A phage display approach to identify highly selective covalent binders

Abstract Molecules that bind macromolecular targets through direct covalent modification have found widespread applications as activity-based probes (ABPs) and as irreversible drugs. Covalent binders can be used to dynamically monitor the activity of enzymes in complex cellular environments, identify targets and induce permanent binding/inhibition of therapeutically important biomolecules. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, robust and unbiased screening method to identify new classes of covalent binding ligands from highly diverse pools of candidate molecules. Here we describe the development of a phage display method to screen for highly selective covalent binding ligands. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that selectively and irreversibly inhibited a cysteine protease with nanomolar potency, exceptional specificity and increased serum stability compared to a linear peptide containing the same electrophile. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent binding ligands for diverse molecular targets.