For VRK2-KD, replacing the 2-amino with a methyl group (26) greatly reduced the observed ΔTm, suggesting that the HB interaction with the hinge carbonyl group is actually productive for VRK2. However, for VRK1-FL, 26 and 5 were equipotent, supporting the hypothesis that the 2-amino moiety contributes little to the binding of 5 to VRK1. This can be further rationalized by the comparison between compounds RS-117 and RS-119 against VRK1 and VRK2. The pair is equipotent against VRK1, as both derivatives would be able to accommodate the difluorophenol group in the back of the binding site by flipping the pose. For VRK2, RS-117 is more potent than RS-119 as only the former would be able to keep the HB interactions with the Glu122 hinge carbonyl and Lys61 catalytic Lys residue, similar to the ones displayed in Figure . VRK1-FL also seemed to be more tolerant to the removal of the 2-amino group than VRK2-KD (25). Those differences between VRK1 and VRK2 were unexpected as both proteins display highly similar ATP-binding sites. Thus, both our DSF results and crystal structures suggest that VRK1, but not VRK2, is capable of binding equally well to the 2-amino pyridine core in two different conformations. The increase in ΔTm observed for compound 26 (2methyl pyridine core) over 25 (pyridine core) might arise from favorable electron-donating properties or conformational effects conferred to the pyridine ring system by the presence of an adjacent methyl group. Alternatively, the lack of a substituent group in the 2-position may incur a free energy penalty associated with 25 inability to fill in a cavity in the protein binding pocket. The 2-amino pyridine moiety is a common feature of many kinase inhibitors, and often plays a role in binding to the kinase hinge region (Supplementary Figure ). We expect that adding a methyl group in the 2-position of the pyridine core will offer an opportunity to increase the selectivity of future VRK1 inhibitors.