Discovery of a Covalent Inhibitor Targeting the PHGDH Dimer Interface with Antitumor Efficacy

Protein oligomerization is functionally essential for many enzymes, yet small-molecule strategies that directly target dimer interfaces remain challenging. Cysteine residues at protein dimer interfaces offer chemically addressable sites for modulating oligomeric assembly. Here, we used covalent DNA-encoded chemical library (CoDEL) screening to identify site-selective covalent hits targeting the PHGDH dimer interface. Covalent hits emerging from CoDEL screening were optimized to yield a selective covalent inhibitor engaging the interfacial Cys281. A representative compound, D5-2, potently disrupts PHGDH dimerization and inhibits its enzymatic activity, restores sensitivity to EGFR tyrosine kinase inhibitors in resistant lung adenocarcinoma cells in vitro, and exhibits antitumor efficacy in mouse xenograft models. Together, these findings establish dimer-interface cysteine targeting as a mechanism-based and therapeutically relevant strategy for modulating PHGDH function, and highlight the potential of CoDEL for discovering covalent inhibitors of protein–protein interfaces.