Protein kinase CK2 is involved in a variety of important cellular physiological processes and aberrant CK2 activity is associated with a wide variety of human diseases [1] 5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945) is the first orally bioavailable and highly selective small molecule inhibitor of CK2α [2].Compound 13 with R3 carboxylate function were demonstrated to be almost 5000-fold less potent than compound 6m (R2=COOH) in vitro [3].Molecular docking [4] and molecular dynamics simulations [s] were employed to elucidate the structural mechanisms through which the R3 carboxylic acid substituent influence binding affinity.The results showed that the structure of CK2α and the orientation of ligand changed to different degrees in CK2α-13 complex.The inappropriate electrostatic interactions between the R3-carboxylate group and the positive region led to improper protein-ligand recognition, which was followed by the reorientation of tricyclic skeletons.For CK2α, the affected positions were distributed over the G-loop, C-loop, and β4/β5 loop.An allosteric pathway between the deviated compound 13 and the affected positions was proposed.Furthermore, the energy analysis [6] also indicates the instability of CK2α-13complex in contrast to CK2α-6m complex.