MM/PBSA binding free energies (kJ/mol) for PHD finger/H3K4me0 complexes.

1<p>experimental binding free energy.</p>2<p>difference between AIRE-PHD1 and PHD finger complex experimental binding free energies.</p>3<p>computational binding free energies.</p>4<p>difference between the computational binding free energy of AIRE-PHD1/H3K4me0 and other PHD/H3K4me0 complex.</p>5<p>coulombic term.</p>6<p>polar solvation term.</p>7<p>polar term (sum of coulombic and polar solvation terms).</p>8<p>van der Waals term.</p>9<p>non-polar solvations term.</p>10<p>non-polar term (sum of van der Waals and non polar solvation terms).</p><p>Standard errors are given in parentheses.</p

Conformational analysis of free and bound AIRE-PHD1.

<p>Cα RMSD from (A) free and (B) bound starting AIRE-PHD1 structure, as a function of time. (C) RMSF of Cα atoms from their time-averaged positions for free (grey) and bound (black) AIRE-PHD1. Secondary structure assignment for free (D) and bound (E) AIRE-PHD1 as defined by <i>do_dssp </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046902#pone.0046902-Kabsch1" target="_blank">[54]</a> as a function of time: black, white and grey denote “β-sheet”, “coil”, and other secondary structure elements, respectively. For this analysis the last 8 ns of each of the five MD simulations were concatenated into a single 40-ns trajectory. Binding of H3K4me0 to AIRE-PHD1 induces the extension of the β1 strand up to residue Glu307. Two representative structures of free (grey) and bound (black) AIRE-PHD1 are shown: white spheres denote Zn²⁺ ions.</p

MM/PBSA calculations of PHD fingers recognizing H3K4me0.

<p>(A) Correlation between the experimental binding free energy (ΔG_exp) and the calculated binding free energy (ΔΔG_comp) of H3K4me0-binding PHD fingers. (B) Representation of the energetic contributions (coulombic and van der Waals energies) associated with peptide-domain intermolecular contacts. For clarity, normalized interaction energies are mapped only on the PHD finger surface in a range from white (no contribution) to green (high contribution). H3K4me0 is represented as orange ribbon. (C) Structural alignment of the H3K4me0-binding PHD fingers generated by MultiSeq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046902#pone.0046902-Roberts1" target="_blank">[55]</a>. Residues interacting with H3R8 and H3K9 are highlighted in red and cyan, respectively; residues interacting with both H3R8 and H3K9 are highlighted in grey. (D) Representation of intermolecular interactions between H3R8 and H3K9 (shown in sticks) and PHD finger residues; dashed lines indicate polar contacts.</p

MM/PBSA calculations of native and mutant AIRE-PHD1/H3K4me0 complexes.

<p>Plot of the experimental binding free energy differences (ΔΔG_exp) versus the calculated binding free energy differences (ΔΔG_comp) of AIRE-PHD1/H3K4me0 alanine mutants.</p

Correlated motions and PCA of free and bound AIRE-PHD1.

<p>Residue based (C_α atoms) correlation maps of (A) free and (B) bound AIRE-PHD1. Correlated (positive) and anticorrelated (negative) motions between atom pairs are represented as color gradients of red and blue, respectively. Above the matrix diagonal only the |Corr_ij|>0.5 are reported. Relevant correlations/anticorrelations discussed in the text are highlighted by numbered boxes and (C–E) reported in red/blue on the AIRE-PHD1 structure. PCA analysis of free (F) and bound (G) AIRE-PHD1. Superimposition of 20 filtered configurations obtained by projecting the Cα motion of free and bound AIRE-PHD1 onto the first 6 and 4 eigenvectors, respectively. The first 6 and 4 eigenvectors obtained from the simulation of free and bound AIRE-PHD1, respectively, capture 70% of the cumulative proportion of the total variance. The Cα atoms of Glu307 and Gly333 are shown as spheres, the H3K4me0 is represented in yellow. (H) Distribution of the distances between the Cα atoms of Glu307 and Gly333 along the dynamics of free (cyan) and bound (blue) AIRE-PHD1.</p

Structural Analysis of the Binding of Type I, I_1/2, and II Inhibitors to Eph Tyrosine Kinases

We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I_1/2 (DFG in) and type II (DFG out). The three structures with type I_1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases

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

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