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Bile acids play crucial roles in host physiology by acting both as detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. Structure-activity relationship (SAR) studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in mouse and human fecal slurry, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo .

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron , we selectively modulated the levels of the bile acid tauro-β-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales., Competing Interests: LY, SN, AA, ND, AM, AB, LB No competing interests declared, SS is currently affiliated with Indigo Agriculture, but the research was conducted when she was a Research Associate at Harvard Medical School. The author has no other competing interests to declare, AD is a consultant for Kintai Therapeutics, (© 2018, Yao et al.)