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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(hPlk1) 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 6.635×103 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 min-1 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 hPlk1-PBD competitors.Conclusion:Thymoquinone and Poloxin are slow-tight ligands to the hPlk1-PBD with kinetic models distinct from each other.Michael addition as the mechanism is excluded.
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 (hPlk1) 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 × 103 L · mol -1 min -1. 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 min-1 to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h.The inhibitors were insignificantly to the presence of Michael donors as hPlk1-PBD competitors.Conclusion: Thymoquinone and Poloxin are slow-tight ligands to the hPlk1-PBD with kinetic models distinct from each other. Micr addition as the mechanism is excluded .