HADDOCK Docking Calculations

Docking calculations were performed using HADDOCK2.4.^30,31 FOXO3-DBD and FOXO4-DBD coordinates were taken as the lowest-energy structure of the PDB entries 2K86 and 1E17. The first two N-terminal amino acid residues of FOXO3-DBD from the thrombin cleavage site were removed from the structure. For all docking runs, active residues of FOXO3-DBD were chosen as residues with CSPs higher than the mean + 2σ_corr⁰. For FOXO3-DBD, residues 151–160 (N-terminus) and 241–251 (C-terminus) were set as flexible. For FOXO4-DBD, residues 96–105 (N-terminus) and 181–185 (C-terminus) were set as flexible. The ligand was set as active and flexible in both cases. Unmentioned running parameters were left on their default values for protein–ligand mode. The resulting structures were clustered by RMSD with a 1.5 Å cutoff and ranked based on their HADDOCK scores. The top-scoring cluster satisfactorily explained the NMR data of all compounds but 5ci. Its data were satisfactorily explained by combining the top and second clusters, as described in the Results section.