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

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

Application of BRET to monitor ligand binding to GPCRs

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs

Resistance to kinase inhibition through shortened target engagement

Imatinib, a selective inhibitor of the breakpoint cluster region (BCR)-ABL kinase, is the poster child for targeted cancer therapeutics. However, its efficacy is limited by resistance mutations. Using a quantitative bioluminescence resonance energy transfer assay in living cells, we identified ABL kinase mutations that could cause imatinib resistance by altering drug residence time

Drugging the kinome

Kinases are perhaps the most successfully drugged target families of the 21st century, with 35 FDA approved medicines. These drugs only target a very small percentage of the human kinome. Tools that allow exploration of the rest of the kinome will no doubt lead to the identification of new validated kinase drug targets and new approved medicines. In this chapter we describe the progress towards drugging the kinome by talking about 4 groups of kinases: kinases currently targeted by FDA approved drugs, kinases targeted by potent and selective chemical probes, kinases for which there are narrow spectrum inhibitors available ("covered" kinases), and then the remaining kinases for which there are currently no narrow spectrum inhibitors available ("uncovered" kinases). We believe that the scientific community has the motivation, opportunity, and ability to parlay the collective experience and wealth of kinase data available into a set of molecules that cover the entire kinome and can be used to identify the next generation of important therapeutic kinase targets25328