2.1. Molecular docking

The Glide module of Schrödinger (Release 2020-3, Schrödinger LLC, New York, NY, https://www.schrodinger.com) was used to dock both the series of pteridinone-, pyrimidine-, purine-, and pyrrolopyrimidine-based compounds (Fig. 1), including the known pan-RSK inhibitors BI-D1870, LJH685, and KJI308, into the ATP-binding site of the N-terminal kinase domain (NTKD) of the RSK2 (PDB: 4NUS) crystal structure. Prior to docking, the water molecules were removed, and the protein was prepared by assigning bond orders, adding hydrogens, and repairing any side chains or missing amino acid sequences. Disulfide bonds were not created, but all other settings in the Protein Preparation Wizard were default. To complete protein preparation a restrained minimization of the protein structure was performed using the default constraint of 0.30Å RMSD and the OPLS_2005 force field [8]. The prepared proteins were subjected to SiteMap analysis [9], to identify top-ranked potential binding sites. Default features of SiteMap were used, requiring at least 15 site points per reported site, reporting up to 5 sites, and using a more restrictive hydrophobicity. Recognized sites from the analysis were cropped at 4Å from the nearest site point. SiteMap analysis identified the ATP-binding site in the NTKD. Docking grids were generated using Receptor Grid Generation by picking a point in the ATP-binding site generated from SiteMap analysis. All other settings for the Receptor Grid Generation were default, with no constraints, rotatable groups, or excluded volumes defined. BI-D1870 and analogs were prepared using LigPrep by generating possible states at the target pH 7.0 ± 2.0 using Epik. Ligands were desalted and tautomers were generated. Specified chiralities of stereoisomers were retained and, at most, 32 stereoisomers were generated per ligand. Finally, ligands were minimized by applying the OPLS_2005 force field [8]. Molecular docking simulations were performed using the Glide ligand docking module in XP (extra precision) mode with default settings and features, and included post-docking minimization [10].