Natural Product-Derived Ianthelliformisamines Inhibit Protein Translation and Block Bacterial Flagellum Assembly

Ianthelliformisamines (Ian) represent a poorly characterized natural product class reported to inhibit Gram-negative bacteria such as Escherichia coli. Given the current antibiotic crisis, revisiting poorly characterized antibacterial natural products may reveal novel modes of action (MoA) as inspiration for drug development. Thus, we elucidated the Ian mode of action and synthesized three Ian analogs along with a chemical probe for activity-based protein profiling (ABPP). All molecules retained antibacterial effects, which were enhanced in the presence of bicarbonate, an abundant ingredient of human serum. Chemical proteomics with the probe unraveled InfA, involved in the initiation of bacterial ribosomal protein biosynthesis, as an essential target, which was confirmed by translation assays. Intriguingly, a virulence-associated target stood out as an additional hit, FliC, with a crucial role in flagellum assembly. The recombinant protein was probe-labeled, and motility assays together with transmission electron microscopy revealed impaired motility and disrupted flagellum assembly, respectively. Consistent with this dual antibacterial/antivirulence profile, Ian treatment reduced invasion of pathogenic E. coli into human host cells. This work illustrates how activity-based chemical proteomics can uncover previously unrecognized cellular targets for natural product scaffolds, thereby revealing distinct modes of action and supporting further therapeutic development.