Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros












Base de dados
Intervalo de ano de publicação
1.
Cell Metab ; 11(2): 101-12, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20096642

RESUMO

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.


Assuntos
Fármacos Antiobesidade/uso terapêutico , Obesidade/tratamento farmacológico , Peptídeos/uso terapêutico , Receptores da Bombesina/agonistas , Receptores da Bombesina/metabolismo , Animais , Fármacos Antiobesidade/farmacocinética , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Peptídeos/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores da Bombesina/antagonistas & inibidores
2.
Mol Pharmacol ; 73(4): 1072-84, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18187582

RESUMO

Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.


Assuntos
Azetidinas/metabolismo , Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Animais , Azetidinas/química , Células CACO-2 , Linhagem Celular , Clonagem Molecular , Cães , Ezetimiba , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Reprodutibilidade dos Testes , Sitosteroides/metabolismo , Sulfonamidas/química , Transfecção , Trítio , beta-Lactamas/metabolismo
4.
Int J Parasitol ; 36(12): 1249-59, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16890941

RESUMO

Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [(3)H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites.


Assuntos
Caseína Quinase I/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Leishmania major/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase I/isolamento & purificação , Imidazóis/metabolismo , Isomerismo , Leishmania major/enzimologia , Ligantes , Espectrometria de Massas/métodos , Proteínas de Protozoários/análise , Piridinas/metabolismo , Pirróis/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos , Trypanosoma brucei brucei/metabolismo
5.
Nucl Med Biol ; 33(4): 555-63, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16720249

RESUMO

We have characterized the interaction of the serotonin transporter ligand [3H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [3H]-DASB, a tritiated version of the widely used [11C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K(d)=0.20+/-0.04 nM). The serotonin transporter density (B(max)) obtained for rhesus frontal cortex was found to be 66+/-8 fmol/mg protein using [3H]-DASB, similar to the B(max) value obtained using the reference radioligand [3H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83+/-22 fmol/mg protein). Specific binding sites of both [3H]-DASB and [3H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [3H]-citalopram binding in a competition autoradiographic study, with K(i) values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [3H]-DASB and [3H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [11C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.


Assuntos
Benzilaminas/farmacocinética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Citalopram/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Autorradiografia , Sítios de Ligação , Técnicas In Vitro , Macaca mulatta , Taxa de Depuração Metabólica , Ligação Proteica , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Trítio/farmacocinética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...