Figure 3. Structural assessment of ROS1^F2004C with molecular dynamic simulation and inhibitor docking studies reveal entrectinib binds both DFG-in and DFG-out ROS1 kinase domain.

A & B, Superimposed wildtype ROS1 (A) and ROS1^F2004C mutant crystal structure models in DFG-in and DFG-out conformations are shown to highlight distinctions in structural features: Activation loop (A-Loop), αC Helix, P-loop, ATP-binding pocket, and the positioning of the aspartic acid (D), phenyalanine (F) and glycine (G), i.e., DFG motif in the two conformations. C & D, Superimposed crystal structures show conformational differences between wildtype ROS1 and ROS1^F2004C in their DFG-in (C) and DFG-out (D) states. E & F, Histograms show binned probability distribution of binding energy (kcal/mol) for ROS1^WT and ROS1^F2004C in the DFG-in (E) and DFG-out (F) kinase conformation. Binding energies were determined with Yasara; in this case the higher binding energies reflect more favorable binding. G, Percentage of docks whose binding energy was greater than the median binding energy of indicated inhibitor docking to ROS1^WT DFG-in and ROS1^WT DFG-out is plotted. The median binding energy ± standard deviation and the total number of docks for the ROS1^WT DFG-in & ROS1^WT DFG-out conformations is indicated inset within graph and panel below graph. H & I, IC_50s of crizotinib, entrectinib, lorlatinib, repotrectinib and cabozantinib as derived from dose-response cell viability assay with Ba/F3 CD74-ROS1 (H) and Ba/F3 EZR-ROS1 (I), wildtype or F2004C mutant cells.