ABSTRACT
The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Animals , Catalytic Domain , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chemistry Techniques, Synthetic , Female , HCT116 Cells , Humans , Inhibitory Concentration 50 , Male , Mice , Minichromosome Maintenance Complex Component 2/metabolism , Molecular Docking Simulation , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Rats , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The human hypoxia-inducible factor prolyl hydroxylases 1, 2, and 3 (HIF-PHD1, -2, and -3) are thought to act as proximal sensors of cellular hypoxia by virtue of their mechanism-based dependence on molecular oxygen. These 2-oxoglutarate (2-OG) and non-heme iron-dependent oxygenases constitutively hydroxylate HIF, resulting in high-affinity binding to Von Hippel-Lindau protein (pVHL). Some reported affinities for the HIF-PHDs for 2-OG and iron approach the estimated physiological concentrations for these cofactors, suggesting that the system as described is not catalytically optimal. Here we report the enzymatic characterization of full-length recombinant human HIF-PHD2 using a novel and sensitive catalytic assay. We demonstrated submicromolar affinities for 2-OG and ferrous iron and HIF-PHD2 Km values for oxygen that are greater than atmospheric oxygen levels, suggesting that molecular oxygen is indeed the key regulator of this pathway. In addition, we observed enhancement of HIF-PHD2 catalytic activity in the presence of ascorbic acid with only minor modifications of HIF-PHD2 requirements for 2-OG, and a detailed pH study demonstrated optimal HIF-PHD2 catalytic activity at pH 6.0. Lastly, we used this sensitive and facile assay to rapidly perform a large high-throughput screen of a chemical library to successfully identify and characterize novel 2-OG competitive inhibitors of HIF-PHD2.
Subject(s)
Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer/methods , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Procollagen-Proline Dioxygenase/chemistry , Ascorbic Acid/metabolism , Enzyme Inhibitors/analysis , Humans , Hydrogen-Ion Concentration , Hypoxia-Inducible Factor-Proline Dioxygenases , Ketoglutaric Acids/metabolism , Kinetics , Models, Biological , Oxygen/metabolism , Procollagen-Proline Dioxygenase/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.
Subject(s)
Aza Compounds/chemical synthesis , Aza Compounds/pharmacology , Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Aza Compounds/chemistry , Basic Helix-Loop-Helix Transcription Factors , Benzimidazoles/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.
