NanoBRET Kinase Inhibition Assay

All BRET assays were performed in white, tissue-culture treated 96-well plates (Corning #3917) using adherent HEK-293 cells at a density of 2 × 10⁴ cells per well. All chemical inhibitors were prepared as concentrated stock solutions in DMSO (Sigma-Aldrich) and diluted in Opti-MEM to prepare working stocks. Cells were equilibrated for 2 h with the appropriate energy transfer probe and test compound prior to BRET measurements. Energy transfer probes were prepared at a working concentration of 20× in Tracer dilution buffer (12.5 mM HEPES, 31.25% PEG-400, pH 7.5). Individual kinase NanoBRET assays used the following energy transfer probes and concentrations: PLK1, probe 11 (0.2 μM); PLK2, probe 11 (1.0 μM); PLK3, probe 11 (1.0 μM); WEE1, tracer K10 (Promega, 0.13 μM). To measure BRET, NanoBRET NanoGlo Substrate and Extracellular NLuc Inhibitor (Promega) were added according to the manufacturer’s recommended protocol, and filtered luminescence was measured on a GloMax Discover luminometer equipped with 450 nm BP filter (donor) and 600 nm LP filter (acceptor), using 0.5 s integration time. BRET ratios are calculated by dividing the acceptor luminescence by the donor luminescence. Milli-BRET (mBRET) units (mBU) are calculated by multiplying the raw BRET ratios by 1,000. Broad spectrum profiling on 192 kinases was performed using the K192 NanoBRET Target Engagement assay (Promega) with tracer K10 using the published protocol [24 (link)].