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