For many years now, targeting deregulation within cancer cells’ metabolism has appeared as a promising strategy for the development of more specific and efficient cancer treatments. Recently, numerous reports highlighted the crucial role of the serine synthetic pathway, and particularly of the phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the pathway, to sustain cancer progression. Yet, because of very weak potencies usually in cell-based settings, the inhibitors reported so far failed to lay ground on the potential of this approach. In this paper, we report a structure–activity relationship study of a series of α-ketothioamides that we have recently identified. Interestingly, this study led to a deeper understanding of the structure–activity relationship (SAR) in this series and to the identification of new PHGDH inhibitors. The activity of the more potent compounds was confirmed by cellular thermal shift assays and in cell-based experiments. We hope that this research will eventually provide a new entry point, based on this promising chemical scaffold, for the development of therapeutic agents targeting PHGDH.

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.