High-throughput chemical diversification using SuFEx chemistry enables the prospective discovery of molecular glue degraders that cooperatively recruit E3 ligases to pre-selected targets through extensive protein-protein contacts, as demonstrated by dHTC1, which degrades ENL through CRBN with 220-fold cooperativity despite minimal independent ligase affinity, and dHTC3, which accesses the previously unexploited SCFFBXO3 pathway for bromodomain-selective BRD4 degradation.

Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4CRBN to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCFFBXO, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.