Chemical Space Expansion of Bromodomain Ligands Guided by in Silico Virtual Couplings (AutoCouple)

Abstract

Expanding the chemical space and simultaneously ensuring synthetic accessibility is of upmost importance, not only for the discovery of effective binders for novel protein classes but, more importantly, for the development of compounds against hard-to-drug proteins. Here, we present AutoCouple, a de novo approach to computational ligand design focused on the diversity-oriented generation of chemical entities via virtual couplings. In a benchmark application, chemically diverse compounds with low-nanomolar potency for the CBP bromodomain and high selectivity against the BRD4(1) bromodomain were achieved by the synthesis of about 50 derivatives of the original fragment. The binding mode was confirmed by X-ray crystallography, target engagement in cells was demonstrated, and antiproliferative activity was showcased in three cancer cell lines. These results reveal AutoCouple as a useful in silico coupling method to expand the chemical space in hit optimization campaigns resulting in potent, selective, and cell permeable bromodomain ligands.

Figure 1

(A) List of current nM inhibitors of the CBP bromodomain.^,−, Dissociation constant (K_d) determined by isothermal titration calorimetry (ITC). Half-maximal inhibitory concentration (IC₅₀) determined by time-resolved fluorescence resonance energy transfer (TR-FRET). Selectivity for CBP over BRD4(1) bromodomains (S₁) determined by the ratio of K_d or IC₅₀ values. (B) Crystal structure of the CBP bromodomain (cyan) in complex with compound 1 (green) (PDB code: 4TQN).^, The acetyl benzene moiety acts as a KAc mimic interacting directly and through a water molecule with the side chains of the conserved residues Asn1168 and Tyr1125, respectively. The carboxylate function of the tail group forms a salt bridge with the guanidinium of Arg1173. The amide linker is involved in two water-bridged hydrogen bonds with the CBP bromodomain. (C) Overlay of the complex of compound 1 (green) with the CBP bromodomain (cyan) and the structure of BRD4(1) (4PCI) shows that the selectivity is due to bumping of the benzoate into the Trp81 side chain (red) of the so-called WPF triad of BRD4(1).

Figure 2

Schematic representation of AutoCouple. A headgroup (here the KAc mimic is shown in orange) is virtually coupled to commercially available building blocks. The resulting library is filtered out to remove any protein-reactive functionalities and subsequently docked while maintaining key interactions of the headgroup inside the target’s binding site. The compounds are ranked according to binding energy calculated by a force field with continuum electrostatic solvation.

Scheme 1. AutoCouple Results for the CBP Bromodomain Using (A) Amide Condensation, (B) Buchwald–Hartwig Amination, and (C) Suzuki Cross-Coupling Reactions from Aniline (2), Bromobenzene (3), and Aryl Boronic Ester (4) as “Headgroups”, Respectively

K_d values (μM) were determined by a competition binding assay in duplicates (BROMOscan). IC₅₀ values for compound 16 are indicated in purple and were determined by amplified luminescent proximity homogeneous assay (Alpha) screen technology (Reaction Biology). Ligand efficiency (LE) values refer to the CBP bromodomain. Selectivity for CBP over BRD4(1) bromodomains (S₁) determined by the ratio of K_d or IC₅₀ values. (D) Chimerization of compounds 5–7. The growing vectors (green arrows) of the different coupling strategies show the similarity between the amide and the C–C coupled products compared to the amine linker in orienting the tail group.

Figure 3

(A) Structural alignment of the crystal structure of the CBP bromodomain (cyan) in complex with ligand 16 (green) (PDB code 5NLK) and the pose of ligand 7 (yellow) as predicted by docking into the CBP structure 4NYX (Arg1173 side chain in yellow). (B) Overlay of the complex of compound 16 (green) with the CBP bromodomain (cyan) and the structure of BRD4(1) (4PCI) shows that the selectivity is due to bumping of the phenyl into the Trp81 side chain (red) of the so-called WPF triad of BRD4(1).

Figure 4

(A,B) FRAP assay for compounds 6, 7, 13, 16, and 17; U2OS cells were transfected with plasmids encoding GFP-fused to wild-type (WT) or mutant (N1168F) multimerized CBP bromodomain, with or without 2.5 μM suberoylanilide hydroxamic acid (SAHA, a deacetylase inhibitor) and indicated compounds at a concentration of 1 μM. (A) Fluorescent recovery curves after photobleaching (normalized to the intensity before bleaching). (B) Half-times of the fluorescence recovery (t_1/2) (n ≥ 7 cells per group, error bars: standard error of the mean). The recovery t_1/2 of the compound-treated cells was compared to that of DMSO-treated cells (bar on the left) within the same experiment setup using Mann–Whitney test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Concentration of compound 16 that results in 50% growth inhibition (GI50). LP1 and Kasumi are human tumor cell lines while the nontransformed fibroblast MRC5 is a negative control. GI50 values were determined by a resazurin assay after 72 h compound incubation. (D) Dose-dependent inhibition of IRF4 and c-Myc mRNA transcription (RT-qPCR) by compound 16 in LP1 cells after 6 h of treatment. (C, D) Values represent the mean of at least three biological replicates ± SD. The curves are fits by a four-parameter logistic function. (E) Selectivity profile of compound 16 in a panel of bromodomains representing all subfamilies of human bromodomains. The K_d values were determined by a competition binding assay. (F, G) FRAP assay for compound 16 in U2OS cells transfected with a plasmid encoding GFP-BRD4. Cells were treated with compound 16 (1 μM) or a BRD4 ligand JQ1 (0.1 μM). (F) Fluorescent recovery curves after photobleaching (normalized to the intensity before bleaching). (G) t_1/2 in the FRAP assay of F (n ≥ 7 cells per group, error bars: standard error of the mean, Mann–Whitney test; ***, P < 0.001; n.s., not significant).