A Chemically and Spatially Tunable Epoxide Warhead Toolkit for On-Demand Covalent Crosslinking at Protein-interaction Interfaces

Abstract: Genetically encoded covalent warheads at the protein-protein interface, enable the locking of interaction from the transient complex into a stable covalent adduct, which could improve the binding affinity, selectivity, and kinetics between a protein binder and its target. Given the diverse chemical microenvironments among different interaction interfaces, achieving efficient and selective cross-linking for each case requires genetically encoded warheads capable of reacting with a broad range of nucleophilic side chains, while offering tunable crosslinking radius and reactivities. Herein, we report a series of novel epoxide bearing unnatural amino acids (EPO-UAAs) that enable precise control over both the chemical reactivity and spatial selectivity of covalent crosslinking at protein interfaces. Our results show that both the chemical reactivity and spatial compatibility of the warhead significantly influence cross-linking efficiency at the binding interface. This enables site-selective cross-linking to drug targets such as PD-L1 or HER2 by a nanobody with precision at the 1.5 Å spatial scale, corresponding to a single C-C bond length difference. Furthermore, we incorporated these EPO-UAAs into the Z domain of protein A (ZPA), an antibody Fc-binding module, to develop covalent binders for the epitope-specific modification of human and murine antibodies. This platform functions as a ‘USB’ interface, enabling rapid functional derivatization of antibodies. In summary, this work provides chemically and spatially tunable epoxide warhead for ‘on-demond’ precision cross-linking at protein interfaces, opening avenues for atomic-scale manipulation of protein interactions.