Targeting subcellular localization through the polo-box domain: non-ATP competitive inhibitors recapitulate a PLK1 phenotype

Abstract

The polo-box domain (PBD) has critical roles in the mitotic functions of polo-like kinase 1 (PLK1). The replacement with partial ligand alternative through computational enrichment (REPLACE) strategy to develop inhibitors of protein-protein interactions has identified alternatives for the N-terminal tripeptide of a Cdc25C substrate. In addition, a peptide structure-activity relationship described key determinants and novel information useful for drug design. Fragment-ligated inhibitory peptides (FLIP) were generated with comparable affinity to peptide PBD inhibitors and possessed antiproliferative phenotypes in cells consistent with the observed decrease in PLK1 centrosomal localization. These FLIPs showed evidence of enhanced PLK1 inhibition in cells relative to peptides and induced monopolar and multipolar spindles, which stands in contrast to previously reported small-molecule PBD inhibitors that display phenotypes only partially representative of PLK1 knockdown. Progress obtained applying REPLACE validates this approach for identifying fragment alternatives for determinants of the Cdc25C-binding motif and extends its applicability of the strategy for discovering protein-protein interaction inhibitors. In addition, the described PBD inhibitors retain high specificity for PLK1 over PLK3 and therefore show promise as isotype selective, non-ATP competitive kinase inhibitors that provide new impetus for the development of PLK1-selective antitumor therapeutics.

Figure 1

Binding mode of the SCCP5594, a benzo[1,4]oxazin-3-one fragment ligated to S[pT]PNGL (generated using Discovery Studio 3.0). H-bonds to the fragment and other non-bonded contacts to the PBD are indicated by black dashed and solid green lines respectively.

Figure 2

Treatment of HeLa cells with PBD inhibitors leads to reduced localization of PLK1 to the centrosomes. The Y-axis represents arbitrary fluorescent intensity units. Control represents fluorescence intensity data from untreated cells. QQ represents the data from cells treated with transfection reagent alone. The remainder represent data from cells transfected with the following peptides: 292 (LLCS[pT]PNGL), 5743 (Ac-PLHS[pT]A), 5781 (Ac-PLHS[pT]A), 5788 (3G1-S[pT]PNGL). Statistically significant decreases in fluorescent intensity were observed for 5743, 5781 and 5788.

Figure 3

Representative images from HeLa cells expressing GFP-H2B and immunostained for PLK1. (a) normal metaphase from an untreated cell, (b) aberrant prometaphase from a cell transfected with 5743, (c) aberrant, quadripolar metaphase from a cell transfected with 5743, (c) aberrant, tripolar metaphase from a cell transfected with 5788.

Figure 4

Treatment with PBD inhibitors induced aberrant mitoses. The y-axis represents the percentage of aberrant mitoses seen in mitotic cells. Control represents data from untreated cells. QQ represents data following treatment with QQ transfection reagent alone. The remainder represent data from the following treatments: 292 (LLCS[pT]PNGL), 5743 (Ac-PLHS[pT]A), 5781 (Ac-LHS[pT]AI), 5788 (3G1-S[pT]PNGL), or 5827 (3G2-S[pT]PNGL).

Figure 5

Induction of apoptosis by PBD inhibitory compounds. The y-axis represents the percentage of cells. PBS represents cells treated with PBS alone. QQ represents cells treated with QQ transfection reagent alone. The remainder represent cells transfected with: 292 (LLCS[pT]PNGL), 5821 (Ac-LLCS[pT]PNGL), 5743 (Ac-PLHS[pT]A), 5781 (Ac-LHS[pT]AI), 5788 (3G1-S[pT]PNGL), or 5827 (3G2-S[pT]PNGL).