Molecular Modeling Studies On Interaction Mechanisms Of Protein Kinase CK2 Inhibitors And Its Structure-based Design

Protein kinase CK2, a serine/threonine kinase, is typically observed in a holoenzyme form that composed of the catalytic α and regulatory β subunits. CK2 over expression is particularly enhanced in various human diseases, such as neurodegenerative diseases, inflammatory conditions and tumors. Hence this kinase has taken into consideration as an attractive target for the development of inhibitors with pharmacological potential. CK2 inhibitors can be schematically categorized into two distinct groups: ATP-competitive inhibitors and CK2α/CK2β interaction inhibitors. ATP-competitive inhibitors are aiming at the CK2 binding pocket which is constructed by hydrophobic region, positive area and hinge region. It locates at the active site primarily through hydrophobic and polar interactions. The CK2β-derived cyclic peptide Pc has been demonstrated to efficiently antagonize the CK2α/CK2β interaction and strongly affect the specificity of the phosphorylation of CK2β-dependent CK2 substrates. The dissertation focuses on elucidating the mechanism of action and inhibitor specificity of CK2 inhibitors, as well as structure-based rational design and biological evaluation for lead compound.The tricyclic quinoline compound has been used in the Phase II clinical trial for advanced solid tumors. This class of compound has favorable pharmacokinetic property and hits the CK2 target without exhibiting toxicity. The 3Q-QSAR and molecular docking were applied to build QSAR models for novel tricyclic quinolone analogs. The detailed interactions of the representative compound with CK2α were indicated by molecular dynamic simulation. The results suggested that the simultaneously existed two polar interactions between the pyridine group and the hinge region as well as the carboxylate substituent and positive regions are essential for guarantee the high potency of this class of compounds. Hydrogen bond donor and Electron-withdrawing group at the substituent of the compound were preferred to improve CK2α inhibitory activity by enhancing the interaction with the positive and hinge region. All these results could provide the rational clues for the tricyclic quinolone analogs with high inhibitory activities.Further molecular docking, molecular dynamics simulations, and energy analysis were adopted to elucidate the structural basic for low-affinity binding of tricyclic quinolone analogs with non-original carboxylate function and pyridine group. Results showed that the changes of carboxylate substituent induced the unreasonable and weakened polar interaction between compound and positive area. Meanwhile, the disappeared hydrogen bond between hinge region and the inhibitor was attributed to the variation of the pyridine group. The changes of two key substituents had a negative effect on the binding mode of compounds, namely the inhibitory activities of tricyclic quinolone analogs would be weakened. Aside from the improper recognition of compounds with CK2α, the structure of CK2α in these systems also undergoes conspicuous conformational changes, and the affected positions were distributed over the G-loop, C-loop, and β4/β5 loop regions. All of these results provided valuable information for further structural modification to develop the highly potent CK2-ATP competitive inhibitors.The ATP-competitive inhibitor, 7-hydroxycoumarin derivative, has been identified as an important anti-cancer lead compounds due to potential antiproliferative and antioxidant activities. Structure-based rational design of the coumarin derivatives was carried out by improving the interaction with the hinge region to enhance the inhibitory activity. As proof of concept, trifluoromethyl derivatives of coumarin were synthesized. The results indicated that the most active compound, 8-chlorin-7-hydroxy-4-trifluoromethylcoumarin(compound 3), exhibits potent inhibitory activity against CK2(IC50=0.4μM) and human lung(A549) cancer cell line(IC50=20.15μM). The biological activities of compound 3 exhibits significant higher potency than the compound 6(8-chlorin-7-hydroxy-4-methylcoumarin)(CK2: IC50=2.2μM and A549: IC50=29.26μM). All these results could provide the rational clues for the novel CK2 agonists design with higher inhibitory activities and better pharmaceutical properties.An alternative strategy that targets the CK2α and CK2β interface was introduced to develop CK2 inhibitor. The CK2β- derived cyclic peptide Pc has been demonstrated to efficiently antagonize the CK2α/CK2β interaction(IC50=3.0 μM) and strongly affect the phosphorylation of CK2β-dependent CK2 substrate specificity. However, the binding affinity of Pc to CK2α is destroyed to different extents by two single-point mutations Y188 A and F190 A. Molecular dynamics(MD) simulations, principal component analysis(PCA), domain cross-correlation map(DCCM) analysis and free energy calculations of molecular mechanics/generalized born-surface area(MM/GBSA) binding were performed to explore the structural mechanism of how the CK2β-competitive Pc disrupting the CK2α/CK2β interaction. The results revealed that the ordered communications between hydrophobic and polar interactions were essential for CK2α–Pc binding in the WT system. Although both mutations resulted in the improper recognition between Pc and CK2α, the detailed structural contributions on the CK2α–Pc complex were different. For the Y188 A mutant, the loss of the H-bond between CK2α and Pc and slightly reduced hydrophobic interactions were responsible for the lower potential of Y188A-mutated Pc. By contrast, the distorted conformation of Pc and disrupted hydrophobic interfaces could explain the unbinding of F190A-mutated Pc to CK2α. These results are fundamental to the development of highly potent CK2 non-ATP-competitive inhibitors.To sum up, this study not only revealed the mechanism of action and inhibitor specificity of CK2 inhibitors, but also carried out the structure-based rational design and biological evaluation for lead compound. These results could provide the rational suggestions for the structure-based design of CK2 inhibitors.