Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells

• Published in: Molecules (Basel, Switzerland) Vol. 28; no. 7; p. 2950
• Main Authors: Yang, Xuan, Smith, Jeffery L, Beck, Michael T, Wilkinson, Jennifer M, Michaud, Ani, Vasta, James D, Robers, Matthew B, Willson, Timothy M
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 25.03.2023; MDPI
• Subjects: Analysis; assay; Assaying; Bacterial infections; Biotechnology industry; Cancer therapies; Cell cycle; Cell Cycle Proteins - metabolism; Cell Proliferation; Clinical trials; Competition; DNA damage; DNA probes; drug; Drug dosages; Energy; Energy transfer; Enzymes; in cell; Infectious diseases; Inhibitors; kinase; Kinases; Measurement; Mitosis; Polo-like kinase 1; Protein Kinase Inhibitors - pharmacology; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Ratios; target engagement; Therapeutic targets; United States; in cell; kinase; target engagement; assay; drug
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules28072950

PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In-cell target engagement for PLK1 was in good agreement with the reported cellular potency for the inhibition of cell proliferation. Probe enabled the investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib via NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses.