title: A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors.
abstract: We have developed a high-throughput fluorescence anisotropy screen, using a 384-well format, to identify small molecules that disrupt the DNA binding of B-ZIP proteins. Binding of a B-ZIP dimer to fluorescently labeled DNA can be monitored by fluorescence anisotropy. We screened the National Cancer Institute diversity set of 1990 compounds to identify small molecules that disrupt the B-ZIP|DNA complex of CREB, C/EBPbeta, VBP, and AP-1 (FOS|JUND) bound to their cognate DNA sequence. We identified 21 compounds that inhibited the DNA binding of at least one B-ZIP protein, and 12 representative compounds were grouped depending on whether they displaced ethidium bromide from DNA. Of the 6 compounds that did not displace ethidium bromide, 2 also inhibited B-ZIP binding to DNA in a secondary electrophoretic mobility shift assay screen with some specificity. Thermal stability monitored by circular dichroism spectroscopy demonstrated that both compounds bound the basic region of the B-ZIP motif. NSC13778 preferentially binds C/EBPalpha 1000-fold better than it binds C/EBPbeta. Chimeric proteins combining C/EBPalpha and C/EBPbeta mapped the binding of NSC13778 to three amino acids immediately N terminal of the leucine zipper of C/EBPalpha. These experiments suggest that the DNA binding of B-ZIP transcription factors is a potential target for clinical intervention.
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Vikas
authors.initials: V
authors.lastname: Rishi
authors.forename: Timothy
authors.initials: T
authors.lastname: Potter
authors.forename: Julie
authors.initials: J
authors.lastname: Laudeman
authors.forename: Russel
authors.initials: R
authors.lastname: Reinhart
authors.forename: Thomas
authors.initials: T
authors.lastname: Silvers
authors.forename: Michael
authors.initials: M
authors.lastname: Selby
authors.forename: Timothy
authors.initials: T
authors.lastname: Stevenson
authors.forename: Paula
authors.initials: P
authors.lastname: Krosky
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
authors.forename: Asha
authors.initials: A
authors.lastname: Acharya
authors.forename: Jon
authors.initials: J
authors.lastname: Moll
authors.forename: Won Jun
authors.initials: WJ
authors.lastname: Oh
authors.forename: Dominic
authors.initials: D
authors.lastname: Scudiero
authors.forename: Robert H
authors.initials: RH
authors.lastname: Shoemaker
authors.forename: Charles
authors.initials: C
authors.lastname: Vinson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019281:Dimerization; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004996:Ethidium / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D006358:Hot Temperature; D016350:Leucine Zippers; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA; D004996:Ethidium