Optimization And Anticancer Activity Evaluation Study Of Novel Cyclic Peptide Inhibitors Based On Protein Kinase CK2 Subunit Interaction

Protein kinase CK2 has been regarded as a valuable target for cancer therapy since this kinase is involvedin the regulation of numerous significant cellular processes including cell proliferation,differentiation and apoptosis.And thus and its inhibitor has become an important source of anticancer lead compounds.However,most of the ATPcompetitive inhibitors failed to become drug candidates due to the defects such as poor selectivity and low druggability.Therefore,the development of anti-cancer CK2 inhibitors targeting non-ATP binding sites has the academic significance and potential application prospects.The CK2 holoenzyme is a tetramer structure consisting of a catalytic subunit CK2α and a regulatory subunit CK2β.It has been demonstrated that the CK2β-derived cyclic peptides exhibited the inhibitory effects on CK2 activityby disturbingthe interaction of the CK2α/CK2β subunit and antagonizing the formation of CK2 holoenzyme.With the computer-aided drug design methods and biological experimental techniques,this thesis focuses on the structure-based design,optimization and biological activity evaluation of novel cyclic peptide inhibitors.1.Structure-based design of cyclic peptide inhibitors of protein kinase CK2 subunit interactionCK2β-derived Pc(cyclic peptide,Pc)has been demonstrated to block the formation of CK2 holoenzyme structure and inhibits its biological function by effectivelyantagonizingthe interaction of α subunit and β subunit.In order to design novel potent peptides inhibitors;the interaction mode of Pc with CK2α and the binding energy calculation result of CK2α-peptide systems were explored.The results showed that hot-spot residues Arg186,Tyr188 and Phe190 were identified as key residues due to their major favorable contribution to the binding affinity,and thus were retained in the novel cyclic peptide design.As for Leu187,Ile192 and His193 residues with the minor contribution,we proposed to increase or extend their side chain to improve the hydrophobic interaction with CK2α.By combining binding energy calculation results and binding modes between CK2α and peptides,five peptides L187V(Leu was mutated to Val),I192F(Ile was mutated to Phe),I192W(Ile was mutated to Trp),H193F(His was mutated to Phe)and H193W(His was mutated to Trp)were selected to synthesize in the following work.2.Synthesis and binding affinity evaluation of cyclic peptide inhibitorsThe cyclic peptide inhibitors of five mutants and wild type Pc were synthesized by manual Fmoc-based solid phase peptide synthesis.The liner peptide chain was assembled according to the sequence of cyclic peptide by themethod of adding Fmocprotected amino acids to Rink resin one by one.After cleavage of the liner peptide from the resin,the peptide was cyclized to form a disulfide bond between two cysteine moieties.And then the purified cyclic peptide was characterized by HPLC and MS.The SPR(Surface plasmon resonance)method was used to detect the binding affinity between cyclic peptide and CK2α.The CM5 sensor chip was selected for detecting protein-peptide interaction.After the running conditions were optimized by preconcentration,CK2α was immobilized on a sensor chip to detect interaction of cyclic peptides and CK2α.The affinity of the peptide to CK2α showed that the affinity of the cyclic peptide inhibitor to CK2α was from strong to weak: I192 F,L187V,Pc,H193 F,I192W,H193 W,I192F,the equilibrium dissociation constant KD was 0.5 μM,in the SPR experiment.It shows the strongest affinity.3.Anticancer activity evaluation of cyclic peptide inhibitorsIn order to further explore the anticancer activity of the synthesized cyclic peptide,CCK8 method,phosphatidylserine valgus analysis method(Annexin-V)and cell scratch methodwere employed to evaluate the anti-proliferation,pro-apoptosis inhibiting cell migration against lung cancer cell A549 and hepatoma cell Hep G2.The results showed that H193 W,H193F and I192 F showed the more potent antiproliferative activity against A549 and Hep G2 than Pc.And H193 W and I192 F also exhibited pro-apoptotic activity against the two cancer cells.In addition,I192 F inhibitedcell migration of A549 and Hep G2.Therefore,I192 F was expected to be the anti-cancer drug lead due to the potentialanti-proliferative,pro-apoptotic and cellinhibiting activity against A549 and HepG2 cells.In summary,strucutre-based novel cyclic peptide inhibitors targeting the CK2 subunit interface were designed,synthesized and evaluated their binding affinity with CK2α and anti-cancer activity.These results provided theoretical basis and experimental guidance for the development of anti-cancer peptide drugs.