Figure 4. Targeted metabolomics analysis to identify intracellular enzyme targets of AGF291, AGF320, and AGF347.

A: A schematic of serine isotope scrambling and dTTP isotope analysis is shown. Heavy (²H) atoms in serine (red circles) enter the C1 flux in the cytosol through reversal of the SHMT1 reaction and TS leading to dTMP and dTTP (as M+2). In the mitochondria, ²H atoms in serine (blue circles) are metabolized via SHMT2, MTHFD2, and MTHFD1 to formate which is converted to 10-formyl-THF and 5,10-me-THF, leading to dTMP and dTTP (as M+1). Most steps are reversible as noted. Adapted from (22). B and C: Total serine pools (B) and the corresponding serine isotope distributions (C) for WT and KO HCT116 sublines, and for inhibitor-treated WT cells, are shown. D and E: Total GAR (D) and AICAR (E) pools are shown for WT and SHMT2 KD H460 sublines, including inhibitor-treated WT cells, with and without 1 mM formate. F and G: Relative adenine nucleotide pools (AMP, ADP, and ATP) and dTTP pools with isotope distributions are shown for untreated and inhibitor-treated MIA PaCa-2 cells. For D and E, results for inhibitor-treated and SHMT2 KD cells were normalized to vehicle-treated WT ± formate or NTC ± formate samples, as appropriate. Data are mean values +/− standard deviations for three technical replicates. #, p < 0.10; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ^ or ^v are used in place of * to specify a significant increase or decrease, respectively. Statistical comparisons were with vehicle-treated WT ± formate or NTC ± formate samples, as appropriate. ns = not significant.