Fig. 4. Compound 3 is selective for ClbP inhibition and active in a community setting.
a, LC–MS measurement of N-myristoyl-d-Asn released from BWpks after treatment with 3 or 5. n = 3 biological replicates, individual replicates shown. b, Volcano plot representation of metabolites detected by LC–MS that are altered in BWpks treated with 1 µM of compound 3 versus untreated (left) and BWΔP versus BWpks (right). Previously characterized colibactin precursor metabolites are labeled with their m/z. n = 5 biological replicates for all conditions. c, Gel-based ABPP using a FP-biotin probe to examine the reactivity of serine hydrolases in E. coli and HEK293T cell lysates does not identify any major targets of compound 3. ClbP is not detected in this assay because of a lack of interaction between it and FP. d, LC–MS measurement of the prodrug scaffold in extracts of E. coli NC101 and E. coli NC101ΔclbP cultured with and without compound 3 and with or without a community of organisms resuspended from fecal pellets of C57BL/6J mice. The levels of prodrug scaffold observed in conditions 3 and 4 are expected as a side product from the upstream enzymes in colibactin biosynthesis. n = 3 biological replicates for all conditions, individual replicates shown. Empty circles are below the limit of quantitation for this protocol (4 nM). ****P < 0.0001; *P < 0.05; NS, P > 0.05; one-way ANOVA and Bonferroni’s multiple comparison test.