Electrophile-First Screening Identifies Covalent Aminothiazole Scaffold Inhibitors for Ubiquitin-Conjugating Enzyme UBE2D2

UBE2 family of E2 ubiquitin-conjugating enzymes occupy central positions in multiple ubiquitination pathways and have been implicated in cancers, motivating efforts to develop chemical probes to interrogate their functions. In this study, we identified XY54 as a covalent modifier of UBE2D2 that engages both C85 and C111 by using an electrophile-first screening approach coupled with X-ray crystallography. Structure-guided structure−activity relationship optimization of XY54 yielded XY54-24, in which modifications of the amide and ester groups of XY54 selectively abolished its binding to C111 while enhancing affinity for C85. XY54-24 inhibited UBE2D2 enzymatic activity in vitro and suppressed the proliferation of A549 lung cancer cells. Collectively, this study identifies covalent aminothiazole scaffold inhibitors for targeting UBE2D2 and provides a structural foundation for further development of selective UBE2D2 chemical probes.