A new genre of fluorescence recovery assay to evaluate polo-like kinase 1 ATP-competitive inhibitors

Abstract

Using a probe consisting of a fluorescein-labeled variant of the potent polo-like kinase 1 (Plk1) inhibitor BI2536 [FITC-PEG-Lys(BI2536) 4], we were able to determine half maximal inhibitory concentration (IC₅₀) of ATP-competitive Type 1 inhibitors of Plk1 by means of a fluorescence recovery assay. This methodology represents a cost-effective and simple alternative to traditional kinase assays for initial screening of potential Plk1 inhibitors.

Figure 1.

Structures of compounds discussed in the text.

Figure 2.

An outline of fluorescence recovery assay based on BI2536 binding to Plk1 KD.

Figure 3.

Results from fluorescence recovery assays to determine an optimum probe. The X axis represents Plk1 concentration (log M) and the Y axis represents fluorescence intensity (Ex: 485 nm, Em: 528 nm). Data points represent average ± SEM from three independent experiments and fit using non-linear regression in GraphPad Prism 7.

Figure 4.

Results from fluorescence recovery assays, which measured the ability of Plk1 inhibitors to compete with 4 for binding to full-length Plk1. The X axis represents inhibitor concentration (log M) and the Y axis represents relative probe binging based on the fluorescence intensity (Ex: 485 nm, Em: 528 nm) of no inhibitor (100%) and blank (no Plk1, 0%). Data points represent average ± SEM from three independent experiments and fit using non-linear regression in GraphPad Prism 7.