Objective: To provide a kinetic model(s) and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 (hPlkl) Polo-box domain (PBD). Methods: The bindi...Objective: To provide a kinetic model(s) and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 (hPlkl) Polo-box domain (PBD). Methods: The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results: Thymoquinone follows a one-step binding with an association rate constant (k1) of 6.635× 10^3 L.mol^-1 min^-1.Poloxin fit a two-step binding with a dissociation constant (Ki) of 118 μmol/L for the intermediate complex and its isomerization rate (k4) of 0.131 5 minJ to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPIkl-PBD competitors. Conclusion: Thymoquinone and Poloxin are slow-tight ligands to the hPlkl-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.展开更多
基金a co-sponsored graduate research project by China Pharmaceutical University and Shanghai Medicilon Inc
文摘Objective: To provide a kinetic model(s) and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 (hPlkl) Polo-box domain (PBD). Methods: The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results: Thymoquinone follows a one-step binding with an association rate constant (k1) of 6.635× 10^3 L.mol^-1 min^-1.Poloxin fit a two-step binding with a dissociation constant (Ki) of 118 μmol/L for the intermediate complex and its isomerization rate (k4) of 0.131 5 minJ to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPIkl-PBD competitors. Conclusion: Thymoquinone and Poloxin are slow-tight ligands to the hPlkl-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.