Azepinopeptides A and B, a new lanthipeptide class, exhibit potent neuroprotective activity via rSAM-catalyzed azepinoindole scaffold formation

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rich source of structurally diverse and bioactive natural products, yet novel enzymatic modifications remain to be explored. Specifically, radical S-adenosylmethionine (rSAM)-catalyzed C-C bond formation has been largely unexplored in lanthipeptide biosynthesis. Discovering new enzymatic pathways for peptide modification can unlock novel chemical scaffolds with therapeutic potential, addressing limitations in current drug discovery by expanding structural diversity. This study explores an unprecedented rSAM-catalyzed cyclization to generate novel lanthipeptides.

Researchers discovered azepinopeptide A, a new lanthipeptide class, through investigation of rSAM-catalyzed C-C bond formation. They performed biochemical reconstitution and mutational analyses to characterize the rSAM enzymes responsible for forming the tetrahydropyrrolo[1',2':1,2]azepino[3,4-b]indole scaffold. Computational structural analysis and in vivo co-expression studies elucidated the enzyme's specialized binding pocket and catalytic mechanism. The neuroprotective activity of Azepinopeptides A and B was assessed, comparing their efficacy to the positive control, 3-n-butylphthalide. Bioinformatic analyses were also conducted to identify related biosynthetic gene clusters.

A novel class of lanthipeptides, azepinopeptides, was discovered, featuring an unprecedented tetrahydropyrrolo[1',2':1,2]azepino[3,4-b]indole scaffold. This unique structure is formed via rSAM-catalyzed cross-linking of adjacent Trp1 and Pro2 residues, representing an unusual sp2-sp3 C-C bond formation previously unobserved in any RiPP rSAM system. Biochemical and mutational studies revealed that these distinct rSAM enzymes exclusively recognize the mature, leader-free lanthipeptide substrate, lacking the RiPP recognition element and auxiliary iron-sulfur cluster found in other RiPP rSAM systems. Computational analysis and in vivo studies identified a specialized binding pocket that positions the catalytic [4Fe-4S] cluster deep within, explaining the observed regioselectivity and strict requirement for leader peptide removal. Bioinformatic analyses further uncovered hundreds of related biosynthetic gene clusters, indicating the widespread nature of this novel biosynthetic strategy. > Azepinopeptides A and B exhibited potent neuroprotective activity at low concentrations, comparable to the positive control, 3-n-butylphthalide.