ABSTRACT
BACKGROUND: Factor Xa (FXa), a key serine protease that converts prothrombin to thrombin in the coagulation cascade, is a promising target enzyme for the prophylaxis and treatment of thromboembolic diseases. DU-176b is a novel antithrombotic agent that directly inhibits FXa activity. OBJECTIVE: To evaluate the in vitro pharmacological profiles and in vivo effects of DU-176b in animal models of thrombosis and bleeding. METHODS: In vitro, FXa inhibition, specificity and anticoagulant activities were examined. Oral absorption was studied in rats and cynomolgus monkeys. In vivo effects were studied in rat and rabbit models of venous thrombosis and tail bleeding. RESULTS: DU-176b inhibited FXa with Ki values of 0.561 nm for free FXa, 2.98 nm for prothrombinase, and exhibited >10 000-fold selectivity for FXa. In human plasma, DU-176b doubled prothrombin time and activated partial thromboplastin time at concentrations of 0.256 and 0.508 microm, respectively. DU-176b did not impair platelet aggregation by ADP, collagen or U46619. DU-176b was highly absorbed in rats and monkeys, as demonstrated by more potent anti-Xa activity and higher drug concentration in plasma following oral administration than a prototype FXa inhibitor, DX-9065a. In vivo, DU-176b dose-dependently inhibited thrombus formation in rat and rabbit thrombosis models, although bleeding time in rats was not significantly prolonged at an antithrombotic dose. CONCLUSIONS: DU-176b is a more potent and selective FXa inhibitor with high oral bioavailability compared with its prototype, DX-9065a. DU-176b represents a promising new anticoagulant for the prophylaxis and treatment of thromboembolic diseases.
Subject(s)
Factor Xa Inhibitors , Pyridines/pharmacology , Serine Proteinase Inhibitors/pharmacology , Thiazoles/pharmacology , Administration, Oral , Animals , Area Under Curve , Blood Coagulation/drug effects , Female , Macaca fascicularis , Male , Platelet Aggregation/drug effects , Pyridines/administration & dosage , Pyridines/pharmacokinetics , Rats , Rats, Wistar , Serine Proteinase Inhibitors/administration & dosage , Serine Proteinase Inhibitors/pharmacokinetics , Thiazoles/administration & dosage , Thiazoles/pharmacokineticsABSTRACT
Asp278 of beta-adrenergic receptor kinase 1 (betaARK1) was suggested to play a key role in substrate recognition of beta2-adrenergic receptors in our previous study, in which a three-dimensional model of betaARK1 was studied in comparison with a crystal structure of PKA-PKI5-24 complex. In the present study, to confirm the molecular recognition mechanism at Asp278 of betaARK1, two mutants of betaARK1, D278R and D278A, were designed based on molecular modeling studies and produced by Sf-9 cells. As predicted by the molecular modeling study, the mutants showed no kinase activities while wild type betaARK1 phosphorylated beta2-adrenergic receptors in a concentration-dependent manner. These results strongly suggest the involvement of Asp278 in substrate recognition by betaARK1. The results also suggest a high reliability of the three-dimensional model of betaARK1.
Subject(s)
Aspartic Acid/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Alanine/genetics , Amino Acid Substitution/genetics , Arginine/genetics , Aspartic Acid/genetics , Conserved Sequence , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Humans , Models, Molecular , Mutagenesis, Site-Directed , Phosphorylation , Substrate Specificity , beta-Adrenergic Receptor KinasesABSTRACT
1. Catecholamine, glucagon, and adrenocorticotropic hormone stimulated 2-deoxyglucose (2-DG) uptake via an increase in glucose transporters in plasma membranes, similarly to insulin. 2. In contrast to the action of insulin, the stimulating effects of these agonists on 2-DG uptake were abolished when Gi was not activated. 3. The mode of the 2-DG uptake stimulation was partially different among these agonists.
