Elsevier

Analytical Biochemistry

Volume 395, Issue 2, 15 December 2009, Pages 189-194
Analytical Biochemistry

Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3

https://doi.org/10.1016/j.ab.2009.08.031Get rights and content

Abstract

The serine/threonine kinases Plk1, Plk2, and Plk3 harbor a protein–protein interaction domain dubbed polo-box domain (PBD). Recently, the inhibition of the PBD of the cancer target Plk1 has been successfully explored as an alternative to the inhibition of the kinase by ATP-competitive ligands. However, because the PBDs of Plk1, Plk2, and Plk3 have very similar optimal binding motifs, absolute specificity for the PBD of Plk1 over the PBDs of Plk2 and Plk3 may also represent a big challenge for a small molecule. To aid in the activity profiling of Plk PBD inhibitors, and to identify selective small molecules that will reveal the cellular consequences of inhibiting the PBDs of Plk2 and Plk3, we have developed high-throughput assays based on fluorescence polarization against the PBDs of Plk2 and Plk3. The assays are stable with regard to time and 10% dimethyl sulfoxide and have Z′ values ⩾0.7, making them well-suited for high-throughput screening. Moreover, our data provide insights into the binding preferences of the PBDs of Plk2 and Plk3.

Section snippets

Peptide synthesis

Peptides were synthesized and purified using standard Fmoc chemistry by the core facility of the Max Planck Institute of Biochemistry (Martinsried, Germany). Coupling to 5-carboxyfluorescein was performed via N,N′-diisopropylcarbodiimide (DIC)/1-hydroxy-benzotriazole (HOBt) activation in N,N-dimethylformamide (DMF) [30]. Unless stated otherwise, peptides were synthesized with an N-terminal amino group and a C-terminal carboxyl group. Peptides were analyzed by high-performance liquid

Results and discussion

To design fluorescent-labeled peptides that can serve as probes in fluorescence polarization assays for the PBDs of Plk2 and Plk3, we turned to the results of peptide library screens for recognition motifs of the PBDs of Plk1, Plk2, and Plk3 [16]. The peptide motif PMQTSpTPK had been reported as a preferred binding motif for the PBD of Plk2 from an immobilized peptide library described by the general sequence MAXXXXSpTXXXXAKK [16]. N-terminal labeling of the preferred binding motif with

Acknowledgments

This work was generously supported by the Department of Molecular Biology (director: Axel Ullrich) at the Max Planck Institute of Biochemistry and by the Bundesministerium für Bildung und Forschung (NGFN-2, Grant 01GS0453 to K.S. and Grant 01GS0451 to T.B.). We extend our thanks to Judith Müller for experimental support.

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