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, PLHSpT (H represents a -(CH)Ph 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 PLHSpT 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 PLHSpT. Tethering BI2536 in these bivalent inhibitors resulted in significant affinity enhancements as compared to the parent monovalent peptides.