Mechanistic dissection of SMARCA2/4 molecular glues reveals programmable switching between DCAF16 and FBXO22

Molecular glue degraders (MGDs) represent a powerful strategy for targeted protein degradation, yet the rules governing ligase selectivity remain elusive. Here, we characterize a class of SMARCA2/4 MGDs with tunable E3 ligase engagement. Compound 1 degraded SMARCA2/4 exclusively via DCAF16, driven by covalent capture of Cys173. Strikingly, 1-carbon analogs rewired ligase preferences: compounds 2 and 3 recruited both DCAF16 and FBXO22, whereas compound 4 remained strictly DCAF16 dependent despite weak FBXO22 binding, although all compounds shared an alkynyl pyridine moiety essential for covalent attachment. Mutational analysis further pinpointed Cys228 and Cys326 in FBXO22 as indispensable for degradation. These findings reveal that subtle scaffold modifications dictate ligase preference and that only productive ternary geometries drive ubiquitination and degradation. By exposing new cysteine liabilities and showcasing precise control over ligase engagement, this work highlights how single-atom edits in MGDs can unlock tunable, switchable degradation mechanisms.