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Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells
Xuan Yang1, Jeffery L Smith1, Michael T Beck2
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract:
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 11 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 11 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.
Insights
Researchers developed a novel probe for live-cell NanoBRET assays to measure Polo-like kinase 1 (PLK1) activity. This probe confirmed PLK1 engagement and revealed adavosertib
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Polo-like kinase 1 (PLK1) is a key regulator of mitosis and a significant target in oncology.
- PLK1 also acts as an antitarget for DNA damage response or anti-infective therapies.
- Existing live-cell assays limit the study of PLK family kinases.
Purpose of the Study:
- To develop a novel energy transfer probe for live-cell NanoBRET target engagement assays targeting PLK1.
- To expand NanoBRET assays to include PLK2 and PLK3.
- To assess the in-cell target engagement and promiscuity of known PLK inhibitors, including adavosertib.
Main Methods:
- Development of an anilino-tetrahydropteridine-based energy transfer probe (Probe 11).
- Configuration of NanoBRET target engagement assays for PLK1, PLK2, and PLK3.
- Measurement of known PLK inhibitor potency in live cells.
- Investigation of adavosertib's target engagement profile.
Main Results:
- Probe 11 successfully enabled NanoBRET assays for PLK1, PLK2, and PLK3.
- In-cell PLK1 engagement correlated well with cellular proliferation inhibition data.
- Adavosertib showed PLK engagement at micromolar concentrations.
- Adavosertib selectively engaged WEE1 at clinically relevant doses, not PLK1.
Conclusions:
- Probe 11 is a valuable tool for live-cell NanoBRET assays of PLK family kinases.
- The study provides new insights into the in-cell target engagement of adavosertib.
- NanoBRET assays reveal differential target engagement of adavosertib at various concentrations and targets.
