ABSTRACT
Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).
Subject(s)
Benzodiazepines/chemistry , Drug Design , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Alkylation , Animals , Benzodiazepines/chemical synthesis , Cell Line, Tumor , Cell Membrane Permeability , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Mice , Models, Molecular , Molecular Structure , Pentanoic Acids/chemistry , Protein Binding , Proto-Oncogene Proteins c-mdm2/chemistry , Structure-Activity Relationship , Tumor Suppressor Protein p53/chemistryABSTRACT
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
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.
Subject(s)
Benzodiazepines/chemical synthesis , Benzodiazepines/pharmacology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Binding Sites , Combinatorial Chemistry Techniques , Fluorescence Polarization , Humans , Inhibitory Concentration 50 , Nuclear Proteins/antagonists & inhibitors , Protein Binding/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2 , Structure-Activity Relationship , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
