Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Abstract

17Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the 18 gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and 19 immune processes by binding to host receptors. There is an unmet need for a potent tool to 20 inhibit BSHs across all gut bacteria in order to study the effects of bile acids on host physiology. 21Here, we report the development of a covalent pan-inhibitor of gut bacterial BSH. From a 22 rationally designed candidate library, we identified a lead compound bearing an alpha-23 fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. Strikingly, this 24 inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose 25 of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels 26 in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid 27 composition in vivo. 28 29 performed exclusively by gut bacterial bile salt hydrolase (BSH) enzymes. 1 BSHs (EC 3.5.1.24) 54 are widespread in human gut bacteria. A recent study identified BSHs in gut species from 117 55 genera and 12 phyla, including the two dominant gut phyla, Bacteroidetes and Firmicutes, as 56 well as Actinobacteria and Proteobacteria. 10 A non-toxic, small molecule pan-inhibitor of gut 57 bacterial BSHs would provide a powerful tool to study how bile acids affect host physiology. 58Such a compound should limit bile acid deconjugation across the vast majority of gut strains 59 without significantly affecting the growth of these bacteria. The use of a pan-inhibitor in vivo 60 would significantly alter bile acid pool composition, shifting the pool toward conjuated bile acids 61 and away from deconjugated bile acids and secondary bile acids (Fig. 1a). This chemical tool 62 would thus allow researchers to investigate previously unanswered questions, including how 63 primary and secondary bile acids differentially affect physiology in a fully colonized host. 64Herein, we report the development of a covalent inhibitor of bacterial BSHs using a 65 rational design approach. Importantly, this compound completely inhibits BSH activity in 66 conventional mouse feces, demonstrating its potential utility as a pan-inhibitor of BSHs. 67 68Results 69 Rational design of covalent small molecule inhibitors of bile salt hydrolases 70In order to generate potent, long-lasting inhibitors of BSHs, we chose to develop covalent 71 inhibitors of these gut bacterial enzymes. Covalent inhibitors have gained renewed interest in the 72 field of drug discovery due to their ability to inactivate their protein target with a high degree of 73 potency and selectivity even in the presence of large concentrations of native substrate. 11 The 74 substrates for BSHs, conjugated bile acids, are found in high concentrations in the colon (1-10 75 mM), 12 suggesting that covalent inhibition could be an effective strategy for targeting these 76 enzymes. In addition, recently develop...

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

“…Using a previously described assay of in vivo BSH activity 25 , we found that cecal BSH activity of CDAHFD-fed rats was significantly increased compared to control rats (Figure 5A). Importantly, no significant differences were seen in expression of BA synthesis or conjugation genes between CDAHFD-fed and control rats at 48 hours after dietary intervention (Figure S8).…”

“…400 µL of equimolar mixtures of bile acids at various concentrations to be tested were added to tubes and rotated overnight at BSH activity assay. BSH activity was quantified using a modified version of a previously described method 25 . Briefly, fresh cecal contents (approximately 20 mg) were diluted in PBS to obtain a concentration of mg/mL µM glycochenodeoxycholic acid-d4 (GCDCA-d4) was added to the mixture and incubated at 37ºC for 30 minutes, then frozen in dry ice for 5 minutes and stored at -80ºC until further analysis.…”

A gut-restricted lithocholic acid analog as an inhibitor of gut bacterial bile salt hydrolases

“…In prior work, sulfonation of AAA-1 ( 1 ) at C3, a position that is exposed to solvent in the co-crystal structure of this compound with the BSH from the gut bacterium Bacteroides thetaiotaomicron ( B. theta ), increased the solubility of this compound and limited its systemic exposure. 24,28 The resultant compound AAA-2 , ( 2 ), however, was less potent than AAA-1 . 24 With the goal of improving potency while still maintaining gut restriction, we decided to append the optimized FMK warhead on naturally occurring bile acid cores found in both the murine and human gut (CDCA, DCA, ursodeoxycholic acid (UDCA), cholic acid (CA), and LCA).…”

“…24,28 The resultant compound AAA-2 , ( 2 ), however, was less potent than AAA-1 . 24 With the goal of improving potency while still maintaining gut restriction, we decided to append the optimized FMK warhead on naturally occurring bile acid cores found in both the murine and human gut (CDCA, DCA, ursodeoxycholic acid (UDCA), cholic acid (CA), and LCA). These compounds could then be sulfonated at the C3 position to produce second-generation inhibitor candidates (Figure 1B).…”

“…In prior work, we took advantage of the nucleophilicity of the highly conserved active site N-terminal cysteine residue (Cys2) in BSHs to develop first-in-class covalent pan-BSH inhibitors (Figure 1B). 24 In this study, we screened electrophilic warheads appended to the core of chenodeoxycholic acid (CDCA), an abundant human bile acid that is recognized by a broad spectrum of BSHs. 20,25,26 This work established an alpha-fluoromethylketone (FMK)-containing molecule, compound 7 (referred to here as AAA-1, 1 ), as a potent and selective pan-BSH inhibitor.…”