Assays were conducted in a standard 384-well plate in a reaction volume of 76 uL assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) as follows: To 25 uL of substrate mix (4 uM NADPH, 1 mM aKG), 1 uL of test compound in DMSO was added. The plate was centrifuged briefly, and then 25 ul of enzyme mix was added (0.1 ug/mL ICDH1 G97D) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 ul of detection mix (30 uM resazurin, 36 ug/ml diaphorase) was added, and the mixture further incubated for 5 minutes at room temperature. The activity readout as a result of the conversion of resazurin to resorufin was detected by fluorescent spectroscopy at excitation 544 nm and emission 590 nm (c/o 590 nm).
