To test our hybridization hypothesis, we prepared prototype compound 5, bearing difluorophenol moieties in both 3-and 5positions of the 2-aminopyridne core (Figure ). We employed a thermal-shift assay (Differential Scanning Fluorimetry, DSF) to identify aminopyridine analogues that could bind to the full-length VRK1 (residues Met1-Lys396, VRK1-FL) and the kinase domain of VRK2 (residues Pro14-His335, VRK2-KD). DSF is a robust method to estimate binding affinities and has been used successfully to triage large compound libraries. For these experiments, we considered positive hits those compounds inducing changes in VRK1-FL and VRK2-KD melting temperatures, ΔTm, larger than 2.0 °C. DSF analysis indicated that 5 induced thermal shifts above the hit threshold of 2 °C for both VRK1 and VRK2 (Figure ).