Comprehensive structural investigation of a potent and selective CXCR4 antagonist via crosslink modification.

Macrocyclization presents a valuable strategy for enhancing the pharmacokinetic and pharmacodynamic profiles of short bioactive peptides. The exploration of various macrocyclic characteristics, such as crosslinking tethers, ring size, and orientation, is generally conducted during the early stages of development. Herein, starting from a potent and selective C-X-C chemokine receptor 4 (CXCR4) cyclic heptapeptide antagonist mimicking the N-terminal region of CXCL12, we demonstrated that the disulfide bridge could be successfully replaced with a side-chain to side-chain lactam bond, which is commonly not enlisted among the conventional disulfide mimetics. An extensive investigation was carried out to explore the chemical space of the resulting peptides, including macrocyclization width, stereochemical configuration, and lactam orientation, all of which were correlated with biochemical activity. We identified a novel heptapeptide that fully replicates the pharmacological profile of the parent peptide on CXCR4, including its potency, selectivity, and antagonistic activity, while demonstrating enhanced stability in a reductive environment. At this stage, computational studies were instructed to shed light on how the lactam cyclization features influenced the overall structure of 21 and, in turn, its ability to interact with the receptor. We envisage that these findings can give new momentum to the use of lactam cyclization as a disulfide bond mimetic and contribute to the enhancement of the repertoire for peptide-based drug development, thereby paving the way for novel avenues in therapeutic innovation.