , Isoxazoles/farmacología , Proteínas de Transporte de Membrana , Factores de Transcripción/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Proteínas Portadoras/genética , Colestasis/metabolismo , Colestasis/patología , Proteínas de Unión al ADN/agonistas , Isocianatos/farmacología , Masculino , Naftalenos/farmacología , Transportadores de Anión Orgánico Sodio-Dependiente , Transportadores de Anión Orgánico Sodio-Independiente/genética , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/fisiología , Esteroide 12-alfa-Hidroxilasa/genética , Simportadores , Taurina/farmacología , Factores de Transcripción/agonistas , Ácido Ursodesoxicólico/farmacologíaRESUMEN
We report the identification of substituted cis-bicyclo[3.3.0]-oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1.
Asunto(s)
Alquenos/síntesis química , Compuestos de Anilina/síntesis química , Compuestos Bicíclicos con Puentes/síntesis química , Proteínas de Unión al ADN/agonistas , Proteínas de Homeodominio/agonistas , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Alquenos/química , Alquenos/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Sitios de Unión , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacología , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ligandos , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Estereoisomerismo , Factor Esteroidogénico 1 , Relación Estructura-ActividadRESUMEN
In addition to its function as a fatty acid hydroxylase, the peroxisome proliferator-activated receptor alpha (PPARalpha) target gene, CYP4A, has been shown to be important in the conversion of arachidonic acid to the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, suggesting a role for this enzyme in mediating vascular tone. In the present study, the cDNA sequence of beagle dog CYP4A37, CYP4A38, and CYP4A39 from the liver was determined. Open reading frame analysis predicted that CYP4A37, CYP4A38, and CYP4A39 each comprised 510 amino acids with approximately 90% sequence identity to one another, and approximately 71 and 78% sequence identity to rat CYP4A1 and human CYP4A11, respectively. PCR analysis revealed that the three dog CYP4A isoforms are expressed in kidney > liver >> lung >> intestine > skeletal muscle > heart. Treatment of primary dog hepatocytes with the PPARalpha agonists GW7647X and clofibric acid resulted in an increase in CYP4A37, CYP4A38, and CYP4A39 mRNA expression (up to fourfold), whereas HMG-CoA synthase mRNA expression was increased to a greater extent (up to 10-fold). These results suggest that dog CYP4A37, CYP4A38, and CYP4A39 are expressed in a tissue-dependent manner and that beagle dog CYP4A is not highly inducible by PPARalpha agonists, similar to the human CYP4A11 gene.