Development of a high-throughput strategy for discovery of potent analogues of antibiotic lysocin E

Lysocin E, a 37-membered natural depsipeptide, induces rapid bacteriolysis in methicillin-resistant Staphylococcus aureus via a unique menaquinone-dependent mechanism, presenting a promising therapeutic lead. Despite the great medical importance, exploring the potential utility of its derivatives as new platform structures for antibiotic development has remained a significant challenge. Here, we report a high-throughput strategy that enabled the preparation of thousands of analogues of lysocin E and large-scale structure-activity relationship analyses. We integrate 26-step total synthesis of 2401 cyclic peptides, tandem mass spectrometry-sequencing, and two microscale activity assays to identify 23 candidate compounds. Re-synthesis of these candidates shows that 11 of them (A1–A11) exhibit antimicrobial activity superior or comparable to that of lysocin E, and that lysocin E and A1–A11 share l-Leu-6 and l-Ile-11. Therefore, the present strategy allows us to efficiently decipher biologically crucial residues and identify potentially useful agents for the treatment of infectious diseases.