Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer.

Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z' factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z' factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.

DITMD-induced mitotic defects and apoptosis in tumor cells by blocking the polo-box domain-dependent functions of polo-like kinase 1.

DITMD (1, 3- Dioxolo[4,5-g] isoquinolinium 5, 6, 7, 8- tetrahydro- 4- methoxy- 6, 6- dimethyl- 5- [2- oxo- 2- (2-pyridinyl)ethyl] - iodide) is a natural product-like compound with a hydrocotarnine moiety. The aim of this study was to investigate the anticancer effects of DITMD including mitotic arrest, apoptosis, radiosensitization, and to further explore its possible mechanism. DITMD (3-30 µM) induced an obvious cell cycle delay at G2/M transition and apoptosis in HeLa cells. In a validation study, DITMD caused chromosome alignment defects and accumulation of mitotic markers such as polo-like kinase 1, cyclin B1, and phospho-histone H3. DITMD pre-treatment for 11 h also significantly decreased the cells' survival after X-ray irradiation. In mechanism studies, DITMD inhibited the polo-box domain of polo-like kinase 1 but not the conserved kinase domain. Molecular modeling also suggests that DITMD binds at the phosphate group recognition site and inhibits the action on phospho-peptide ligands. In addition, DITMD was analyzed as a PLHSpT competitive inhibitor with an IC50 value of 2.1 µM and exhibited good selectivity against 105 distinct kinases. Taken together, these results indicate that DITMD induced chromosome alignment defects, apoptosis and radio-sensitization, and suggest that one mechanism underlying these anticancer effects involves inhibiting the polo-box domain-dependent functions of polo-like kinase 1.