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Updated: Jun 3, 2025
Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Published on: October 21, 2016
Fine-tuning probes for fluorescence polarization binding assays of bivalent ligands against polo-like kinase 1 using
Kohei Tsuji1, Hirokazu Tamamura2, Terrence R Burke3
1Department of Medicinal Chemistry, Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan; Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 1050 Boyles St., Frederick, MD 21702, USA.
Abstract:
Polo-like kinase 1 (Plk1) is an important cell cycle regulator that is a recognized target for development of anti-cancer therapeutics. Plk1 is composed of a catalytic kinase domain (KD), a flexible interdomain linker and a polo-box domain (PBD). Intramolecular protein-protein interactions (PPIs) between the PBD and KD result in "auto-inhibition" that is an essential component of proper Plk1 function. Recently, we developed high-affinity PBD-binding inhibitors using a bivalent approach. These ligands contain the low-nanomolar affinity Plk1 KD-binding inhibitors BI2536 or Wortmannin tethered to the PBD-binding peptide, PLH*SpT (H* represents a -(CH2)8Ph group on the histidine side chain π-nitrogen). Due to the extremely high affinity of these bivalent inhibitors, to avoid bottoming out in competitive binding assays, it was necessary to use PLH*SpT in the affinity probe. As reported herein, we have developed fluorescence polarization assays using a new fluorescent probe based on the Plk1 PBD-binding peptide, FDPPLHSpTA. We applied the assay to evaluate the affinities of bivalent inhibitors that possess a variety of PBD-binding peptides having much lower PBD-affinities than PLH*SpT. Tethering BI2536 in these bivalent inhibitors resulted in significant affinity enhancements as compared to the parent monovalent peptides.
Insights
We developed a new fluorescence polarization assay to measure Polo-like kinase 1 (Plk1) interactions. This assay allows for the evaluation of bivalent inhibitors, showing significant affinity enhancements when tethered to Plk1.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator and a key target for anti-cancer drug development.
- Plk1's activity is regulated by intramolecular interactions between its kinase domain (KD) and polo-box domain (PBD).
- Bivalent inhibitors have been developed by tethering Plk1 KD-binding agents to PBD-binding peptides for enhanced affinity.
Purpose of the Study:
- To develop a novel fluorescence polarization assay for evaluating Plk1 PBD-binding affinities.
- To assess the impact of tethering a Plk1 KD-binding inhibitor (BI2536) to various PBD-binding peptides on overall inhibitor affinity.
- To characterize the binding affinities of novel bivalent Plk1 inhibitors.
Main Methods:
- Development of a new fluorescent probe, FDPPLHSpTA, based on a Plk1 PBD-binding peptide.
- Establishment of fluorescence polarization (FP) assays utilizing the FDPPLHSpTA probe.
- Application of the FP assay to quantify the binding affinities of bivalent inhibitors with varying PBD-binding peptide affinities.
Main Results:
- The developed FP assay successfully measured Plk1 PBD-binding affinities.
- Bivalent inhibitors incorporating the BI2536 moiety demonstrated significantly enhanced binding affinities compared to their monovalent peptide counterparts.
- The affinity enhancement was observed across a range of PBD-binding peptides with differing intrinsic affinities.
Conclusions:
- A robust fluorescence polarization assay was established for studying Plk1 PBD interactions.
- Bivalent inhibition strategies effectively enhance the affinity of Plk1-targeting compounds.
- These findings support the development of novel bivalent inhibitors for Plk1-targeted cancer therapeutics.
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