The co-structures helped us rationalize the relevance of the difluorophenol moiety for binding. Regardless of the compound binding pose, this group facilitated an HB network with polar side chains from a conserved and catalytic-important lysine (Lys71 in VRK1 or Lys61 in VRK2). Interestingly, the difluorophenol group participating in these contacts displayed distinct dihedral angles to the 2-amino core depending on its attachment position: ~45° in R1 and ~9° in R2. In VRK1-KD, these different orientations of the difluorophenol group are accommodated by a corresponding movement of the side chain from residue Met131, which occupies the gatekeeper position in this protein. Consequently, the difluorophenol group fits tightly between the αC and the gatekeeper residue in both poses. These observations might explain why we could not find substituents that improved binding over the difluorophenol group, showing its pharmacophoric feature.