A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases

Abstract

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.

Inhibition of microbial deconjugation of micellar bile acids protects against intestinal permeability and liver injury

“…AAA-10 administration led to ~20-30 µM AAA-10 in cecal contents 48 hours and 1 week post-gavage (Figure 4C). No AAA-10 was detected in peripheral blood (Figure S13A), a finding that is consistent with our previous results (Adhikari et al, 2021) and indicates that this BSH inhibitor exhibits low systemic exposure.…”

“…We then show that this sequestration of unconjugated bile acids protects epithelial cells from damage and permeability in vitro. Furthermore, using a small molecule inhibitor of bacterial BSHs that we recently developed (Adhikari et al, 2021;Adhikari et al, 2020), we show that inhibition of BSH activity increases conjugated BA abundance in vivo and prevents the development of increased intestinal permeability, hepatic steatosis, and hepatic inflammation in diseased rats. Finally, we demonstrate that glyco-conjugated BAs, the predominant BAs in humans, also protect against epithelial damage caused by unconjugated BAs in vitro.…”

“…Because conjugated BAs appear to protect against intestinal epithelial damage, we hypothesized that a decrease in microbial BA deconjugation by BSH might attenuate intestinal permeability in CDAHFD-fed rats. We recently reported the development of a covalent, gut-restricted, small molecule inhibitor of gut bacterial BSHs, AAA-10, a compound that effectively inhibits BSH activity and increases the abundance of conjugated BAs in vivo (Figure 4A) (Adhikari et al, 2021). We thus hypothesized that treatment of CDAHFD-fed rats with AAA-10 would prevent increased intestinal permeability.…”

Inhibition of microbial deconjugation of micellar bile acids protects against intestinal permeability and liver injury

“…We recently reported the development of a covalent, gut-restricted, small-molecule inhibitor of gut bacterial BSHs, AAA-10, a compound that effectively inhibits BSH activity and increases the abundance of conjugated BAs in vivo ( Fig. 4A ) ( 26 ). We thus hypothesized that treatment of CDAHFD-fed rats with AAA-10 would prevent increased intestinal permeability.…”

“…We then show that this physicochemical sequestration of unconjugated BAs protects epithelial cells from damage and permeability in vitro, thus providing a signaling-independent role for micellar BAs in maintaining gut barrier integrity. Furthermore, using a small-molecule inhibitor of bacterial BSHs that we recently developed (26,27), we show that inhibition of BSH activity increases conjugated BA abundance and prevents the development of increased small intestine permeability, hepatic steatosis, and hepatic inflammation in diseased rats. Last, we demonstrate that glyco-conjugated BAs, the predominant BAs in humans, also protect against epithelial damage caused by unconjugated BAs in vitro.…”