Extended Data Fig. 1: TH9619 and TH9975 are inhibitors of MTHFD1(DC) in vitro.
From: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells
(a) Chemical structures of MTHFD1/2 inhibitors TH9619 and TH9975 and IC50 values for inhibition of MTHFD1(DC) and MTHFD2 enzymatic activities for each inhibitor. (b) Enzymatic activity of TYMS, SHMT1 and SHMT2 upon incubation with 100 µM TH9975 compared to their known inhibitors lometrexol (LMTX) and raltitrexed (RLTX), means ± SD (n = 3). (c, d) Expression of the indicated proteins was analyzed by Western Blot in SW620 WT, MTHFD2-/- and SHMT1-/- cells. β-actin served as loading control. Immunoblots are representative of 3 independent repeats. Signal intensity was quantified and normalized to loading control and global mean, means ± SD (n = 3). (e) Verification of the purity of mitochondrial and nuclear fractions for CETSA analysis. Shown are representative immunoblots of one out of two independent experiments. (f, g) Serine and ATP levels normalized to packed cell volume (PCV) in SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); paired, two-tailed t-test. (h) [U-13C]serine-derived MID of ATP in SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); one-way ANOVA with Šídák’s multiple comparison test for M + 0. (i) [U-13C]serine-derived release rate of formate M + 1 isotopologue from SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); paired, two-tailed t-test. (j, k) DARTS analysis of MTHFD1 stabilization in SW620 cells upon TH9619 (j) or DS18561882 (k) treatment. Intact cells (j) or cell lysates (k) were incubated with 10 µM TH9619 or vehicle for 3 h (j) or 30 min (k), followed by protein digestion at increasing pronase concentration and assessment of MTHFD1 degradation by Western blot. MTHFD1 intensities were normalized to SOD1; one representative immunoblot of two independent experiments. (l) Design of MTHFD1/2 inhibitor resistant MTHFD1 protein based on the crystal structure of structurally similar MTHFD2 bound to LY345899 (PDB ID: 5TC4). Highlighted is the residue Q100 that was mutated to alanine (Q100A) and tested in downstream biochemical assays. (m) Enzymatic activity of purified MTHFD1(DC) WT and MTHFD1(DC) mutant (Q100A), means ± SD (n = 3).
