The expression of
polo-like kinase 1 (Plk1) correlates with
malignancy and is thus recognized as a target for
cancer therapy. In addition to the development of
ATP-competitive Plk1 inhibitors, the polo-box domain (PBD), a unique functional domain of PLKs, is being targeted to develop Plk1-specific inhibitors. However, the action mechanisms of these two classes of Plk1 inhibitors have not been thoroughly evaluated. Here, we evaluate the differences in cellular effects of
ATP-binding domain inhibitors (
BI 2536,
GSK 461364) and PBD inhibitors (
poloxin,
thymoquinone) to determine their mechanisms of Plk1 inhibition. Our data show that
BI 2536 and
GSK461364 increased the population of cells in the G2/M phase compared with controls, while treatment with
poloxin and
thymoquinone increased cell population in the S phase as well as in G2/M, in a p53-independent manner. The population of cells staining positively for p-
Histone H3 and MPM2, mitotic index, was increased by treatment with
BI 2536 or
GSK461364, but not by treatment with
poloxin or
thymoquinone. Furthermore, treatment with
BI 2536 or
GSK461364 resulted in activation of the BubR1 spindle checkpoint
kinase, suggesting that treatment with
ATP-binding domain inhibitors induces metaphase arrest. However, the administration of
poloxin and
thymoquinone resulted in an increase in p21(WAF1) and S arrest, indicating that PBD inhibitors also affected interphase before mitotic entry. Taken together, these data suggest that the PDB of Plk1 plays a role in S phase progression through interaction with other
proteins, while its
ATP-binding domain is important for regulating mitotic progression mediated by its catalytic activity involving consumption of
ATP.