| Assay Method Information | |
| | Human DHODH Inhibition Assay |
| Description: | The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described in J. Med. Chem. 2006; 49:1239. Briefly, the hDHODH concentration was adjusted in a way that an average slope of approximately 0.2 AU/min served as the positive control (e.g. without inhibitor). The standard assay mixture contained 60 UM 2,6-dichloroindophenol, 50 μM decylubiquinone and 100 UM dihydroorotate. The hDHODH enzyme with or without at least six different concentrations of the compounds was added and measurements were performed in 50 mM TrisHCI, 150 mM KCl and 0.1% Triton X-100 at pH 8.0 and at 30° C. The reaction was started by adding dihydroorotate and measuring the absorption at 600 nm for 2 min. For the determination of the IC50 values, each data point was recorded in triplicate. For the determination of the inhibitory constant Ki, the KM values for DHO and decylubichinon were determined. Afterwards, the compounds were diluted in a dilution series depending on their IC50 values in DMSO. |
| Affinity data for this assay | |
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