ABSTRACT
AIM: To analyze the metabolic parameters and adipose tissue inflammation via NLRP3 inflammasome following chronic treatment of mouse models of obesity with AJ5018 as the peripherally restricted cannabinoid 1 receptor (CB1R) antagonist. MATERIALS AND METHODS: The selectivity for CB1R over CB2R, brain/plasma concentration ratio, and centrally mediated neurobehavioural effects of AJ5018, were assessed. The long-term effects of AJ5018 and rimonabant on the metabolic parameters and adipose tissue inflammation were analyzed in diet-induced obese (DIO) mice and diabetic db/db mice. RESULTS: AJ5018 had a higher degree of selectivity for CB1R over CB2R and markedly reduced brain penetrance, as reflected by the lower brain/plasma concentration ratio and the attenuated centrally mediated neurobehavioural effects, compared with its brain-penetrant parent compound rimonabant. In DIO and db/db mice, AJ5018 exhibited comparable effects to rimonabant in improving metabolic abnormalities and suppressing macrophage infiltration into white adipose tissue, activation of the NLRP3 inflammasome, and production of proinflammatory cytokines. CONCLUSIONS: These results suggest that peripheral CB1R blockade improves obesity-induced insulin resistance by suppressing adipose tissue inflammation via the NLRP3 inflammasome.
Subject(s)
Adipose Tissue/metabolism , Cannabinoid Receptor Antagonists/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Inflammasomes/metabolism , Inflammation/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Obesity/drug therapy , Animals , Brain/metabolism , Diabetes Mellitus, Experimental/metabolism , Insulin Resistance/physiology , Macrophages/metabolism , Male , Mice , Mice, Obese , Obesity/etiology , Obesity/metabolism , Rimonabant/pharmacologyABSTRACT
Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Hyperargininemia/therapy , Amino Acids/blood , Ammonia/blood , Animals , Arginase/genetics , Arginase/metabolism , Blotting, Western , Female , Fertility/genetics , Gene Expression Regulation , Gene Transfer Techniques , Genotype , Glutamine/blood , Hyperargininemia/genetics , Immunohistochemistry , Male , Mice , Mice, KnockoutABSTRACT
Starting with a high-throughput screening lead, a novel series of CCR5 antagonists was developed utilizing an information-based approach. Improvement of pharmacokinetic properties for the series was pursued by SAR exploration of the lead template. The synthesis, SAR and biological profiles of the series are described.
Subject(s)
Anti-HIV Agents/chemistry , Benzamides/chemistry , CCR5 Receptor Antagonists , HIV Fusion Inhibitors/chemistry , Pyrroles/chemistry , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacokinetics , Benzamides/chemical synthesis , Benzamides/pharmacology , HIV Fusion Inhibitors/chemical synthesis , HIV Fusion Inhibitors/pharmacokinetics , Humans , Microsomes, Liver/metabolism , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Rats , Receptors, CCR5/metabolism , Structure-Activity RelationshipABSTRACT
Human Cripto-1, a membrane-bound protein, plays an important role during early embryogenesis and has oncogenic properties, including cell transformation and enhancement of invasion. Cripto-1 is up-regulated in various malignant tissues and premalignant lesions. However, Cripto-1 expression in intraductal papillary mucinous neoplasms (IPMNs) has yet to be reported. This study aimed to investigate Cripto-1 expression in IPMNs and evaluate the expression patterns according to the histological grade or phenotypic subclassification. Cripto-1 expression was evaluated by immunohistochemistry using 37 IPMN tissue samples and real-time RT-PCR analysis of seven frozen samples. Cripto-1 was up-regulated in 59.5% of IPMNs. Cripto-1 was positively stained in 3 of 4 (75%) adenomas, 12 of 19 (63.2%) borderline neoplasms, 5 of 11 (45.5%) non-invasive carcinomas and 2 of 3 (66.7%) invasive carcinomas. There was no correlation between Cripto-1 overexpression and the histological grade (P>0.05). Cripto-1 expression was significantly increased in pancreatobiliary- (4/5, 80%) and gastric-type (13/19, 68.4.2%) IPMNs compared with those of the intestinal type (2/10, 20%; P<0.01). Cripto-1 mRNA expression was higher in gastric- and pancreatobiliary-type IPMNs than in intestinal ones, supporting the immunohistochemical results. It is concluded that Cripto-1 overexpression is involved in the tumorigenesis of gastric- and pancreatobiliary-type IPMNs.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Pancreatic Ductal/metabolism , Epidermal Growth Factor/biosynthesis , Membrane Glycoproteins/biosynthesis , Neoplasm Proteins/biosynthesis , Pancreatic Neoplasms/metabolism , Adenocarcinoma, Mucinous/metabolism , Adenocarcinoma, Mucinous/pathology , Adenocarcinoma, Papillary/metabolism , Adenocarcinoma, Papillary/pathology , Adult , Aged , Carcinoma, Pancreatic Ductal/pathology , Female , GPI-Linked Proteins , Gene Expression , Humans , Immunohistochemistry , Intercellular Signaling Peptides and Proteins , Male , Middle Aged , Pancreatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The mechanisms involving the inhibitory effects of ascorbic acid (AA) on carcinogenesis have not fully defined, except for its free-radical scavenging activity against oxidative DNA damage. In this study, we examined the effects of AA on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene cytochrome P4501A1 (CYP1A1), which catalyzes the activation of genotoxic metabolites that can lead to mutagenesis. Cultured human HepG2 cells were incubated with AA with or without the potent AhR agonist/CYP1A1 inducer 2,3,7,8-tetrachloridibenxo-p-dioxin (TCDD). AA was highly effective at suppressing CYP1A1 induction following coincubation of the cells with 1nM TCDD. The preventive effects of AA were seen at the level of mRNA and protein expression as well as CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. A transient transfection assay using a dioxin response element (DRE)-linked luciferase reporter and an electrophoretic mobility shift assay revealed that AA reduced the amount of AhR that could form a complex with the DRE sequence in the promoter region of the CYP1A1 gene. In addition, AA inhibited the TCDD-induced Ecto-ATPase activity, which is known to be requiring for AhR translocation to the nucleus. These results suggest that AA may exert at least part of its anticarcinogenesis effect by controlling the expression of CYP1A1 at the transcription level.
Subject(s)
Ascorbic Acid/pharmacology , Cytochrome P-450 CYP1A1/genetics , Polychlorinated Dibenzodioxins/toxicity , Antigens, CD/metabolism , Apyrase/metabolism , Cells, Cultured , Cytochrome P-450 CYP1A1/metabolism , Humans , Promoter Regions, Genetic , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/physiologyABSTRACT
Histone deacetylase 2 (HDAC2) is aberrantly regulated and plays a pivotal role in the development of hepatocellular carcinoma (HCC) through regulation of cell-cycle components at the transcriptional level, but the underlying mechanism leading to oncogenic HDAC2 remains unknown. In this study, we show that expression of CK2α (casein kinase II α subunit) was up-regulated in a large cohort of human HCC patients, and that high expression of CK2α was significantly associated with poor prognosis of HCC patients in terms of five-year overall survival. It was also found that CK2α over-expression positively correlated with HDAC2 over-expression in a subset of HCCs. We observed that treatment with epidermal growth factor (EGF) elicited an increase in CK2α expression and Akt phosphorylation, causing induction of HDAC2 expression in liver cancer cells. It was also observed that ectopic expression of dominant-negative CK2α blocked EGF-induced HDAC2 expression, and that ectopic CK2α expression attenuated the suppressive effect of Akt knockdown on HDAC2 expression in liver cancer cells. Targeted disruption of CK2α influenced the cell cycle, causing a significant increase in the number of liver cancer cells remaining in G2/M phase, and suppressed growth via repression of Cdc25c and cyclin B in liver cancer cells. Taken together, our findings suggest the oncogenic potential of CK2α in liver tumorigenesis. Furthermore, a regulatory mechanism for HDAC2 expression is proposed whereby EGF induces transcriptional activation of HDAC2 by CK2α/Akt activation in liver cancer cells. Therefore, this makes CK2α a promising target in cancer therapy.
Subject(s)
Carcinogenesis , Epidermal Growth Factor/metabolism , ErbB Receptors/agonists , Gene Expression Regulation, Neoplastic , Histone Deacetylase 2/metabolism , Liver Neoplasms/metabolism , Carcinoma, Hepatocellular/metabolism , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Line, Tumor , Cell Proliferation , Cohort Studies , ErbB Receptors/metabolism , Histone Deacetylase 2/genetics , Humans , Liver/metabolism , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphorylation , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction , Survival AnalysisABSTRACT
In the past few years, there have been many advances in the efforts to cure patients with hepatitis C virus (HCV). The ultimate goal of these efforts is to develop a combination therapy consisting of only direct-antiviral agents (DAAs). In this paper, we discuss our efforts that led to the identification of a bicyclic template with potent activity against the NS5B polymerase, a critical enzyme on the life cycle of HCV. In continuation of our exploration to improve the stilbene series, the 3,5,6,8-tetrasubstituted quinoline core was identified as replacement of the stilbene moiety. 6-Methoxy-2(1H)-pyridone was identified among several heterocyclic headgroups to have the best potency. Solubility of the template was improved by replacing a planar aryl linker with a saturated pyrrolidine. Profiling of the most promising compounds led to the identification of quinoline 41 (RG7109), which was selected for advancement to clinical development.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Quinolines/pharmacology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Dogs , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hepacivirus/enzymology , Humans , Models, Molecular , Quinolines/chemistry , Quinolines/pharmacokinetics , Rats , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
Hepatitis C virus (HCV) is a major global public health problem. While the current standard of care, a direct-acting antiviral (DAA) protease inhibitor taken in combination with pegylated interferon and ribavirin, represents a major advancement in recent years, an unmet medical need still exists for treatment modalities that improve upon both efficacy and tolerability. Toward those ends, much effort has continued to focus on the discovery of new DAAs, with the ultimate goal to provide interferon-free combinations. The RNA-dependent RNA polymerase enzyme NS5B represents one such DAA therapeutic target for inhibition that has attracted much interest over the past decade. Herein, we report the discovery and optimization of a novel series of inhibitors of HCV NS5B, through the use of structure-based design applied to a fragment-derived starting point. Issues of potency, pharmacokinetics, and early safety were addressed in order to provide a clinical candidate in fluoropyridone 19.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Area Under Curve , Cell Line, Tumor , Dogs , Drug Discovery/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hepacivirus/drug effects , Hepacivirus/enzymology , Hepacivirus/physiology , Hepatitis C/prevention & control , Hepatitis C/virology , Host-Pathogen Interactions/genetics , Humans , Models, Molecular , Molecular Targeted Therapy/methods , Protein Binding , Protein Structure, Tertiary , Pyridones/chemical synthesis , Pyridones/pharmacokinetics , Pyridones/pharmacology , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Rats , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolismABSTRACT
Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM. CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM. CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM. CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.