Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery
- 1University of Bath
Abstract
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.
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BibTeX
@misc{sawtell2026expanding,
title = {{Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery}},
author = {E. Sawtell and M. Barrueco and J. R. Whiteside and C. Williams and M. Laabei and S. Lovell},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.04.749435},
url = {https://www.biorxiv.org/content/10.64898/2026.09.04.749435v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery
AU - E. Sawtell
AU - M. Barrueco
AU - J. R. Whiteside
AU - C. Williams
AU - M. Laabei
AU - S. Lovell
PY - 2026
DA - 2026/09/04
PB - bioRxiv
DO - 10.64898/2026.09.04.749435
UR - https://www.biorxiv.org/content/10.64898/2026.09.04.749435v1
AB - 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.
ER -