Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics^1-3. Molecular Glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs^4-6. The discovery and development of Molecular Glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4^DCAF16 ligase to the second bromodomain of BRD4 (BRD4_BD2). BRD4_BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4_BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16_Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4_BD2 revealed that the loop conformation around BRD4_His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent Molecular Glues, which opens a new path to proximity driven pharmacology.