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

Macrocyclic peptides are an attractive therapeutic modality capable of engaging challenging protein targets while retaining many favourable drug-like properties. Their high-affinity binding also provides an ideal framework for proximity-driven covalent inhibition through incorporation of latent electrophiles. Phage display enables the high-throughput screening of billion-member macrocyclic peptide libraries; however, existing libraries rely predominantly on cysteine-mediated cyclisation, restricting the range of macrocyclic topologies available for ligand discovery.

Here, we report a mild and efficient cyclisation strategy based on a bromomethyl picolinaldehyde (BMP) linker that reacts with a cysteine side chain and the peptide N-terminus to generate a previously unexplored macrocyclic topology incorporating neighbouring pyridine and imidazolidinone rings. The chemistry is compatible with phage display and enabled screening of BMP-cyclised peptide libraries against plasma kallikrein, yielding a potent macrocyclic inhibitor. The BMP-cyclised peptide displayed substantially greater potency than analogous peptides cyclised through either a disulfide bond or the widely used linker 1,4-bis(bromomethyl)benzene (DBMB). Furthermore, comparison with an equivalent DBMB-cyclised library demonstrated that BMP-mediated cyclisation enabled access to binding motifs not identified by conventional cysteine-to-cysteine cyclisation.

Finally, positional sulfur(VI) fluoride exchange (SuFEx) electrophile scanning converted the BMP-derived hit into a selective covalent macrocyclic activity-based probe capable of labelling plasma kallikrein in human plasma. Together, these findings establish BMP-mediated cyclisation as a versatile strategy for expanding the topological diversity of phage-displayed macrocycles and accelerating the discovery of both reversible and covalent macrocyclic peptide ligands.