Alignment of BET bromodomain mutants.

<p>(A) Secondary structure elements are shown at the top of the sequence alignment. Mutated residues are highlighted in red and studied mutations are listed. The conserved asparagine (N391in BRD3(2) numbering) is highlighted in yellow. The green dots represent the residues involved in binding with inhibitor JQ1 (PDB ID: 3ONI, 3S92, 3MXF). The residues underlined in blue are involved in PFI-1binding (PDB ID: 4E96). (B) Location of the mutations. Shown are the first (left) and second bromodomain of BRD2. The mutated residues are highlighted in ball and stick and the position of Cα atoms are shown as a sphere. The main structural elements are labelled.</p

Far-UV CD spectra of wild type bromodomains and mutants.

<p>Far-UV CD spectra were recorded at 20°C in a 0.1-cm quartz cuvette in 20 mM Tris/HCl, pH 7.5 containing 0.20 M NaCl and 0.40 mM DTT, as described in Materials and Methods. Wild type spectra are shown as black solid lines and mutants are coloured as indicated in the figure.</p

Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of BRDs wild type and mutants measured by far-UV CD and fluorescence spectroscopy.

<p>Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of BRDs wild type and mutants measured by far-UV CD and fluorescence spectroscopy.</p

Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors

CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation of gene transcription. Both proteins contain a bromodomain flanking the HAT catalytic domain that is important for the targeting of CBP/P300 to chromatin and which offeres an opportunity for the development of protein–protein interaction inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an <i>N</i>-acetyl-lysine mimetic scaffold that led to the recent development of the chemical probe I-CBP112. We present comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective inhibitor TPOP146, which showed 134 nM affinity for CBP with excellent selectivity over other bromodomains

Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors

CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation of gene transcription. Both proteins contain a bromodomain flanking the HAT catalytic domain that is important for the targeting of CBP/P300 to chromatin and which offeres an opportunity for the development of protein–protein interaction inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an <i>N</i>-acetyl-lysine mimetic scaffold that led to the recent development of the chemical probe I-CBP112. We present comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective inhibitor TPOP146, which showed 134 nM affinity for CBP with excellent selectivity over other bromodomains

Structure of BET mutants and tertiary structure of mutants in solution.

<p>(A) Superimposition of wild type BRD2(1) shown as a ribbon diagram with the mutants BRD2(1) R100L and D161Y shown as protein worm in green and magenta, respectively. The mutated residues are shown in ball and stick representation and main structural elements are labelled. (B) Details of interactions formed by R100 in the wild type compared to the mutated residue. (C) Detailed view of BRD2(1) wild type and D161Y. (D) Superimposition of BRD2(2) shown as a ribbon diagram and the mutant BRD2(2) Q443H shown as protein worm in blue. (E) Comparison of the near UV CD spectra of wild type BRD2(1) and all generated mutants. (F) Comparison of the near UV CD spectra of wild type BRD4(1) and the mutants BRD4(1) A89V. (G) Comparison of the near UV CD spectra of wild type BRD2(2) and the mutants BRD2(2) R419W and Q443H. (H) Comparison of the near UV CD spectra of wild type BRD3(2) and the mutants BRD3(2) H395R. (I) Comparison of the near UV CD spectra of wild type BRD4(2) and the mutants BRD4(2) A420D. Near-UV CD spectra were recorded at 20°C in a 1.0-cm quartz cuvette in 20 mM Tris/HCl, pH 7.5 containing 0.20 M NaCl and 2.00 mM DTT, as described in Materials and Methods.</p

Structure of holo-HmuY.

<p>(A) The protein mimics a right hand, whose thumb and fingers trap the heme co-factor (surface model in orange with an inserted green sphere for the iron ion). (B) Topology scheme of HmuY, which consists of 15 β-strands, each labeled with the protein residues it spans. The heme group is shown as in (A). (C) Richardson plot of holo-HmuY Glu35-Lys216 with the bound heme as an orange stick model and an inserted sphere for the iron. The view was chosen to match (A). (D) Close-up view in stereo of (C) after a horizontal 90° rotation. Protein residue side chains engaged in shaping the heme-binding cavity and in interactions with the co-factor are shown and labeled. The four pyrrole rings of the protoporphyrin IX moiety are also labeled (a–d). (E) Tetrameric quaternary arrangement of holo-HmuY. Each constituting heme/HmuY complex is shown in one color. (F) Richardson plot of rabbit serum hemopexin (PDB access code 1QHU; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000419#ppat.1000419-Paoli1" target="_blank">[32]</a>). The two hemopexin-like β-propeller domains contain central channels to bind ions (colored spheres), and the heme-binding site is at the domain intersection.</p

Protein∶co-factor and protein tetramerization contacts.

a<p>Atom CBB is the terminal vinyl methylene carbon of pyrrole ring b, and atom CMA is the methyl carbon of pyrrole ring a (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000419#ppat-1000419-g004" target="_blank">Figure 4D</a> for pyrrole naming within the heme protoporphyrin ring). Atoms O1A, O2A, and O1D are carboxylate oxygens of the propionate substituents of rings a and d, respectively.</p>b<p>All the interactions between molecule 1 and molecule 2 are symmetric. Distances 1-2 and 2-1 are given separated by a slash.</p>c<p>All the interactions between molecule 1 and molecule 3 are symmetric and made by identical atoms.</p

Proposed working mechanism for the extracellular hmu components.

<p>HmuY and HmuR are shown with their proposed roles in heme uptake, as well as the other suggested players. Hb stands for hemoglobin; Hp for hemopexin; Ab for serum albumin; Rgp for gingipains R types A and B; Kgp for gingipain K; Lp for lipid; and Hm for heme (represented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000419#ppat-1000419-g004" target="_blank">Figure 4A</a>).</p

Crystallographic data.

a<p>Friedel mates were treated as independent reflections in the derivative dataset.</p>b<p>Values in parentheses refer to the outermost resolution shell.</p>c<p> and .</p>d<p>Mean figure or merit computed for data to 1.8 Å before and after density modification with program DM within CCP4.</p>e<p>Crystallographic R_factor = Σ_hkl ||F_obs| − k |F_calc||/Σ_hkl |F_obs|; free R_factor, same for a test set of reflections not used during refinement.</p>f<p>Including atoms in alternate conformation.</p