| Assay Method Information | |
| | TNFα Binding Assay |
| Description: | Table 15: TNFα binding was measured in black 96-well plates using a time-resolved FRET assay using a previously described fluorescence acceptor TNFα probe (Xiao et al J. Med. Chem. 2020). Biotinylated TNFα was purchased from Acro Biosystems. Binding reactions were conducted in 20 mM Hepes (pH 7.5) 10 mM MgCl2 0.015% Tween-20 0.05 mg/ml BSA 2% DMSO with 1 nM TNFα 0.5 nM Streptavidin-Tb and 80 nM probe. Test compounds were resuspended in DMSO and 3-fold serial dilutions were made at 100× final concentrations. Fluorescence was measured using λex=340 nm, λem=490, 520 nm on an SpectraMax M5e plate reader (Molecular Devices) after 16 hr incubation time. Dose responses were conducted in duplicate and normalized to the response with DMSO (high) and 100 nM (R)-3-(1-((3-chloro-7-fluoro-6-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-2-methyl-1,5-naphthyridin-4-yl)amino)ethyl)-4-fluorobenzonitrile (low) on each plate. IC50 values were determined by fitting to 4-parameter curves in GraphPad Prism. |
| Affinity data for this assay | |
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