PROJECT SUMMARY/ABSTRACT: Bacteria on and within the body (the microbiota) influence human physiology, therapeutic responses, and dis- ease states. Many of these effects are mediated by small molecules, but determining the structures and func- tions of these small molecule mediators is challenging due to the complex metabolic and genetic background of the microbiota. Certain strains of E. coli in the human gut contain a gene cluster (referred to as “clb” or “pks”) that encodes the biosynthesis of a metabolite known as colibactin. Several independent studies have demon- strated that the clb cluster is epidemiologically-correlated with colorectal cancer (CRC) in humans, and recently a casual role for clb+ bacteria in tumorigenesis has been established. However, it has been impossible to link these effects directly to colibactin because the molecule is unstable and cannot be isolated from the bacteria. During the prior funding period we indirectly elucidated the overall structure of colibactin by a combination of chemical synthesis, genetics, stable isotope labeling, mass spectrometry (MS) and enzymology. This work es- tablished colibactin as a heterodimeric molecule capable of generating interstrand cross-links (ICLs) in DNA via two-fold nucleotide addition to electrophilic cyclopropane residues. Nonetheless, because the structure was based largely on MS analysis, alternative colibactin isomers are possible and we now have evidence that the right-hand warhead of colibactin undergoes ring–chain isomerization to form more complex isomers. Addition- ally, the central α-aminoketone of colibactin is unstable toward oxidative degradation and it is not yet known whether or not this residue is present at the time of DNA cross-linking. Here we will carry out the synthesis and biological evaluation of synthetic colibactins with the goal of resolving these final structural ambiguities. We have designed and will synthesize colibactin derivatives locked in the ring and chain isoforms and carry out studies to evaluate the genotoxicity of each of these compounds. We have successfully synthesized the α-aminoketone core of colibactin and will use this chemistry to prepare colibactin 771, the immediate product of the clb pathway. We will carry out crystallographic studies to determine the mode of binding of colibactin to DNA, and the mode of interaction of colibactin precursors with the maturation enzyme ClbP, the self-resistance enzyme ClbS, and the transmembrane transporter ClbM. Significantly, the synthetic chemistry we have developed allows us to probe for a causal link between colibactin and tumorigenesis through elucidation of the mutational signature triggered by the molecule. We will map out the functional host response to colibactin exposure using a high- throughput CRISPR genomic screen. This work is essential in that it provides the only means to study colibactin since it cannot be obtained from natural sources. It will test the hypothesis that colibactin...