Fine-tuning probes for fluorescence polarization binding assays of bivalent ligands against polo-like kinase 1 using full-length protein

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.