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.