Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery

Macrocyclic peptides possess the ability to interact with difficult protein targets while maintaining desirable drug-like characteristics. These peptides exhibit strong binding affinity, making them suitable for proximity-driven covalent inhibition via latent electrophiles. Phage display allows for the screening of vast macrocyclic peptide libraries; yet, current libraries predominantly utilize cysteine-mediated cyclisation, constraining the variety of macrocyclic conformations available for ligand exploration.

Researchers have now devised a gentle and efficient cyclisation method utilizing bromomethyl picolinaldehyde (BMP) linker that reacts with both a cysteine side chain and the peptide N-terminus, creating a previously unexplored macrocyclic structure with pyridine and imidazolidinone rings. This chemistry is compatible with phage display, enabling the screening of BMP-cyclised peptide libraries against plasma kallikrein, resulting in a highly effective macrocyclic inhibitor.

The BMP-cyclised peptide exhibited considerably higher potency compared to peptides cyclised through disulfide bonds or the commonly used linker 1,4-bis(bromomethyl)benzene (DBMB). Additionally, when compared to a DBMB-cyclised library, BMP-mediated cyclisation revealed binding motifs that were not identified through traditional cysteine-to-cysteine cyclisation.

Lastly, the application of positional sulfur(VI) fluoride exchange (SuFEx) electrophile scanning transformed the BMP-derived hit into a selective covalent macrocyclic activity-based probe, capable of labeling plasma kallikrein in human plasma. Overall, these discoveries demonstrate BMP-mediated cyclisation as a flexible approach for broadening the topological variety of phage-displayed macrocycles and expediting the identification of both reversible and covalent macrocyclic peptide ligands.