Acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors

Abstract

Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50=25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors.

Figure 1

Crystal structures of BRD4-1 in complex with kinase inhibitors. Complexes were identified by co-crystallization screening against the Selleck and GSK kinase inhibitor libraries. All inhibitors bind to the KAc site of BRD4. Inhibitor is shown in yellow and the 2F_o–F_c electron density map (contoured at 1σ) is indicated as blue mesh. F_o–F_c electron density maps omitting the inhibitor during the refinement are shown in Supplementary Figure S1.

Figure 2

Inhibitory activity and binding potential of identified kinase inhibitors against BRD4-1 and BRDT-1. (A) IC₅₀ values were determined by Alpha Screen assay and differential melting temperatures (ΔT_m) by DSF as described under Methods. Compound JQ1 served as a positive control. Dose–response graphs are shown in Supplementary Figures S2, S3. (B) Logarithmic relationship between inhibitory potency and stability of the protein-inhibitor complex for BRD4-1 (○) and BRDT-1 (●). Data were fit to y = y₀ + a(ln x) (solid and dashed lines for BRD4 and BRDT, respectively).

Figure 3

Differential binding modes of BI2536 and TG101209 in BRD4 and kinases. (A) General architecture of the KAc site in BRD4-1. The ZA channel consists of a network of tightly bound water molecules (cyan spheres) from Glu85 to Tyr97. The WPF shelf (green) constitutes one flank of the binding cleft opposite to Leu92. Asn140 is colored in magenta. The right panel shows the potential hydrogen bonding (black dotted lines) and VDW interactions (green dotted lines) of BI2536 in the KAc site of BRD4. (B) Hydrogen bonding interactions of BI2536 in BRD4 (left) and in PLK1 (right, PDB 2RKU) along with schematic presentations of the binding modes. Atoms colored in magenta interact with Asn140, and those colored in orange interact with the hinge region. The gatekeeper residue is indicated in red. (C) Hydrogen bonding interactions of TG101209 in BRD4 (left) and JAK2 (right, PDB 4JI9). Stereo presentations of the binding interactions of all identified BRD4 inhibitors are shown in Supplementary Figure S1.

Figure 4

Hydrogen bonding interactions of kinase inhibitors in BRD4-1 with micromolar inhibitory activities. The color code is the same as in Figure 3.

Figure 5

Profiling of BI2536 and TG101209 against a panel of 32 human BRDs. The binding potential of BI2536 and TG101209 toward other human BRDs was determined in duplicate at a single compound concentration of 2 μM using a qPCR-based binding assay by Discoverx Corp. Binding activity is expressed as a percentage of the positive control, with lower values indicating higher binding affinity (larger circles). Shown is an artistic representation of the human BRD phylogenetic tree highlighting the potency and selectivity of these kinase inhibitors against BET BRDs (Family II). The other BRDs affected were TAF1L bromodomain 2 (TAF1L-2) and TAF1 bromodomain 2 (TAF1-2) (Family VII), and CREBBP and EP300 (Family III). The experimental values against each BRD are shown in Supplementary Table S3.