RESUMO
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Assuntos
Benzodiazepinas/síntese química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2 , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/biossínteseRESUMO
MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4-10mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1-370, removal of the C-terminal autoinhibitory peptide in 1-338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1-338 by p38alpha further increased activity 12-fold. A putative constitutively active mutant, 1-370/T222E/T334E, was 6.3-fold less active than phosphorylated 1-370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg(2+)-dependent. Residues 1-40 were required for high-affinity binding of ADP, ATPgammaS, staurosporine, and K252a. A mutation M138A rendered 1-370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 microM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.
Assuntos
Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/química , Sítios de Ligação , Ativação Enzimática , Estabilidade Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/análise , Isoenzimas/química , Ligantes , Ligação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Temperatura , TermodinâmicaRESUMO
A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.