A high-throughput fluorescence–anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors
Section snippets
Proteins
The majority of the B-ZIP domain proteins used in this study have been described elsewhere: VBP (vitellogenin gene binding protein) [17], C/EBP [18], CREB [19], c-FOS [20], and SREBP-1 (sterol regulatory element binding protein) [21]. All proteins used in this study have 13 amino acid Phi 10 epitopes at their N terminal. The c-JUND protein has the following amino acid sequence: SPIDMESQERIKAERKRMRNRIAASKCRKR KLER IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLM LTQQLQTF. The sequence of C/EBPβ
High-throughput screen
Fluorescein-labeled DNA containing a consensus-binding site for each B-ZIP protein was added at 5 nM to a black 384-well polypropylene plate with a Biomek 2000 automated pipetting instrument. The corresponding B-ZIP protein was added at 300 nM for FOS|JUND, 400 nM for CREB, and 1000 nM for both C/EBPβ and VBP by the Biomek 2000. The test compounds were diluted to the required concentrations and transferred from the 96-well storage plates to the test plate by the Biomek 2000. After incubation for 60
Results
Fig. 1A presents the X-ray structure of a B-ZIP homodimer bound to DNA [24]. Fig. 1B presents the increase in fluorescence polarization after a 28-bp double-stranded DNA, labeled with fluorescein at the 5′ ends of both DNA strands, binds to a FOS|JUND heterodimer. This indicates a slowing of the tumbling rate of the DNA on protein binding. Normally, the binding between two entities of similar mass does not produce an easily observable change in fluorescent anisotropy. However, in the case of
Discussion
We have developed a high-throughput fluorescence polarization screen to identify small molecules that disrupt the DNA binding of B-ZIP proteins to DNA. We screened the NCI diversity set of 1990 compounds using four B-ZIP dimers (CREB, C/EBPβ, VBP, and FOS|JUND) and identified 39 compounds that were active against at least one protein|DNA complex. Confirmation titrations identified 21 compounds that disrupted DNA binding in the micromolar range. We used three secondary screens to evaluate 12
References (30)
- et al.
A fluorescence polarization competition immunoassay for tyrosine kinases
Anal. Biochem.
(1998) - et al.
BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization
Anal. Biochem.
(1996) - et al.
A high-throughput STAT binding assay using fluorescence polarization
Anal. Biochem.
(1997) - et al.
A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay
Anal. Biochem.
(2003) - et al.
Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies
J. Biol. Chem.
(2000) - et al.
A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis
J. Biol. Chem.
(1997) - et al.
The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α helices: crystal structure of the protein–DNA complex
Cell
(1992) - et al.
A simple statistical parameter for use in evaluation and validation of high throughput screening assays
J. Biomol. Screen.
(1999) - et al.
Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids
J. Mol. Biol.
(1998) - et al.
Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha
J. Biol. Chem.
(2003)
In vivo activation of the p53 pathway by small-molecule antagonists of MDM2
Science
Classification of human B-ZIP proteins based on dimerization properties
Mol. Cell. Biol.
AP-1: a double-edged sword in tumorigenesis
Nat. Rev. Cancer
Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion
Proc. Natl. Acad. Sci. USA
CREB regulates hepatic gluconeogenesis through the coactivator PGC-1
Nature
Cited by (51)
Potential in vitro and ex vivo targeting of bZIP53 involved in stress response and seed maturation in Arabidopsis thaliana by five designed peptide inhibitors
2018, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :Given the significance of the bZIPs in various biological processes, it is important to use the molecules that selectively disrupt the dimer formation or the bZIP-DNA interactions [13–15]. The availability of the high-throughput screening (HTS), computer-aided molecular docking and other approaches have contributed in the designing of small molecules that may work as an inhibitor of the bZIP-DNA complex or affect the bZIP dimerization. [16–18]. Currently, various classes of inhibitory molecules are available, including small molecules, polyamides, and effector domains but their efficacy and specificity in disrupting the bZIP dimerization or bZIP-DNA interactions are debatable [19–21].
C/EBPβ (CEBPB) protein binding to the C/EBP|CRE DNA 8-mer TTGC|GTCA is inhibited by 5hmC and enhanced by 5mC, 5fC, and 5caC in the CG dinucleotide
2015, Biochimica et Biophysica Acta - Gene Regulatory MechanismsCitation Excerpt :The C/EBPβ homodimer monitored at 222 nm has a Tm of 61.6 °C. With heating, the C/EBPβ homodimer cooperatively loses ellipticity at 222 nm as observed previously [41]. The denaturation is well fit using a two-state model of α-helical dimers becoming un-helical monomers.
Identification of influenza virus inhibitors targeting NS1A utilizing fluorescence polarization-based high-throughput assay
2012, Journal of Biomolecular ScreeningCitation Excerpt :EB displacement assays were performed to evaluate nonspecific binding characteristics of small chemical compounds with oligonucleotides. This assay was modified from a protocol described by Rishi et al.18 Instead of hairpin DNA, unlabeled 16-nucleotide dsRNA, which has the same sequence used for the FP assay, was used. In detail, hit compounds identified from primary screening were diluted in hybridization buffer and were added to a mixture of EB and unlabeled dsRNA diluted in hybridization buffer.
Targeting MgrA-mediated virulence regulation in Staphylococcus aureus
2011, Chemistry and BiologyCitation Excerpt :Protein-DNA interaction is central to transcriptional regulation in biology. Despite a growing number of examples of small molecules targeting transcription (Chau et al., 2005; Darnell, 2002; Dervan et al., 2005; Kiessling et al., 2007; Lauth et al., 2007; Lu et al., 2005; Rishi et al., 2005; Vassilev et al., 2004), the process of identifying effective small molecules that exhibit activity inside cells is still challenging. In very few examples have small molecules been identified that effectively disrupt protein-DNA interactions.
Synthesis and evaluation of quinoxaline derivatives as potential influenza NS1A protein inhibitors
2011, Bioorganic and Medicinal Chemistry Letters