A high-throughput fluorescence-anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors.
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.
Rishi
Vikas
V
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Potter
Timothy
T
Laudeman
Julie
J
Reinhart
Russel
R
Silvers
Thomas
T
Selby
Michael
M
Stevenson
Timothy
T
Krosky
Paula
P
Stephen
Andrew G
AG
Acharya
Asha
A
Moll
Jon
J
Oh
Won Jun
WJ
Scudiero
Dominic
D
Shoemaker
Robert H
RH
Vinson
Charles
C
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*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA; D004996:Ethidium