The B-RAF kinase plays an important role both in tumor induction and maintenance in several cancers. The molecular basis of the inactive B-RAF(WT) and B-RAF(V600E) inhibition and selectivity of a series of inhibitors was examined with a combination of molecular dynamics (MD), free energy MM-PBSA and local-binding energy (LBE) approaches. The conformational stability of the unbounded kinases and in particular the processes of the B-RAF (V600E) mutant activation were analyzed. A unique salt bridge network formed mainly by the catalytic residues was identified in the unbounded B-RAFs. The reorganization of this network and the restriction of the active segment flexibility upon ligand binding inhibit both B-RAF(WT) and B-RAF (V600E), thus appearing as an important factor for ligand selectivity. A significant correlation between the binding energies of the compounds in B-RAF(WT) and their inhibition effects on B-RAF (V600E) was revealed, which can explain the low mutant selectivity observed for numerous inhibitors. Our results suggest that the interactions between the activation segment and the alpha C-helix, as well as between the residues in the salt bridge network, are the major mechanism of the B-RAF (V600E) activation. Overall data revealed the important role of Lys601 for ligand activity, selectivity and protein stabilization, proposing an explanation of the observed strong kinase activation in the K601E mutated form.

The structural and dynamical properties of the peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor have been broadly studied in its agonist state but little is known about the key features required for the receptor antagonistic activity. Here we report a series of molecular dynamics (MD) simulations in combination with free energy estimation of the recently discovered class of non-covalent PPARγ antagonists. Their binding modes and dynamical behavior are described in details. Two key interactions have been detected within the cavity between helices H3, H11 and the activation helix H12, as well as with H12. The strength of the ligand-amino acid residues interactions has been analyzed in relation to the specificity of the ligand dynamical and antagonistic features. According to our results, the PPARγ activation helix does not undergo dramatic conformational changes, as seen in other nuclear receptors, but rather perturbations that occur through a significant ligand-induced reshaping of the ligand-receptor and the receptor-coactivator binding pockets. The H12 residue Tyr473 and the charge clamp residue Glu471 play a central role for the receptor transformations. Our results also demonstrate that MD can be a helpful tool for the compound phenotype characterization (full agonists, partial agonists or antagonists) when insufficient experimental data are available.

Cypher/ZASP (LDB3 gene) is known to interact with a network of proteins. It binds to α-actinin and the calcium voltage channels (LTCC) via its PDZ domain. Here we report the identification of a highly conserved ZASP G54S mutation classified as a variant of unknown significance in a sample of an adult with hypertrophic cardiomyopathy (HCM). The initial bioinformatics calculations strongly evaluated G54S as damaging. Furthermore, we employed accelerated and classical molecular dynamics and free energy calculations to study the structural impact of this mutation on the ZASP apo form and to address the question of whether it can be linked to HCM. Seventeen independent MD runs and simulations of 2.5 μs total were performed and showed that G54S perturbs the α2 helix position via destabilization of the adjacent loop linked to the β5 sheet. This also leads to the formation of a strong H-bond between peptide target residues Leu17 and Gln66, thus restricting both the α-actinin2 and LTCC C-terminal peptides to access their natural binding site and reducing in this way their binding capacity. On the basis of these observations and the adult's clinical data, we propose that ZASP(G54S) and presumably other ZASP PDZ domain mutations can cause HCM. To the best of our knowledge, this is the first reported ZASP PDZ domain mutation that might be linked to HCM. The integrated workflow used in this study can be applied for the identification and description of other mutations that might be related to particular diseases.

3D-QSAR, Docking, Local Binding Energy (LBE) and GRID methods were integrated as a tool for predicting toxicity and studying mechanisms of action. The method was tested on a set of 73 allelochemical-like pesticides, for which acute toxicity (LD(50)) for the rat was available. 3D-QSAR gave a model with high predictive ability and the regression maps indicated the important toxic chemical substituents. Significant ligand-protein residue interactions and oxidation positions in the binding site were found by docking analysis using CYP1A2 homology modelling. The binding energies of the compounds and the important substituents (Local Binding Energy, LBE) were calculated in order to demonstrate quantitatively the substituent contributions in the metabolism and toxicity. The GRID examination identified the CYP1A2 binding pocket feature. Finally, a 3D-QSAR map was compared to the GRID map, showing good overlaps and confirming the important role of CYP1A2 in allelochemical-like compounds toxicity.