To confirm binding and estimate compound potency, we employed Isothermal Titration Calorimetry (ITC). Our ITC results indicated that the top compounds by DSF (26 for VRK1-FL and 18, 19 and 13 for VRK2-KD) had KD values in the sub-micromolar range. The most potent compound for VRK1-FL, 26, had a KD of 190 nM, whereas for VRK2-KD, 18 had a KD of 401 nM (Figure ). For both proteins, compound binding was mostly enthalpy-driven (|ΔH| > |TΔS|), although binding of 18 to VRK2-KD did have a more pronounced entropic contribution than the one observed for compounds 19 and 13 (Supplementary Table ). At this point, we do not have an explanation for the observed discrepancy between DSF and ITC results for compound 19.