CHEMBL5732220

IC50 Measurements for Inhibitors Using CECR2 TR-FRET Binding Assay: His/Flag epitope tagged CECR2424-538 was cloned, expressed, and purified to homogeneity. CECR2 binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound 1001 (Example 125) with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CECR2 (1.5 nM final) was combined with biotin-ligand (25 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer AD0110) and SureLight¿ Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 12.5 nMolar APC-SA, respectively. After forty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit. Novel compound 1001 and the CECR2 TR-FRET Binding Assay described above represent additional embodiments of the invention.