Molecular Basis of Inactive B-RAF^WT and B-RAF^V600E Ligand Inhibition, Selectivity and Conformational Stability: An in Silico Study

Abstract

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 phosphorylation specificity of B-RAFWT, B-RAFD594V, B-RAFV600E and B-RAFK601E kinases: An in silico study

“…The corresponding value for the interactions between the Lys601 and Glu501 residues was À93.8 AE 3.5 kcal/mol, comprising about 47% of the total A-loop-protein electrostatic interactions [16]. Similar results were obtained from previous analysis of a 10 ns MD simulation [15]. The strong interactions between Lys601 and the catalytic and ATP the coordinating residues Asp594, Glu501 and Lys483 suggest the important role of this lysine in the B-RAF WT function and stability, which can explain the observed strong kinase activation in the presence of the K601E mutation.…”

“…The structures were refined by 15 ns molecular dynamics simulations, and the phosphorylation specificity of each kinase was analysed with the GRID method. Previous studies of the effect of the different mutations on especially the electrostatic interactions and on the formation of networks of hydrogen bonds within the binding cavity were used to provide additional support to our results [15,16].…”

“…As described in detail in two recent publications, the two interlinked water-mediated hydrogen bond networks (HBNs) identified in B-RAF WT , remained stable throughout the MD simulation [15,16]. One of these HBNs (HBN1) was formed between Lys483, Glu501, Asp594, Gly596, Lys601, Ser607 and water molecules, and Asp594 and Lys601 were also a part of a second water-mediated HBN (HBN2) with Lys578, Asn581, Asp576 and Thr599.…”

“…Before carrying out the MD simulations and the GRID analysis, the structures of B-RAF WT , B-RAF V600E , B-RAF K601E and B-RAF D594V were prepared as previously described [15]. For the two B-RAF forms, B-RAF WT and B-RAF V600E , for which crystal structures were available in the protein data bank [17] (pdb codes: 1uwh and 1uwj), the structures were prepared by removing the inhibitor Sorafenib and keeping the crystal waters.…”

“…The full structural models of these four B-RAF forms were based on the available crystal structures, supplemented by models for the missing A-loop portions [15,16]. The structures were refined by 15 ns molecular dynamics simulations, and the phosphorylation specificity of each kinase was analysed with the GRID method.…”