In vitro TR-FRET kinase assay

Assays were performed in white ProxiPlate-384 Plus™ 384-well microplates (Perkin Elmer). Incubation steps were performed at 21 °C in kinase reaction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, 2 mM DTT, 0.01% Tween-20). Purified proteins were diluted in kinase reaction buffer prior to use. All measurements were performed in triplicate.

Plates were read on a TECAN Infinite M1000 Pro plate reader, using fluorescence top reading settings (λ_ex = 320/20 nm, λ_em,donor = 615/10 nm, λ_em,acceptor = 665/10 nm, 100 μs integration time, 50 μs lag time). Emission ratios were calculated as fluorescence of acceptor / fluorescence of donor. Z′-factors were determined for each individual assay plate as Z′ = 1–3(σ_pc + σ_nc)/(μ_pc − μ_nc), with σ = standard deviation and μ = mean of measured replicates, WT or untreated samples as positive control (pc) and samples incubated with no ATP as negative control (nc). All plates met the requirement of Z′ > 0.7. Emission ratios were corrected for background signal of samples incubated with no ATP. Corrected ratios were averaged and normalized to WT or untreated signal as 100% reference.

For relative activity determination of mutants, purified WT or mutant FES (12.5 ng per well) or FER (5 ng per well) was incubated with 50 nM ULight-TK peptide (Perkin Elmer) and 100 μM ATP for 60 min at 21 °C in a total volume of 10 μL. The reaction was quenched by addition of 10 μL development solution (20 mM EDTA, 4 nM Europium-anti-phosphotyrosine antibody (PT66, Perkin Elmer) and incubated for 60 min before fluorescence was measured.

For K_M determinations, assay was performed as described above, but with variable ATP concentrations in a dilution series of 1 mM to 100 nM. For IC₅₀ determinations, serial dilutions of inhibitor were prepared in DMSO, followed by further dilution in kinase reaction buffer. The inhibitors were premixed with peptide and ATP (5 μM final concentration, unless indicated otherwise), after which WT or mutant kinase was added to initiate the reaction. The final DMSO concentration during the reaction was 1%. K_M and IC₅₀ curves were fitted using GraphPad Prism® 7/8 (GraphPad Software Inc.).