Dihydroorotate

The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described previously [36 (link)]. 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 µM 2,6-dichloroindophenol (Sigma, St. Louis, MO, USA), 50 µM decylubiquinone (Sigma), and 100 µM dihydroorotate (Sigma). The hDHODH enzyme with or without at least six different concentrations of the compounds was added, and measurements were performed in 50 mM TrisHCl, 150 mM potassium chloride (KCl) (Merck), and 0.1% Triton X-100 (Sigma) at pH 8.0 and at 30 °C [37 (link)]. 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. DHODH inhibitors Cmp 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5 were provided by Immunic Therapeutics. Brequinar, uridine, 2′-deoxyuridine, cytidine, and 2′-deoxycytidine were purchased from Sigma. UCK2 inhibitor, UCK2-IN-20874830 was obtained from ChemDiv (San Diego, CA, USA).

DHODH activity and complex III activity were measured by spectrophotometry as previously described 59 (link). DHODH activity was determined spectrophotometrically at 37 °C by monitoring the decrease in absorbance at 600 nm of reduced 2,6-dichlorophenol-indophenol (DCPIP). Briefly, the reaction was initiated with 20 mM dihydroorotate (Sigma) in 1 ml of standard reaction buffer supplemented with 50 μM DCPIP (Sigma), 2 μg of rotenone (Sigma), 2 μg of antimycin A (Sigma), 5 mM NaN₃ (Sigma) and 0.1 mg of whole-cell lysate. The reaction was stopped by the addition of 2 μg leflunomide. Complex III activity was evaluated spectrophotometrically at 37 °C by monitoring the increase in absorbance at 550 nm of cytochrome c. Briefly, the reaction was initiated with 5 mM decylubiquinone (Sigma) to 1 mL of standard reaction buffer supplemented with 2 μg of rotenone (Sigma), 5 mM NaN₃, 60 μM cytochrome c (Sigma) and 0.1 mg of whole-cell lysate. The reaction was stopped by the addition of 2 μg antimycin A (Sigma). One unit was defined as nmol· min ^-1· μg ^-1 of protein. The fold changes were calculated using baseline values of control cells as a reference (set to 1).

Antibodies recognizing Tubulin, COX IV, SOX2, c-Myc, were obtained from Cell Signaling Technology. Antibodies recognizing DHODH, and MIC19 were obtained from Abcam. Antibody against Flag, anti-Flag M2 agarose beads, dihydroorotate, and bovine serum albumin were purchased from Sigma. Antibody recognizing MIC60 was obtained from NOVUS. Antibody recognizing MIC10 was obtained from PROSPEC. Antibody recognizing MIC27, horseradish peroxidase-conjugated goat anti-mouse, or rabbit secondary antibodies were obtained from Thermo Fisher Scientific. [¹⁵N₂]-glutamine was purchased from Cambridge Isotope Laboratories. BAY 2402234 and Vidofludimus were obtained from TOPSCIENCE. Lipofectamine 2000 was obtained from Thermo Fisher Scientific.