ABSTRACT
A series of 4-amino-6-piperazin-1-yl-pyrimidine-5-carbaldehyde oximes has been discovered and developed as potent FLT3 tyrosine kinase inhibitors. The series exhibited potent antiproliferative activity against both an FLT3 ITD-mutated human leukemic cell line as well as a wild-type FLT3 BaF(3) expressed cell line. The structure-activity relationship of this class of compounds is described.
Subject(s)
Aldehydes/chemistry , Chemistry, Pharmaceutical/methods , Oximes/chemical synthesis , Oximes/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation , Drug Design , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Models, Chemical , Mutation , Oximes/chemistry , Structure-Activity RelationshipABSTRACT
A series of 2-acylaminothiophene-3-carboxamides has been identified which exhibit potent inhibitory activity against the FLT3 tyrosine kinase. Compound 44 inhibits the isolated enzyme (IC50 = 0.027 microM) and blocks the proliferation of MV4-11 cells (IC50 = 0.41 microM). Structure-activity relationship studies within this series are described in the context of a proposed binding model within the ATP binding site of the enzyme.
Subject(s)
Amides/chemistry , Amides/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Thiophenes/chemistry , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Binding Sites , Cell Line , Models, Molecular , Molecular Structure , Structure-Activity Relationship , fms-Like Tyrosine Kinase 3/chemistry , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.
Subject(s)
Chemistry, Pharmaceutical/methods , Crystallography, X-Ray/methods , Dipeptidyl Peptidase 4/chemistry , Enzyme Inhibitors/pharmacology , Protease Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Binding Sites , Blood Glucose/metabolism , Caco-2 Cells , Dipeptidyl Peptidase 4/metabolism , Drug Design , Drug Screening Assays, Antitumor , Humans , Kinetics , Mice , Models, Chemical , Models, Molecular , Rats , Structure-Activity RelationshipABSTRACT
2-Hydroxy-4,6-diamino-[1,3,5]triazines are described which are a novel class of potent inhibitors of the VEGF-R2 (flk-1/KDR) tyrosine kinase. 4-(Benzothiazol-6-ylamino)-6-(benzyl-isopropyl-amino)-[1,3,5]triazin-2-ol (14d) exhibited low nanomolar potency in the in vitro enzyme inhibition assay (IC(50) = 18 nM) and submicromolar inhibitory activity in a KDR-induced MAP kinase autophosphorylation assay in HUVEC cells (IC(50) = 280 nM), and also demonstrated good in vitro selectivity against a panel of growth factor receptor tyrosine kinases. Further, 14d showed antiangiogenic activity in an aortic ring explant assay by blocking endothelial outgrowths in rat aortas with an IC(50) of 1 microM.
Subject(s)
Angiogenesis Inhibitors/chemical synthesis , Thiazoles/chemical synthesis , Triazines/chemical synthesis , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/chemistry , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Aorta/drug effects , Aorta/physiology , Benzothiazoles , Capillaries/drug effects , Capillaries/physiology , Cell Line , Combinatorial Chemistry Techniques , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Humans , Organ Culture Techniques , Phosphorylation , Rats , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Triazines/chemistry , Triazines/pharmacology , Umbilical Veins/cytologyABSTRACT
Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Receptors, Colony-Stimulating Factor/chemistry , Automation , Biochemistry/methods , Cell Line , Detergents/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Epitopes/chemistry , Genetic Vectors , Histidine/chemistry , Humans , Immunoblotting/methods , Immunoprecipitation , Inhibitory Concentration 50 , Octoxynol/pharmacology , Phosphorylation , Phosphotyrosine/chemistry , Signal Transduction , Time FactorsABSTRACT
APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.
