Extended Data Fig. 2: Inhibitory effects of DHODH inhibitors against FSP1 enzyme activity.
From: DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition
a. (Left) Scheme of the FSP1 enzyme activity assay. Resazurin (100 μM), a substrate of FSP1, is reduced to resorufin by incubation with recombinant FSP1 protein (50 and 40 nM of human and mouse FSP1, respectively) and NADH (200 μM). The amount of resorufin evaluated by fluorescent intensity (ex 540/em 590 nm) indicates FSP1 enzymatic activity. (Right) Scheme of the DHODH enzyme activity assay. Enzyme reaction of recombinant human DHODH (25 nM), dihydroorotate (DHO, 500 μM) and CoQ0 (100 μM) reduces an electron acceptor 2, 6-dichlorophenolindophenol (DCIP, 120 μM) to DCIPH2. The change in absorbance of DCIP (at absorbance 600 nm) indicates DHODH enzyme activity. b. NADH consumption assay using recombinant human FSP1 protein (25 nM) in combination with or without brequinar (BQR, 300 μM). Menadione (50 μM) was used as a substrate of FSP1. Brequinar inhibited the FSP1-dependent NADH consumption. c. The inhibitory effect of BQR and iFSP1 on mouse FSP1 enzymatic activity. d. Heatmaps showing the viability and immunoblotting of hFSP1-overexpressed (OE) and Dhodh KO-Pfa1 cells with or without overexpression of hDHODH. Viability was measured after treatment with RSL3 for 24 h. Combination of RSL3 with BQR synergistically induced cell death in both cell lines. e. The inhibitory effect of known DHODH inhibitors on human and mouse FSP1 enzyme activity. f. Calculated IC50 values of iFSP1 and DHODH inhibitors against recombinant human and mouse FSP1. g. The inhibitory effect of DHODH inhibitors and iFSP1 against human DHODH enzymatic activity. h. Heatmaps showing the viability of HT-1080 cells (5,000 cells per well) treated with RSL3 in combination with vidofludimus or BAY-2402234 for 24 h. The values of the groups treated with zero or 0.01 μM of RSL3 are also shown in the right graphs. i. The binding prediction of iFSP1 in human FSP1 protein. Data is mean ± s.d. of n = 3 (b). Data is representative of three (b, c and e) and two independent experiments (d, g and h), respectively.
