Probing Microbiome Genotoxicity: A Stable Colibactin Provides Insight into Structure-Activity Relationships and Facilitates Mechanism of Action Studies

Colibactin is a genotoxic metabolite produced by commensal-pathogenic members of the human microbiome that possess the clb (aka pks) biosynthetic gene cluster. clb⁺ bacteria induce tumorigenesis in models of intestinal inflammation and have been causally linked to oncogenesis in humans. While colibactin is believed underlie these effects, it has not been possible to study the molecule directly due to its instability. Herein, we report the synthesis and biological studies of colibactin 742 (4), a stable colibactin derivative. We show that colibactin 742 (4) induces DNA interstrand-cross-links, activation of the Fanconi Anemia DNA repair pathway, and G₂/M arrest in a manner similar to clb⁺E. coli. The linear precursor 9, which mimics the biosynthetic precursor to colibactin, also recapitulates the Bacterial phenotype. In the course of this work, we discovered a novel cyclization pathway that was previously undetected in MS-based studies of colibactin, suggesting a refinement to the natural product structure and its mode of DNA binding. Colibactin 742 (4) and its precursor 9 will allow researchers to study colibactin's genotoxic effects independent of the producing organism for the first time.