Finally, our co-structure revealed that 18 did not induce the expected folded P-loop conformation in VRK2-KD (Supplementary Figure ). The P-loop is a conserved (GxGxF/YG motif) but flexible region of the kinase domain. Indeed, in VRK2-KD co-crystal structure, poor electron density maps for this region prevented us building P-loop residues Gly38 and Gly39 into the final protein model. By contrast, the P-loop of VRK1 bound to 5 was found to be folded over the ligand, regardless of the ligand binding pose. The folded P-loop conformation is often stabilized by the interaction of the aromatic residue within this motif (Phe48 in VRK1) and appropriate groups from the ligand. In VRK1-KD, P-loop residue Phe48 is accommodated by the ligand three ring system. Such interaction cannot be facilitated by the equivalent P-loop residue in VRK2-KD (Phe40). A structural alignment between the two VRK structures revealed that the position of the two conserved aromatic residues in the protein P-loop are substantially different, with Phe40 in VRK2 much closer to the protein αC than its structural equivalent in VRK1 (Supplementary Figure ).