brd4 bromodomain plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Chemical structure of the JQ1-oligo-Cy5 conjugate used as a fluorescent probe (BP). JQ1, a selective BRD4 ligand, is covalently linked to a DNA oligonucleotide and a Cy5 fluorophore, allowing fluorescence-based detection of BRD4 binding. (B) Schematic illustrations (top) and corresponding fluorescence images (bottom) of agarose μ-droplets containing magnetic beads only, BRD4-bound magnetic beads, agarose alone, intact HeLa cells, or permeabilized HeLa cells after incubation with JQ1-oligo-Cy5. Strong Cy5 fluorescence was observed in droplets containing BRD4-bound magnetic beads and permeabilized HeLa cells, whereas no detectable Cy5 fluorescence was observed in magnetic beads only, intact HeLa cells, and agarose-only droplets. Scale bars, 50μm. (C) Schematic illustration of two-color Exchange-PAINT imaging using orthogonal DNA docking-imager strand pairs. An R6 docking strand conjugated to an anti-GFP nanobody was used to label GFP-BRD4, while an R2 docking strand was incorporated into the JQ1-based probe (JQ1-BP). Sequential super-resolution imaging was performed using R6* and R2* imager strands to independently localize BRD4 and bound JQ1, respectively. (D) Two-color Exchange-PAINT super-resolution images of GFP-BRD4-expressing Cos7 cells showing GFP-BRD4 (green) and the JQ1-based probe (JQ1-BP, red). Left, whole-nucleus view; middle, magnified view of the boxed region highlighting nanoscale clustering of BRD4 and JQ1. Right, the same region after Q-PAINT-based cluster filtering (K > 10), revealing higher-order nanoclusters containing both BRD4 and JQ1. Enrichment of JQ1-BP localizations at GFP-BRD4 clusters indicates that JQ1 preferentially localizes to BRD4-enriched nuclear regions.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Fluorescence, Binding Assay, Magnetic Beads, Incubation, Imaging, Expressing

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Composition of the four-compound library used for proof-of-concept DEL screening, including JQ1 as a positive control, GL-CBS and methotrexate (MTX) as off-target binders, and benzoic acid as a negative control. (B) Enrichment ratios (Target / Control) derived from nanopore sequencing data following small-scale DEL screening in agarose μ-droplets containing BRD4-bound magnetic beads (target) or magnetic beads only (control). JQ1 exhibited strong enrichment above the threshold (Target / Control = 1), whereas other compounds showed minimal enrichment. (C) Quantitative analysis of small-scale DEL screening performed in cell-containing agarose μ-droplets, in which DEL enrichment was quantified by qPCR following screening. Agarose-only μ-droplets were used as a control condition, while μ-droplets containing permeabilized HeLa cells or BRD4-overexpressing HeLa cells were treated as target conditions. JQ1 showed the highest enrichment among tested compounds and displayed a pronounced preference for BRD4-overexpressing HeLa cells compared to permeabilized cells. Representative bright field, emGFP, and Cy5 fluorescence images are shown on the right. Scale bars, 50μm. emGFP fluorescence was observed specifically in BRD4-overexpressing HeLa cells. As both HeLa cell conditions were permeabilized, Cy5 fluorescence originating from BP was detected in droplets containing either permeabilized or BRD4-overexpressing HeLa cells.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Drug discovery, Positive Control, Negative Control, Control, Derivative Assay, Nanopore Sequencing, Magnetic Beads, Fluorescence

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Aggregated nanopore sequencing read count distributions for each core scaffold across four screening conditions: magnetic bead–only droplets, BRD4-bound magnetic bead droplets, permeabilized HeLa cell droplets, and BRD4-overexpressing HeLa cell droplets. The DNA-encoded library consisted of three distinct core scaffolds (pyrimidine, trifunctional benzene, and chiral proline cores), each comprising a 96 × 96 × 96 combinatorial spaces. (B) Three-dimensional enrichment maps for each core scaffold across the four screening conditions, displaying the top 200 BB1–BB2–BB3 combinations ranked by nanopore sequencing read count. Each axis (x, y, and z) corresponds to one building block position (BB1, BB2, and BB3), and individual points represent distinct BB1– BB2–BB3 combinations. Point color indicates read count abundance, while combinations exceeding a read count threshold of 65 are additionally highlighted by increased marker size to emphasize highly enriched species. (C) Chemical structures of the top enriched compounds identified from large-scale DEL screening. Enrichment was calculated as the ratio of nanopore sequencing read counts between target and control samples. For each core scaffold, the two compounds with the highest target-to-control enrichment are shown for both magnetic bead droplet and HeLa cell droplet screening conditions. The corresponding BB1-BB2-BB3-derived structures are displayed.

Article Snippet: The BRD4 bromodomain plasmid (pHis-BRD4 BD1, item #196544), GFP-BRD4 expression plasmid (GFP-BRD4, item #65378) was obtained from Addgene (Watertown, MA, USA).

Techniques: Nanopore Sequencing, Blocking Assay, Marker, Control, Derivative Assay

Journal: Biochemistry

Article Title: Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

doi: 10.1021/acs.biochem.2c00157

Figure Lengend Snippet: Figure 1. Acetylated histone-binding site, “WPF” shelf tryptophan (red), and a basic interaction site (blue) for (A) BRDT−BD1 (W50, K37, K41, and K45) and (B) BRD4−BD1 (W81, R68, K72, and K76). (C) Sequence similarity in each bromodomain of BRDT (left) and BRD4 (right) relative to BRD4−BD1. Percent similarities were relative to BRD4−BD1 calculated by the PDB sequence and structure alignment (PDB ID: 7L73 for BRDT−BD1 and 3UVW for BRD4−BD1).

Article Snippet: The pNIC28-Bsa4 plasmid containing the first bromodomain of BRD4 (Addgene #38943), first bromodomain of BRDT (Addgene #38898), second bromodomain of BRD2 (Addgene https://doi.org/10.1021/acs.biochem.2c00157 Biochemistry 2022, 61, 1260−1272 1261 #39074), bromodomain of CREB-binding protein (Addgene #38977), and bromodomain of BPTF (Addgene #39111) were a kind gift from Nicola Burgess-Brown.

Techniques: Binding Assay, Sequencing

Journal: Biochemistry

Article Title: Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

doi: 10.1021/acs.biochem.2c00157

Figure Lengend Snippet: Figure 6. (A) Bromodomain phylogenetic tree adapted from Pomerantz et al..37 BDs tested in the PrOF NMR screen are highlighted. (B) Table showing PrOF NMR Kd values from the titration of 40 bp dsDNA with 5FW-labeled proteins. N.B. = binding affinity could not be determined. a: Kd is reported as the mean ± SD of two experimental replicates. b: Kd is reported as the mean ± SD of three experimental replicates.

Article Snippet: The pNIC28-Bsa4 plasmid containing the first bromodomain of BRD4 (Addgene #38943), first bromodomain of BRDT (Addgene #38898), second bromodomain of BRD2 (Addgene https://doi.org/10.1021/acs.biochem.2c00157 Biochemistry 2022, 61, 1260−1272 1261 #39074), bromodomain of CREB-binding protein (Addgene #38977), and bromodomain of BPTF (Addgene #39111) were a kind gift from Nicola Burgess-Brown.

Techniques: Titration, Labeling, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: Design of three new subseries I, II, and III of compounds as potential novel BRD4 inhibitors based on our lead BRD4 inhibitor 7.

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: Binding Affinities of Selected Active Compounds for the BET Bromodomains and Non-BET Protein CBP (IC 50 , nM) a

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques: Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: Dose–response curves and binding affinities of selected compounds 52 and 53 for the BRD4 bromodomains (with the positive controls compounds 1 and 3). TR-FRET assays were performed using recombinant BRD4 BD1 and BD2, and IC50 values are calculated using the Four Parameters Regression method (https://www.aatbio.com/tools/ic50-calculator/).

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques: Binding Assay, Recombinant

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: (A) Ribbon representation of crystal structure (PDB 6U0D) of BRD4 BD1 (sequence with residues Ser42 to Glu168) in complex with compound 52 (CPK representation in magenta). (B) Overlay of crystal structures BRD4 BD1 (in yellow) in complex with compound 52 (in magenta) and MS436 (in light-blue, PBD 4nud). The surface of traditional KAc pocket is colored in gray, and the new binding site is highlighted in light-blue. (C) New binding site of compound 52 (magenta) in ribbon representation. A solvent DMSO molecule occupies in the traditional KAc-binding pocket (red dashed circle area) with key residues of the classic KAc site shown in sticks. (D) Unbiased omit map for the KAc pocket, calculated without the coordinates for the DMSO molecule. The Fo-FcWT difference map electron density is shown as a green (positive density) or red (negative density) mesh, respectively, contoured at 3σ. (E) Detailed interactions of compound 52 with BRD4 BD1. Salt bridge with Glu151, direct hydrogen bond with Gly143, and indirect hydrogen bonds with Glu154, Tyr137, and Asp144 are highlighted in dashed line (black). (F) Overlay of BRD4 BD1 (PBD 4nud), BRD4 BD2 (PBD 6c7q), and BRD2 BD1 (PBD 3aqa). Key residues from BRD4 BD1 are in yellow, residues from BRD4 BD2 are in green, and residues from BRD2 BD2 are in magenta.

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques: Sequencing, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: 52 binds selectively to WT BRD4-BD1 but not WT BRD4-BD2 nor E151-mutated BRD4-BD1. (A) Purified human BRD4 BD1 and BD2 and the domain features of human BRD4 protein. E151A, glutamate 151 to alanine mutation. Numbers indicate the positions of amino acid residues in the full-length protein. The lower right panel is a Coomassie Brilliant Blue-stained SDS-PAGE gel image showing the purity of purified BD1 and BD2 domains used for TSA analysis. (B) TSA of 52 and JQ1 binding to WT BRD4 BD1 (left panel) and BRD4 BD2 (right panel). The graphs (top) show ΔTm upon compound binding (versus DMSO) at different compound/protein ratios (mean ± SEM) and the tables (bottom) show ΔTm values deduced from JQ1 or 52 binding at different compound/protein ratios with experimental replicates n = 9 for BD1 and n = 7 for BD2. Each experimental replicate was also analyzed in triplicate. (C) Superimposed BD1 structures showing the locations of E151 and JQ1-binding pocket (see PDB 3MXF and 6U0D). (D) TSA showing JQ1 but not 52 binding to E151A mutant of BRD4 BD1 similarly conducted as described in (B). Experimental replicates n = 9, each in triplicate.

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques: Purification, Mutagenesis, Staining, SDS Page, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site

doi: 10.1021/acs.jmedchem.1c01851

Figure Lengend Snippet: BRD4 inhibitors 52 and 53 significantly blocked poly(I:C)-induced H3K122Ac levels in hSAECs (A) and in lung tissue of mice (B). (A) Immunofluorescence staining of H3K122Ac (green color) was performed in hSAECs. (B) Immunofluorescence staining of H3K122Ac (red) was performed on paraffin-embedded lung sections of mice. Right panel, quantification of total fluorescence intensity shown as fold changes on immunofluorescence. #p < 0.01, compared with control; *p < 0.01 compared with poly(I:C) only, n = 5. Compound 3 was included as a positive control.

Article Snippet: Plasmid DNA encoding the canonical human BRD4 bromodomain (42–168) protein was purchased from Addgene.

Techniques: Immunofluorescence, Staining, Fluorescence, Positive Control