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

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.

Keywords: Bivalent inhibitor; Fluorescence Polarization (FP) assay; Phosphorylated threonine (pT); Polo-box domain (PBD); Polo-like kinase 1 (Plk1).

Fig. 1.

Structures of the reported Plk1 inhibitors BI2536, Lys(BI2536), PLH*SpT, PLHSpT, and bivalent inhibitors 1–8.^,,

Fig. 2.

Plk1 PBD-binding assays. (a) ELISA assay using NeutrAvidin coated plate, biotin-labeled PMQSpTPLN, myc-tagged Plk1, anti-myc antibody and HRP-fused antibody^,,; (b) FP assays using full-length Plk1 or isolated Plk1 PBD and FITC-labeled PLH*SpT, FITC-labeled FDPPLHSpTA, or 5CF-GPMQSpTPLNG. Ahx: 6-aminohexanoic Acid, HRP: horseradish peroxidase, FITC: fluorescein isothiocyanate, miniPEG: 8-amino-3,6-dioxaoctanoyl, 5CF: 5-carboxyfluorescein.

Fig. 3.

Structures of fluorescent probes used in FP assays.^,

Fig. 4.

FP assays with different fluorescent probes. (a) Results from full-length Plk1assays using FITC-miniPEG-PLH*SpT as a probe. Binding curves and IC₅₀ values for PLH*SpT, PLH*SpT + 1 μM BI2536, 1, 4, 5, and 8 have been previously reported. The X axis represents log (inhibitor concentration) and the Y axis represents % binding based on probe alone as 0% probe binding and probe with Plk1 as 100% probe binding. (b) Determination of K_D values of each probe (10 nM). The X axis represents Plk1 concentration, and the Y axis represents FP signal intensity. The K_D values for FITC-miniPEG-PLH*SpT (8.3 ± 0.80 nM) and FITC-miniPEG-FDPPLHSpTA (210 ± 16 nM) were determined from two independent experiments and shown as the average K_D value ± SEM (nM).

Fig. 5.

Structures of pentapeptide-based bivalent ligands.

Fig. 6.

Structures of the tripeptide-based bivalent ligands.