ABSTRACT
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.
Subject(s)
Eating/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Growth Differentiation Factor 15/genetics , Obesity/metabolism , Weight Loss/drug effects , Animals , Diet, High-Fat , Eating/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Growth Differentiation Factor 15/metabolism , Growth Differentiation Factor 15/pharmacology , Humans , Macaca fascicularis , Mice , Mice, Knockout , Weight Loss/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Protein Kinases/chemistry , Pyridines/chemical synthesis , Thiazoles/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Membrane Permeability , Checkpoint Kinase 1 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/chemistry , Drug Design , Halogenation , Humans , Kinetics , Molecular Structure , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyridines/pharmacology , Structure-Activity Relationship , Thiazoles/pharmacology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
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.
Subject(s)
Drug Delivery Systems/methods , Endosomes/drug effects , Peptide Fragments/pharmacology , RNA Stability/drug effects , RNA, Messenger/metabolism , RNA, Small Interfering/administration & dosage , Autoantigens/genetics , Autoantigens/metabolism , Biological Transport/drug effects , Biological Transport/genetics , Cells, Cultured , Efficiency , Endosomes/metabolism , Gene Targeting/methods , Gene Transfer Techniques , HeLa Cells , Humans , Hydrogen-Ion Concentration , Peptide Fragments/metabolism , RNA Interference/drug effects , RNA Interference/physiology , RNA Stability/genetics , RNA, Small Interfering/pharmacology , Ribonucleoproteins/antagonists & inhibitors , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , SS-B AntigenABSTRACT
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.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Binding Sites , Checkpoint Kinase 1 , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Quinazolines/chemistryABSTRACT
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.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Kinases/drug effects , Quinolones/chemical synthesis , Quinolones/pharmacology , Apoptosis/drug effects , Binding Sites , Cell Line, Tumor , Checkpoint Kinase 1 , Crystallography, X-Ray , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Photochemistry , Protein Kinases/chemistry , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
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).
Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinolones/pharmacology , Checkpoint Kinase 1 , Crystallography, X-Ray , Models, Molecular , Protein Kinase Inhibitors/chemistry , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
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.