Time-Resolved Fluorescence Resonance Energy Transfer Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Inhibition of N-terminal His-tagged recombinant human BTK (393 to 659 residues) expressed in baculovirus infected Sf9 cells using Biotin-AVLESEEELYSSARQ-NH2 as substrate preincubated for 1 hrs followed by substrate addition in presence of ATP at Km concentration and measured after 1 hrs by TR-FRET assay
Inhibition of EGFR (unknown origin) preincubated for 1 hrs followed by substrate addition and measured after 1 hrs in the presence of ATP at Km concentration by TR-FRET assay
Inhibition of TEC (unknown origin) preincubated for 1 hrs followed by substrate addition and measured after 1 hrs in the presence of ATP at Km concentration by TR-FRET assay
Selectivity ratio of IC50 for inhibition of TEC (unknown origin) to IC50 for inhibition of N-terminal His-tagged recombinant human BTK (393 to 659 residues) expressed in baculovirus infected Sf9 cells
Inhibition of human TXK preincubated for 1 hrs followed by substrate addition and measured after 1 hrs in the presence of ATP at Km concentration by TR-FRET assay
Inhibition of human BMX preincubated for 1 hrs followed by substrate addition and measured after 1 hrs in the presence of ATP at Km concentration by TR-FRET assay
Selectivity index, ratio of IC50 for inhibition of human TXK to IC50 for inhibition of N-terminal His-tagged recombinant human BTK (393 to 659 residues) expressed in baculovirus infected Sf9 cells
Selectivity index, ratio of IC50 for inhibition of human BMX to IC50 for inhibition of N-terminal His-tagged recombinant human BTK (393 to 659 residues) expressed in baculovirus infected Sf9 cells
Potency index, ratio of BGB-3111 IC50 to test compound IC50 for inhibition of EGFR (unknown origin) preincubated for 1 hrs followed by substrate addition and measured after 1 hrs in the presence of ATP at Km concentration by TR-FRET assay