The first rate-limiting
enzyme of the
serine synthesis pathway (SSP),
phosphoglycerate dehydrogenase (PHGDH), is hyperactive in multiple
tumors, which leads to the activation of SSP and promotes
tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified
withangulatin A (WA), a natural small molecule, as a novel covalent inhibitor of PHGDH. Affinity-based
protein profiling identified that WA could directly bind to PHGDH and inactivate the
enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the
cysteine 295 residue (Cys295) of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain (SBD) of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the
glutathione synthesis was decreased and intracellular levels of
reactive oxygen species (ROS) were elevated, leading to the inhibition of
tumor proliferation. This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-
tumor agents for targeting PHGDH.