Protein　kinase　CK2　is　a　novel　potential　target　for　cancer　treatment.　The　tricyclic　quinoline　compound　CX-4945　(R2=COOH)　is　the　first　bioavailable　CK2　inhibitor　used　in　human　clinical　trials　for　advanced　solid　tumors.　CX-4945　analogs　with　non-R2　carboxylate　function　were　demonstrated　to　be　approximately　5000-fold　less　potent　than　compound　12　(R2=COOH)　in　vitro.　Molecular　docking　and　molecular　dynamics　simulations　were　employed　to　elucidate　the　structural　mechanisms　through　which　the　R2　non-ionizable　and　R3　carboxylic　acid　substituents　influence　binding　affinity.　Results　show　that　the　structure　of　CK2　and　the　orientation　of　ligands　changed　to　different　degrees　in　non-R2　carboxylate　function　systems.　The　inappropriate　electrostatic　interactions　between　the　non-R2　carboxylate　group　and　the　positive　region　lead　to　improper　protein-ligand　recognition,　which　is　followed　by　the　reorientation　of　tricyclic　skeletons.　For　CK2,　the　affected　positions　are　distributed　over　the　glycine-rich　loop　(G-loop),　C-loop,　and　the　4/5　loop.　The　allosteric　mechanisms　between　the　deviated　ligands　and　the　changed　regions　are　proposed.　Detailed　energy　calculation　and　residue-based　energy　decomposition　indicate　the　energetic　influences　on　the　contributions　of　the　critical　residues.　These　results　are　in　accordance　with　one　another　and　could　provide　rational　clues　to　the　design　of　more　potent　CK2　inhibitors.