Phenotypic high-throughput screening identifies modulators of gut microbial choline metabolism

Anaerobic metabolism of dietary choline to trimethylamine (TMA) by the human gut microbiome is a disease-associated pathway. The host's impaired ability to oxidize TMA to trimethylamine-<i>N</i>-oxide (TMAO) results in trimethylaminuria (TMAU), while elevated serum TMAO levels have been positively correlated with cardiometabolic disease. Small molecule inhibition of gut bacterial choline metabolism attenuates the development of disease in mice, highlighting the therapeutic potential of modulating this metabolism. Inhibitors previously developed to target this pathway are often designed to mimic choline, the substrate of the key TMA-generating enzyme choline trimethylamine-lyase (CutC). Here, we use a growth-based phenotypic high-throughput screen and medicinal chemistry to identify distinct chemical scaffolds that can modulate anaerobic microbial choline metabolism and lower TMAO levels <i>in vivo</i>. These results illustrate the potential of using phenotypic screening to rapidly discover new inhibitors of gut microbial metabolic activities.IMPORTANCEGut microbial metabolic activities play important roles in human health, prompting interest in the discovery of gut microbiome-targeted small molecule inhibitors as potential therapeutics. Anaerobic choline metabolism by the gut microbiome generates trimethylamine and its downstream metabolite trimethylamine-<i>N</i>-oxide (TMAO), which cause trimethylaminuria and are correlated with cardiometabolic diseases, respectively. Current strategies for modulating microbial metabolism with small molecule inhibitors typically require having a target enzyme. Here, we show that a growth-based phenotypic screen can identify inhibitors of choline metabolism with chemical scaffolds that are structurally distinct from choline and existing inhibitors. The resulting optimized compounds lower serum TMAO in gnotobiotic mice without significantly perturbing gut microbiome composition. This work highlights the potential of using phenotypic screening to rapidly discover additional inhibitors of microbial metabolic activities, which would accelerate mechanistic studies of the microbiome and deepen our understanding of disease biology from correlation to causation.