DHODH inhibition Assay
DHODH enzymatic activity was determined using 2, 6-dichloroindophenol
(DCIP) method as previously described(Diao et al., 2012). In brief,
recombinant human DHODH (15 nM) was incubated with varied concentrations
of indicated compounds for 10 min at room temperature. The assay buffer
was composed of 50 mM Tris (pH 8.0), 120 μM DCIP, 100 μM
Coenzyme
Q0 (CoQ0), 0.1% Triton X-100. The enzymatic reaction was initiated by
adding dihydroorotate (DHO) with a final concentration of 500 μM and
absorption at 600 nm was continuously recorded for 6 min with
EnVision Multimode Plate Reader
(Perkin
Elmer, Waltham, MA).
The
IC50 values were calculated by GraphPad Prism 7
(GraphPad software, CA).