FF tags-SynbioDEL: Biosynthetic and Proteome Profiling-Guided Discovery of PHGDH Inhibitor from Reversible DNA-Encoded Libraries Selection Platform

DNA-encoded libraries (DELs) have emerged as an effective and efficient selection strategy for lead compound discovery in academia and industry over the past decades. Despite recent advancements in this field, DEL is still limited by sensitive DNA-based constructs, especially low selection success rates from random selection of targets. Here, we describe a biosynthesis on-DNA reaction for DEL syntheses and develop a proteome profiling-guided reversible DEL selection platform. This platform, termed FF tags-SynbioDEL, integrates DEL technology, biocatalysis, and fully-functionalized (FF) chemical tags to match DELs with selection targets, even with limited information on ligandable hotspots. Using a diazirine-based FF indole probe, we comprehensively surveyed binding partners in live cells and identified phosphoglycerate dehydrogenase (PHGDH) as a potential target for DEL selection. DEL01 and DEL02 were designed, synthesized, and selected against PHGDH, leading to the identification of a novel enzyme-active compound with an IC50 value of 2.4 μM. Our strategy, utilizing FF tags-SynbioDEL, establishes a generalizable workflow for rapid target hunting, ligand discovery, and optimization. This approach provides a transformative method for matching DELs with potential targets, demonstrating its significant potential in drug discovery.