Assembly‐Glued Ligands and Warheads for Hypoxia‐Activatable Supramolecular Covalent Inhibitors

Abstract Targeted covalent inhibitors are promising for drug discovery and challenged by the strict structural features for target proteins to sustain proximity‐induced conjugations. Herein, an assembly‐glued strategy is reported to create surface‐displayed supramolecular covalent inhibitors that leverage the display‐induced proximity between noncovalent ligands and warheads. The ligand and warhead are obtained via attaching an epidermal growth factor receptor (EGFR)‐binding segment or nitroreductase (NTR)‐activated moiety to bola‐amphiphilic peptides, respectively. Co‐assembling the ligand and warhead with a filler peptide glues them and forms the surface‐displayed covalent inhibitor. While the ligand associates with EGFR, the warhead undergoes NTR‐induced cysteine conjugation facilitated by the ligand‐EGFR association. In vitro studies show the reliable hypoxia‐activated inhibition of EGFR phosphorylation and enhanced cytotoxicity of the covalent inhibitor under hypoxic condition. In addition, this strategy also allows for creating covalent inhibitors targeting proteins without small molecular covalent drugs, due to the display‐induced proximity between ligands and warheads. In vivo results illustrate the prolonged retention of the covalent inhibitor and its efficacy in inhibiting tumor growth. These findings demonstrate that simultaneous surface display of ligands and warheads via assembly adhesives allows for implementation of covalent inhibition functions, thus providing a new strategy for developing covalent inhibitors in the future.