Discovery of Bile Salt Hydrolase Inhibitors Using an Efficient High-Throughput Screening System

Smith, Katie; Zeng, Xianying; Lin, Jun

doi:10.1371/journal.pone.0085344

Cited by 42 publications

(59 citation statements)

References 55 publications

(70 reference statements)

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“…We also tested riboflavin ( 10 ) and caffeic acid phenethyl ester (CAPE, 11 ), compounds that had been previously identified in a high throughput screen for inhibition of a BSH from a Lactobacillus salivarius chicken gut isolate. 33 (Fig. 1e).…”

Section: Resultsmentioning

confidence: 96%

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Adhikari

Seegar

Ficarro

et al. 2019

Preprint

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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...

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Section: Resultsmentioning

confidence: 96%

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Adhikari

Seegar

Ficarro

et al. 2019

Preprint

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“…CAPE is a potential BSH inhibitor identified by high-throughput screening in vitro (10). To verify the effects of CAPE on BSH in vivo, the cecum content BSH activities were measured in mice on an HFD after they were subjected to CAPE treatment, which was found to substantially attenuate the BSH activities (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, inhibition of bacterial BSH could block intestinal FXR signaling by increasing intestinal T-β-MCA. To this end, treatment with caffeic acid phenethyl ester (CAPE), a BSH inhibitor (10), was used in the current study. BSH increased T-β-MCA, resulting in inhibition of intestinal FXR signaling and decreased ceramide synthesis.…”

Section: Introductionmentioning

confidence: 99%

An Intestinal Farnesoid X Receptor–Ceramide Signaling Axis Modulates Hepatic Gluconeogenesis in Mice

et al. 2016

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Increasing evidence supports the view that intestinal farnesoid X receptor (FXR) is involved in glucose tolerance and that FXR signaling can be profoundly impacted by the gut microbiota. Selective manipulation of the gut microbiota–FXR signaling axis was reported to significantly impact glucose intolerance, but the precise molecular mechanism remains largely unknown. Here, caffeic acid phenethyl ester (CAPE), an over-the-counter dietary supplement and an inhibitor of bacterial bile salt hydrolase, increased levels of intestinal tauro-β-muricholic acid, which selectively suppresses intestinal FXR signaling. Intestinal FXR inhibition decreased ceramide levels by suppressing expression of genes involved in ceramide synthesis specifically in the intestinal ileum epithelial cells. The lower serum ceramides mediated decreased hepatic mitochondrial acetyl-CoA levels and pyruvate carboxylase (PC) activities and attenuated hepatic gluconeogenesis, independent of body weight change and hepatic insulin signaling in vivo; this was reversed by treatment of mice with ceramides or the FXR agonist GW4064. Ceramides substantially attenuated mitochondrial citrate synthase activities primarily through the induction of endoplasmic reticulum stress, which triggers increased hepatic mitochondrial acetyl-CoA levels and PC activities. These results reveal a mechanism by which the dietary supplement CAPE and intestinal FXR regulates hepatic gluconeogenesis and suggest that inhibiting intestinal FXR is a strategy for treating hyperglycemia.

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“…Bile salt hydrolase activity assay. Faecal water was prepared and total faecal protein quantified similarly to a method previously-described 41 , but with the addition of bacterial and mammalian protease inhibitor cocktails (G Biosciences, Uttar Pradesh, India), as well as Dithiothreitol to 1 mM final concentration (Roche, Basel, Switzerland) (to minimise enzyme oxidation 42 ).…”

Section: Methodsmentioning

confidence: 99%

Ursodeoxycholic acid enriches intestinal bile salt hydrolase-expressing Bacteroidetes in cholestatic pregnancy

Ovadia

Perdones-Montero

Fan

et al. 2020

Sci Rep

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Ursodeoxycholic acid (UDcA) treatment can reduce itch and lower endogenous serum bile acids in intrahepatic cholestasis of pregnancy (ICP). We sought to determine how it could influence the gut environment in icp to alter enterohepatic signalling. the gut microbiota and bile acid content were determined in faeces from 35 pregnant women (14 with uncomplicated pregnancies and 21 with ICP, 17 receiving UDCA). Faecal bile salt hydrolase activity was measured using a precipitation assay. Serum fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) concentrations were measured following a standardised diet for 21 hours. Women with a high ratio of Bacteroidetes to Firmicutes were more likely to be treated with UDcA (fisher's exact test p = 0.0178) than those with a lower ratio. Bile salt hydrolase activity was reduced in women with low Bacteroidetes:Firmicutes. Women taking UDcA had higher faecal lithocholic acid (p < 0.0001), with more unconjugated bile acids than women with untreated ICP or uncomplicated pregnancy. UDCA-treatment increased serum FGF19, and reduced C4 (reflecting lower bile acid synthesis). During ICP, UDCA treatment can be associated with enrichment of the gut microbiota with Bacteroidetes. these demonstrate high bile salt hydrolase activity, which deconjugates bile acids enabling secondary modification to FXR agonists, enhancing enterohepatic feedback via FGF19.The serum and faecal bile acid composition is intimately related to biotransformation of bile acids by intestinal bacteria, and their subsequent enterohepatic circulation. Deconjugation of primary bile acids by bacterial bile salt hydrolase (BSH) enables unconjugated bile acids to be modified to secondary bile acids. Bile acids act as signalling molecules for many different end organs (e.g. liver, pancreas, adipose tissue, inflammatory cells), with individual bile acid species of differing ligand potency for different receptors (e.g. farnesoid X receptor (FXR), Takeda G-protein-coupled receptor 5 (TGR5)) 1-3 .Intrahepatic cholestasis of pregnancy (ICP) is predominantly a liver disorder specific to pregnancy, defined by pruritus and elevated serum bile acids beyond the normal asymptomatic hypercholanaemia of pregnancy. Fetal adverse outcomes are related to the extent of elevation of serum concentrations of total bile acids 4,5 . Women with ICP have increased rates of impaired glucose tolerance, gestational diabetes mellitus, and dyslipidaemia 6,7 .

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Discovery of Bile Salt Hydrolase Inhibitors Using an Efficient High-Throughput Screening System

Cited by 42 publications

References 55 publications

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

Development of a Covalent Inhibitor of Gut Bacterial Bile Salt Hydrolases

An Intestinal Farnesoid X Receptor–Ceramide Signaling Axis Modulates Hepatic Gluconeogenesis in Mice

Ursodeoxycholic acid enriches intestinal bile salt hydrolase-expressing Bacteroidetes in cholestatic pregnancy

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