Identification of Natural Product Inhibitors Against Colibactin-Mediated Early Onset Colorectal Cancer - Abstract This proposal aligns with the NCI’s priority areas in cancer interception and microbiome-based prevention. The rising incidence of early-onset colorectal cancer (EOCRC) has been causally linked to colibactin, a genotoxin produced by colibactin-producing Enterobacteriaceae. To date, colibactin represents one of the most extensively studied microbial metabolites associated with CRC. Colibactin-producing bacteria carry the polyketide synthase (pks) gene island, and recent studies have demonstrated a high prevalence of pks+ bacteria in 55–67% of CRC patients. Colibactin induces DNA damage, senescence, and genomic mutations in the host intestinal epithelium. The causality between colibactin and CRC has been validated in mouse CRC models, human colon organoids, and CRC tumor specimens. Despite strong evidence connecting colibactin to CRC, no FDA-approved agents exist to selectively neutralize this metabolite. Our objective is to identify and validate first-in-class natural product inhibitors of ClbQ, an upstream thioesterase essential for colibactin biosynthesis, and to assess their efficacy and safety in preclinical cancer prevention models. This UG3/UH3 proposal is structured in two phases based on the hypothesis that ClbQ inhibition will prevent colibactin biosynthesis at an early stage, thereby reducing host DNA damage and interrupting CRC initiation. In the UG3 phase, Aim 1 will establish a high-throughput screening (HTS) platform to identify ClbQ inhibitors from a unique microbial natural product library using in silico docking, enzymatic assays, and functional validation in bacterial and human cell-based models. Hits will undergo orthogonal screening to assess selectivity, host toxicity, and microbial specificity. The UH3 phase (Aims 2 and 3) will prioritize and optimize lead compounds by structure– activity relationship studies, orthogonal functional assays in colon organoids, and in vivo testing in the CDX2P- CreERT2-Apcfl/+ model of early-stage CRC. Benchmarking against existing inhibitors targeting a downstream colibactin biosynthesis enzyme, ClbP, will clarify the therapeutic advantage of inhibiting an upstream enzyme, ClbQ. Efficacy endpoints include suppression of DNA damage, tumor burden, and colibactin-specific mutational signatures, alongside pharmacokinetic and microbiome safety evaluations. This proposal will be led by Drs. Sherman, Li, Tripathi and Shah, who bring complementary expertise in natural product drug discovery, medicinal chemistry and cancer biology in CRC. Our approach integrates in silico and biochemical screening, organoid models, and mouse models of early CRC. The expected outcome is to deliver first-in-class natural product-based inhibitors of colibactin, thereby complementing current CRC screening efforts, particularly for at-risk populations carrying high levels of pks⁺ bacteria. Our long-term goal is to address an unmet need in cancer prevention by targeting microbiome-derived genotoxins at the root of disease initiation.
