ABSTRACT
Fibroblast growth factor receptor 1 (FGFR1) plays an important role in tumorigenesis and is therefore an attractive target for anticancer therapy. Using molecular docking approach we have identified inhibitor of FGFR1 belonging to 5-amino-4-(1H-benzoimidazol-2-yl)-phenyl-1,2-dihydro-pyrrol-3-ones with IC50 value of 3.5 µM. A series of derivatives of this chemical scaffold has been synthesized and evaluated for inhibition of FGFR1 kinase activity. It was revealed that the most promising compounds 5-amino-1-(3-hydroxy-phenyl)-4-(6-methyl-1H-benzoimidazol-2-yl)-1,2-dihydro-pyrrol-3-one and 5-amino-4-(1H-benzoimidazol-2-yl)-1-(3-hydroxy-phenyl)-1,2-dihydro-pyrrol-3-one inhibit FGFR1 with IC50 values of 0.63 and 0.32 µM, respectively, and posses antiproliferative activity against KG1 myeloma cell line with IC50 values of 5.6 and 9.3 µM. Structure-activity relationships have been studied and binding mode of this chemical class has been proposed.
Subject(s)
Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Drug Design , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Benzimidazoles/chemistry , Humans , Protein Kinase Inhibitors/chemistryABSTRACT
Protein kinase ASK1 (Apoptosis signal-regulating kinase 1) plays a key role in cell differentiation, aging and apoptosis. High activity of the kinase is associated with several pathologies. The ASK1 inhibitors might be therapeutic for patients with neurodegenerative, cardiovascular diseases and fibrous histiocytoma. In this work the identification of ASK1 inhibitors was performed by the methods of computer modeling and biochemical testing in vitro. The virtual screening experiments were carried out targeting the ATP binding site of ASK1 by browsing the database which contained 164 840 compounds of diverse chemical classes. The best-scored 300 ligands have been taken for the kinase assay analysis. In vitro tests revealed that derivatives of 2-thioxo-thiazolidin-4-one exhibited inhibitory activity against ASK1. The most active compound was 5-bromo-3-(4-oxo-2-thioxo-thiazolidin-5-ylidene)-1,3-dihydro-indol-2-one (IC50 = 2 microM). Binding mode for inhibitors of this class with ASK1 ATP-binding site was proposed. Our results can be used for further optimization and developing more potent and selective inhibitors of ASK1.
Subject(s)
MAP Kinase Kinase Kinase 5/metabolism , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/metabolism , Small Molecule Libraries/pharmacology , Thiazolidinediones/pharmacology , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , Binding Sites , Cardiovascular Diseases/drug therapy , Cell Differentiation/drug effects , Cell Line , High-Throughput Screening Assays , Histiocytoma, Benign Fibrous/drug therapy , Humans , Inhibitory Concentration 50 , MAP Kinase Kinase Kinase 5/genetics , Models, Molecular , Neoplasms/drug therapy , Neurodegenerative Diseases/drug therapy , Phosphorus/analysis , Phosphorus/metabolism , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Quantitative Structure-Activity Relationship , Radioactive Tracers , Recombinant Proteins/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic use , Thiazolidinediones/chemistry , Thiazolidinediones/therapeutic useABSTRACT
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca(2+) concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca(2+)-independent step, termed docking and followed by fusion step that is triggered by Ca(2+). The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.
