Targeting the Selectivity Barrier in One-Carbon Metabolism: Rutaecarpine Preferentially Inhibits MTHFD2 over MTHFD1

Folate-mediated one-carbon metabolism supports nucleotide biosynthesis, methylation reactions, and redox homeostasis, and the mitochondrial enzyme MTHFD2 has emerged as a cancer-associated metabolic target. However, the development of MTHFD2-preferential inhibitors remains challenging because MTHFD2 is structurally and functionally related to other folate-metabolism enzymes, including the cytosolic enzyme MTHFD1 and the mitochondrial isoenzyme MTHFD2L. In this study, a curated library of 115 phytochemicals was screened against the folate/substrate-binding pockets of MTHFD2 and MTHFD1 using a redocking-controlled molecular-docking protocol. Three compounds, dehydrotrametenolic acid, eburicoic acid, and rutaecarpine, met the docking-based prioritization criteria for predicted MTHFD2-preferential binding. Subsequent ADME, drug-likeness, and toxicity profiling indicated that the two triterpenoids had unfavorable physicochemical and pharmacokinetic properties and were therefore not advanced to biochemical testing, whereas rutaecarpine showed the most balanced predicted profile. In vitro enzyme-inhibition assays showed that rutaecarpine inhibited both MTHFD2 and MTHFD1 in a concentration-dependent manner. Rutaecarpine inhibited MTHFD2 with an apparent IC50 of 31.35 μM and MTHFD1 with an apparent IC50 of 54.20 μM, corresponding to a modest 1.73-fold preference for MTHFD2. In contrast, the reference antifolate LY345899 preferentially inhibited MTHFD1. These findings identify rutaecarpine as a natural-product scaffold with statistically supported but modest MTHFD2-preferential activity and support its further structure-guided optimization, including future profiling against MTHFD2L. These results should be interpreted as evidence of exploratory scaffold-level activity rather than identification of a potent, optimized, or broadly validated MTHFD2 inhibitor.