Development of Chemical Tools to Investigate Colibactin, a Genotoxin from the Human Microbiota

Abstract

The bacterial genotoxin colibactin is produced by certain commensal and pathogenic strains of E. coli and other Proteobacteria and may be a key driver of colorectal cancer (CRC) development. Colibactin induces mutations and genomic instability in mammalian cells by forming DNA interstrand crosslinks (ICLs) and double-strand breaks (DSBs). However, understanding colibactin’s biological effects remains difficult due to the instability of the proposed genotoxic agent and the complexity of the gut microbiota. Colibactin forms ICLs via double alkylation of DNA using its two electrophilic cyclopropane warheads. The biosynthesis of colibactin employs a prodrug resistance mechanism in which a precursor, precolibactin, is hydrolytically activated to form colibactin. This final activation step is performed by an unusual inner membrane-bound periplasmic serine peptidase, ClbP. This enzyme presents an ideal target for the development of chemical tools to monitor and modulate colibactin biosynthesis. Through our detailed characterization of ClbP, we have developed such tools, which will enable us to interrogate colibactin’s biosynthesis and genotoxic effects in unprecedented detail. Building on work initiated by past members of our research group, Chapter 2 discusses our detailed in vitro biochemical characterization of catalytically active, full-length ClbP. Through a structure-activity relationship (SAR) study, we elucidated ClbP’s high degree of selectivity for D-asparagine-containing peptide substrates with large, hydrophobic N-terminal acyl groups. Using this information, we developed a fluorogenic activity probe which can be activated by ClbP in a manner analogous to the activation of precolibactin. This activity probe detected ClbP activity both in vitro and in live colibactin-producing E. coli. We designed and optimized a high-throughput screening assay to discover ClbP inhibitors, but discovered that none of the commercially available compounds tested can inhibit ClbP. Chapter 3 describes our efforts to rationally design the first true inhibitors of ClbP. Using the results from our SAR study as a guide, we explored the synthesis of a variety of different putative inhibitors which were designed with electrophilic groups to covalently engage ClbP’s catalytic nucleophile, S95. Ultimately, a boron-based inhibitor proved to be the most effective and accessible lead. We developed an improved route to access this compound via a stereoselective copper-catalyzed hydroboration and prepared a panel of four electrophilic substrate analogs. These compounds all inhibited ClbP in vitro and in live bacteria with IC50 values below 50 nM. They also retained efficacy in the context of complex bacterial communities, recapitulated the metabolic changes observed in a ΔclbP mutant, and were highly selective for ClbP over other serine hydrolases. Chapter 4 explores potential applications for these inhibitors in studying colibactin and related natural products. We showed that our inhibitors blocked the genotoxic effects of colibactin on human cell lines using a variety of indicators of DNA damage. We then used these inhibitors to study the activity of other prodrug activating peptidases related to ClbP. We used this inhibitory activity to identify cryptic intermediates in the biosynthesis of zwittermicin and the edeines. The availability of these ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC and providing a starting point for possible therapeutic intervention. The application of our inhibitors to related peptidase-encoding pathways highlights the power and generalizability of chemical tools in studies of natural product biosynthesis.