ABSTRACT
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.
Subject(s)
Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/chemistry , Binding Sites , Enzyme Activation , Enzyme Stability , Intracellular Signaling Peptides and Proteins , Isoenzymes/analysis , Isoenzymes/chemistry , Ligands , Protein Binding , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Structure-Activity Relationship , Temperature , ThermodynamicsABSTRACT
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.
Subject(s)
Benzodiazepines/chemical synthesis , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/agonists , Benzodiazepines/chemistry , Benzodiazepines/pharmacology , Binding Sites , Cell Line, Tumor , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Mimicry , Molecular Structure , Proto-Oncogene Proteins c-mdm2 , Stereoisomerism , Structure-Activity Relationship , Tumor Suppressor Protein p53/biosynthesisABSTRACT
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.
