Homogeneous time-resolved fluorescence assays

Homogeneous time-resolved fluorescence (HTRF) assays were performed with a HTRF^® KinEASE™-TK kit (Trevigen). Briefly, the compounds dissolved in DMSO, BTK kinase, ATP (at K_m), and TK peptide were added to 384-well plates at a volume of 10 µL per well and incubated at 23 °C for 120 min. Eu³⁺ cryptate-labeled anti-phosphotyrosine antibody and Streptavidin-XL-665 were added to the mixture after the reaction was stopped. After an additional 1 h incubation, the fluorescence signals (excitation at 320 nm, and emission at 615 and 665 nm) were read with an EnVision multilabel plate reader (PerkinElmer). Half-maximal inhibitory concentration (IC₅₀) values of the compounds were generated in GraFit software (Version 6.0) and are presented at the mean of 3 independent experiments performed in duplicate.