A Synergistic Inhibitor Development Strategy Against Human UDP‐Galactose‐4‐Epimerase

Abstract

ABSTRACT O‐GalNAc ( N ‐acetylgalactosaminyl) glycosylation is an abundant posttranslational modification in mammalian cells. Dysregulation of O‐GalNAc glycosylation is implicated in cancer metastasis and immune evasion; however, our mechanistic understanding remains limited due to the lack of small‐molecule tools. O‐GalNAc biosynthesis depends heavily on the availability of UDP‐GalNAc that is biosynthesised by the cytosolic enzyme UDP‐galactose‐4‐epimerase (GalE). Knockout studies have demonstrated that loss of GalE severely impairs O‐GalNAc glycosylation, positioning GalE as a promising enzymatic therapeutic target in oncology. Here, we present an efficient workflow that combines both covalent and high‐throughput crystallographic non‐covalent fragment screening with structure‐based design to identify GalE inhibitors. Using these strategies, we discovered a ligandable pocket adjacent to a reactive tyrosine, enabling the development of a potent, “beyond cysteine” sulfonyl fluoride covalent inhibitor as well as a derived covalent alkyne probe. Structurally‐enabled fragment screening methodologies yielded nanomolar non‐covalent as well as covalent binders within no more than 22 elaborated compounds. Our work demonstrates synergism in next‐generation delivery of chemical matter for GalE inhibition, with the broader potential for targeting non‐cysteine residues in chemical biology and therapeutic applications.