| Assay Method Information | |
| | Inhibition of Human Dihydroorotate Dehydrogenase (DHODH) |
| Description: | DHODH enzyme assays were performed with 6 nM recombinant human DHODH (purified essentially as described by Walse et al., Biochemistry, 47, 8929-8936 (2008)). The reaction mixture consisted of 1 mM DL-dihydroorotic acid (#D7003, Sigma-Aldrich), 100 μM 3,4-dimethoxy-5-methyl-p-benzoquinone (#D9150, Sigma-Aldrich), and 100 μM 2,6-dichlorophenolindophenol sodium salt (DCIP) (#D1878, Sigma-Aldrich) in enzyme buffer (50 mM Tris-HCl pH 8.0, 0.1% Triton X-100, 150 mM KCl). A stock solution of 20 mM DCIP was prepared in enzyme buffer and filtered through Whatman paper just before use. Reactions were performed in a total volume of 100 μL, consisting of 1 μL of compound, 49 μL of enzyme and 50 μL of reaction mixture. Loss in absorbance by chromogen DCIP was measured at 595 nm following a 70 minute incubation at 32° C. |
| Affinity data for this assay | |
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