Chemical biology. A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes

Abstract

Small molecules are useful tools for probing the biological function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The Bromo and Extra-Terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-molecule inhibitors, but these inhibitors lack selectivity for individual family members. We developed an ethyl derivative of an existing small-molecule inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiology and disease.

Fig. 1. BET bromodomains, pan-selective inhibitors, and bump-and-hole approach

(A) Domain organization of BET proteins. The name and length of the proteins are shown together with the position of their first and second bromodomains. (B) Chemical structures of BET bromodomain inhibitors I-BET, JQ1, and GW841819X that share a common triazolodiazepine scaffold. (C) Dissociation constants (K_d, in nanomolar) determined by means of ITC are shown for I-BET binding to the eight individual BET bromodomains distributed as subfamily branch in a human bromodomain phylogenetic tree. (D) Rationale for a bump-and-hole approach to engineer selectivity of BET bromodomain inhibitors.

Fig. 2. Identification of L94 as mutational position and bumped ligand design

(A) Sequence alignment of the eight BET bromodomains. Conserved residues (blue) and a conserved asparagine (orange) that directly hydrogen bonds to acetyl-lysine are highlighted, and positions of α-helices are shown. Conserved and nonconserved residues making contacts with I-BET are highlighted with black and green dots, respectively.The targeted leucine residue is highlighted in red.The sequence numbering shown on top of the alignment is for Brd4(1). (B) Stick representation of I-BET (yellow carbons) and binding site residues of Brd4(1) [PDB 3P5O (2)]. L94 is highlighted in red. (C) Chemical structure of a methylester derivative of I-BET and position selected for derivatization to target a hole introduced by mutation at L94. (D) Synthetic route to introduce bumps at position 2 of the methylester side chain. R, alkyl; X, I; KHDMS = potassium bis(trimethylsilyl)amide.

Fig. 3. Bumped ligand ET binds to engineered L/A bromodomains with high selectivity

(A) Thermal stabilization (ΔT_m, in degrees Celsius) of WT Brd2 bromodomains and their L/A mutants by I-BET, ME, and ET measured by means of DSF. The data shown are mean ± SD of three measurements. (B) Dissociation constants (K_d, in nanomolar) determined by means of ITC for I-BET binding to WT and for ME and ET binding to WT and L/A mutant Brd2 bromodomains. The error bars reflect the quality of the fit between the nonlinear least-squares curve and the experimental points. (C and D) Cocrystal structures of Brd2(2)L383A (green, surface representation) in complex with ME [(C) stick, yellow carbons] and ET [(D) stick, pink carbons].The L/A mutation is shown in red, and A383 is shown as stick, green carbons. (E and F) Affinity profile (K_d, in nanomolar) of ET binding to (E) wild-type and (F) L/A mutant BET bromodomains measured by means of ITC.

Fig. 4. ET is highly selective for L/A BET bromodomains in vitro and in cells

(A) ITC titrations of I-BET against WT tandem bromodomain construct of Brd2 (black) and of ET against the wild type (purple) and the same construct containing the L/A mutation in the first only (blue), second only (green), and both first and second bromodomains (red) at 30°C. Stoichiometry n and dissociation constants K_d are given in the inset table. (B) Evaluation of the selectivity of ET in human osteosarcoma cells (U2OS) cells by using FRAP. Quantitative comparison of half-time of fluorescence recovery are shown for cells transfected with full-length human green fluorescent protein (GFP)–Brd4 and treated with dimethyl sulfoxide (white, vehicle control) or 1 μM I-BET (black), and for cells expressing wild type (purple), L/A-WT (blue), WT-L/A (green), or double L/A (red) GFP-Brd4 and treated with 1 μM ET.The data shown represent the mean ± SEM (n = 16 to 21 biological replicates). Statistical significance was determined with one-tailed t tests: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant.