Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1

Jung, Marie; Philpott, Martin; Müller, Susanne; Schulze, Jessica; Badock, Volker; Eberspächer, Uwe; Moosmayer, Dieter; Bader, Benjamin; Schmees, Norbert; Fernández-Montalván, Amaury E.; Haendler, Bernard

doi:10.1074/jbc.m113.523019

Cited by 128 publications

(142 citation statements)

References 66 publications

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“…Indeed, the EC 50 values of JQ1 for the mutant BRD4 variants in our cellular assay matched well with the affinities obtained in biochemical and biophysical assays performed with these mutants. 12 Moreover, we showed that the loss of stabilization induced by JQ1 for each mutant was similar to the loss of binding affinity of BRD4 to acetylated H4 peptide and that the highest impact was found with mutants Y97F and N140A. These residues are known to recognize acetylated histones by forming direct (N140) and watermediated (Y97) hydrogen bonds with acetylated peptides and to interact with inhibitors mimicking acetyl-lysines such as JQ1 and I-BET-762.…”

mentioning

confidence: 90%

“…We selected W81, P82, Y97, N140, and M149 as mutational sites for this study, in view of their role in the recognition by acetylated histone peptides and JQ1. 12 In total, 48 small molecules were tested against wild-type and five different mutant BRD4 BD1 constructs.…”

Section: Impact Of Brd4 Bd1 Mutations On Intracellular Stability Andmentioning

confidence: 99%

“…A biochemical TR-FRET assay described previously 12 was used to assess inhibitor affinity. The method quantifies the inhibition by test compounds of the binding between N-terminal His6-tagged human BRD4 bromodomain 1 and a synthetic tetra-acetylated peptide derived from human histone 4.…”

Section: Biochemical Binding Assaymentioning

confidence: 99%

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Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

et al. 2015

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Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay.

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confidence: 90%

Section: Impact Of Brd4 Bd1 Mutations On Intracellular Stability Andmentioning

confidence: 99%

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Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

et al. 2015

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“…Thet etra-acetylated histone H4-mimicking peptide H4 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] [36] and 4.8 mm (ITC). [38] BRD4 (1) can recognize two adjacent KAc residues on the same peptide at any one time. [39] X-ray crystal structures reveal the N-terminal KAc forms ah ydrogen-bonding interaction with the conserved asparagine residue,w hile flexible glycine residues allow ap eptide conformation where the C-terminal KAc fits into the grooves created by the ZA and BC loops (PDB:3 UVW,3 UVX, 3UVY).…”

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confidence: 99%

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

et al. 2016

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Ar ange of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains.Three of these amino acids were incorporated into ah istone H4-mimicking peptide and their affinity for BRD4 (1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of ahyperacetylated histone H4-mimicking cognate peptide,a nd demonstrated ad ependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of ahistone H4-mimicking peptide,containing alysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate aK 18C mutant of histone H3.

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“…BRD4 binds Kac of histone via its bromodomains, and then recruits the positive transcription elongation factor b (P-TEFb), which is a heterodimer of cyclin-dependent kinase 9 (CDK9) and its regulator cyclin T, to the transcription start site, leading to transcription elongation through phosphorylation of the carboxyl-terminal of RNA polymerase II. [11][12][13] BRD4 therefore acts as a transcriptional coactivator of many cellular genes. For example, the interaction of BRD4 with P-TEFb promotes G 1 gene expression and cell cycle progression.…”

Section: Features Of Bet Family Proteinsmentioning

confidence: 99%

BET Bromodomain as a Target of Epigenetic Therapy

Noguchi‐Yachide

2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Acetylation of histone is a key epigenetic modification, and contributes to many DNA-dependent cellular processes. The bromodomain structure, which consists of approximately 110 amino acid residues, serves as a 'reader' that recognizes acetylated lysine in histones, leading to recruitment of positive transcriptional elongation factor b (P-TEFb), and thereby promoting transcriptional activity and chromatin remodeling.

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Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1

Cited by 128 publications

References 66 publications

Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

BET Bromodomain as a Target of Epigenetic Therapy

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