Fig. 2.

Crystal structure of human RalA in covalent complex with 1. (A) Stereo image of the composite omit electron density map (blue mesh) of compound 1 (sticks with yellow carbon, red oxygen, blue nitrogen, and orange sulfur) covalently bound to RalA Tyr-82 (sticks with green carbon) (PDB ID code: 6P0I). (B) Stereo image of the binding pocket of 1 shown in solvent-accessible surface representation, colored by hydrophobicity (darker brown indicates more hydrophobicity). (C) Same as in B, zoomed out to show location of the pocket with respect to GDP. (D) Stereo image of RalA-1 binding pocket (carbons and loop in green) superimposed with apo RalA (carbons and loop in pink) where the switch II loop of apo RalA is in a conformation that has the pocket more open (PDB ID codes: 6P0I and 6P0O). (E) Stereo image of RalA-1 binding pocket (carbons and loop in green) superimposed with apo RalA (carbons and loop in pink) where the switch II loop of apo RalA is in a conformation that has the pocket closed (PDB ID codes: 6P0I and 6P0J). (F) Druggability scores and volume of representative RalA structures at the binding pocket of 1. Pocket scores and volumes were calculated by Sitemap (49). In comparison, the scores of a FDA-approved kinase inhibitor Abemaciclib and CPI-0610, a compound targeting the bromodomain BRD4 in clinical trials, are shown. (G) Stereo image of RalA–1 complex, highlighting the binding interactions between 1 (sticks with yellow carbons, blue nitrogens, red oxygens, orange sulfurs) and the RalA pocket (stick with green carbons). Hydrogen bonds are displayed in dashes and labeled with distance. (H) Two-dimensional ligand interaction map of covalently bound compound 1 in the pocket at the switch II loop of RalA generated using Maestro. (I) Percent inhibition of Rgl2-mediated guanine nucleotide exchange of RalB, RalB Tyr82Phe mutant, RalB Ser85Ala mutant, Thr69Ala mutant, and RalA by 100 µM 1 after 24-h incubation at 4 °C (mean ± SD, n = 2).