RESUMO
Growth differentiation factor 15 (GDF15), a distant member of the transforming growth factor (TGF)-ß family, is a secreted protein that circulates as a 25-kDa dimer. In humans, elevated GDF15 correlates with weight loss, and the administration of GDF15 to mice with obesity reduces body weight, at least in part, by decreasing food intake. The mechanisms through which GDF15 reduces body weight remain poorly understood, because the cognate receptor for GDF15 is unknown. Here we show that recombinant GDF15 induces weight loss in mice fed a high-fat diet and in nonhuman primates with spontaneous obesity. Furthermore, we find that GDF15 binds with high affinity to GDNF family receptor α-like (GFRAL), a distant relative of receptors for a distinct class of the TGF-ß superfamily ligands. Gfral is expressed in neurons of the area postrema and nucleus of the solitary tract in mice and humans, and genetic deletion of the receptor abrogates the ability of GDF15 to decrease food intake and body weight in mice. In addition, diet-induced obesity and insulin resistance are exacerbated in GFRAL-deficient mice, suggesting a homeostatic role for this receptor in metabolism. Finally, we demonstrate that GDF15-induced cell signaling requires the interaction of GFRAL with the coreceptor RET. Our data identify GFRAL as a new regulator of body weight and as the bona fide receptor mediating the metabolic effects of GDF15, enabling a more comprehensive assessment of GDF15 as a potential pharmacotherapy for the treatment of obesity.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Obesidade/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Ingestão de Alimentos/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/farmacologia , Humanos , Macaca fascicularis , Camundongos , Camundongos Knockout , Redução de Peso/genéticaRESUMO
Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.
Assuntos
Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/química , Piridinas/síntese química , Tiazóis/síntese química , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Desenho de Fármacos , Halogenação , Humanos , Cinética , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Tiazóis/farmacologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endossomos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , Autoantígenos/genética , Autoantígenos/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Eficiência , Endossomos/metabolismo , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/metabolismo , Interferência de RNA/efeitos dos fármacos , Interferência de RNA/fisiologia , Estabilidade de RNA/genética , RNA Interferente Pequeno/farmacologia , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Quinazolinas/síntese química , Quinazolinas/farmacologia , Sítios de Ligação , Quinase 1 do Ponto de Checagem , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Conformação Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Quinazolinas/químicaRESUMO
From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Quinases/efeitos dos fármacos , Quinolonas/síntese química , Quinolonas/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Fotoquímica , Proteínas Quinases/química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Quinolonas/farmacologia , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.