Small molecule regulators of intestinal bacterial bile acid metabolism

Abstract

Described herein are methods and compositions and uses thereof involving inhibition of bile salt hydrolase (BSH). Provided herein are methods and compositions for treating metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases (e.g., gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis), gastroesophageal reflux disease (GERD) in a subject in need thereof. ); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., digestive cancer, liver cancer), or inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis), the method comprising administering to a subject a compound of formula (I)-(XVIII).

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 846,457, filed May 10, 2019, and U.S.S.N. 62 / 962,048, filed January 16, 2020, under 35 U.S.C.§119(e), the disclosures of which are incorporated herein by reference in their entireties.

[0003] Government Support

[0004] This invention was made with government support under Grant Nos. R35 GM128618 and 5P30DK034854-32 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0005] The technology described herein relates to compounds, compositions, and methods for inhibiting bile salt hydrolase (BSH). Background Art

[0006] Bile salt hydrolase (BSH) is widely expressed by human gut bacteria and catalyzes a gateway reaction that results in the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Both primary and secondary bile acids regulate key metabolic and immune processes in the host by acting as ligands for host receptors. There is currently an unmet need for effective selective agents that inhibit BSH for the treatment of diseases such as cancer, inflammation, obesity, diabetes, and gastrointestinal diseases, and for use as tools to understand bile acid physiology in host subjects. Summary of the Invention

[0007] In one aspect, provided herein is a compound of formula (I):

[0008]

[0009] Wherein:

[0010] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0011] m is 1, 2, 3, or 4;

[0012] X is an electrophilic group;

[0013] R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R11 , R 12 , R 15 , R 16 and R 17 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 18 , N(R 18 ), 2 , SR 18 , halogen, CN, -CHO, -CO 2 H, -CO 2 R 18 , -NO 2 , -ONO 2 , -SO 2 Cl, -SO 3 - , -OSO 3 - , -NR 18 SO 3 - , -PO 3 2- , -OPO 3 2- , -OSO 2 R 18 , -SO 2 N(R 18 ), 2 , -OSO 2 N(R 18 ), 2 , -NR 18 SO 2 R 18 , -SO 2 N(R 18 ), 2 , -NHNH 2 , -ONH 2 , or -NHC(O)NHNH 2 ;

[0014] Each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0015] or a pharmaceutically acceptable salt thereof.

[0016] In one aspect, the compound of formula (I) is of formula (I’):

[0017]

[0018] Wherein:

[0019] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0020] m is 1, 2, 3 or 4;

[0021] X is an electrophilic group;

[0022] R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 18 , N(R 18 ) 2 , SR 18 , halogen, CN, -CHO, -CO 2 H, -CO 2 R 18 , -NO 2 , -ONO 2 , -SO 2 Cl, -SO 3 H, -OSO 3 H, -NR 18 SO 3 , -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 R 18 , -SO 2 , N(R 18 ) 2 , -OSO 2 , N(R 18 ) 2 , -NR 18 SO 2 R 18 , -SO 2 , N(R 18 ) 2 , -NHNH 2 , -ONH 2 , or -NHC(O)NHNH2 , wherein each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0023] or a pharmaceutically acceptable salt thereof.

[0024] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound provided herein and a pharmaceutically acceptable carrier or excipient.

[0025] In another aspect, the present disclosure provides a method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound provided herein.

[0026] In another aspect, the present disclosure provides a method for inhibiting bile acid dissociation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein.

[0027] In another aspect, the present disclosure provides a method for promoting bile acid conjugation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein.

[0028] In another aspect, the present disclosure provides a method of modulating bile acids in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein. In another aspect, the present disclosure provides a method for treating a metabolic disorder (such as diabetes, obesity), a gastrointestinal disease (such as gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancer; liver malignancy; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancer), or an inflammatory disease such as (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis), the method comprising administering to a subject in need thereof a compound of formula (I)-(XVIII), a genetically engineered microorganism that secretes cholate 7-sulfate, or a population thereof.

[0029] On the other hand, there are provided compounds of formula (I)-(XVIII), or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising a compound of formula (I)-(XVIII) for treating metabolic disorders (such as diabetes, obesity), gastrointestinal diseases (such as gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or inflammatory diseases such as (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver diseases, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0030] On the other hand, there are provided kits that comprise a compound of formula (I)-(XVIII), or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising a compound of formula (I)-(XVII). In certain embodiments, the kit further comprises instructions for administration (such as human administration) and / or use.

[0031] Details of particular embodiments of the invention are set forth in the Detailed Description of Certain Embodiments below. Other features, objects, and advantages of the invention will be apparent from the definition, examples, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0033] Figure 1A and 1B shows the chemical and biological effects of the intestinal bacterial bile salt hydrolase (BSH). Figure 1AIt is shown that BSH is the gateway enzyme in the conversion of primary (host-produced) to secondary (bacterial-produced) bile acids. Removal or inhibition of BSH should lead to a decrease in the dissociated primary and secondary bile acids. Figure 1B It is shown that specific primary and secondary bile acids are ligands of host nuclear hormone receptors (NhRs) and G protein-coupled receptors (GPCRs). By acting as agonists or antagonists of these receptors, these bile acids affect host processes, including metabolic control and immune responses.

[0034] Figures 2A to 2E The rational design of small molecule broad-spectrum BSH inhibitors is demonstrated. Figure 2A The mechanism of BSH enzymatic amide bond cleavage is shown. Figure 2B The co-crystal structure of BSH from the Gram-positive gut bacterium Clostridium perfringens with taurodeoxycholic acid (TDCA) (PDB 2BJF) that guides inhibitor design is shown. When hydrophobic interactions orient the bile acid core towards the active site (magenta residue), the D-ring side chain and amino acids are exposed to the solvent. Figure 2C The representative mechanism of BSH inhibition by the rationally designed inhibitors is shown. Attack on the catalytic nucleophilic cysteine residue in the BSH active site can lead to covalent binding to the inhibitor. Figure 2D A library of synthetic inhibitors is shown. The electrophilic warheads successfully introduced in the design of kinase and protease inhibitors are appended to the chenodeoxycholic acid bile acid core, thus generating broad-spectrum BSH inhibitors. Figure 2E The most potent BSH inhibitors identified from high-throughput screening, namely riboflavin and caffeic acid phenethyl ester (CAPE), which are also included in this study, are shown.

[0035] Figures 3A to 3B The screening that identified inhibitor 7 as an effective and persistent inhibitor of recombinant BSH is demonstrated. Figure 3A The screening of the inhibitor library against BSH of Bacteroides thetaiotaomicron (B.theta) is shown, which shows the % dissociation at 2 and 21 hours. Figure 3B The screening of compounds 1, 7, and CAPE against BSH of Bifidobacterium longum (B.Longum) is shown, which shows the % dissociation at 2 and 21 hours. The inhibitor (100 μM) was incubated with 200 nM rBSH for 30 minutes, and then the taurine-conjugated bile acid substrates (TβMCA, TCA, TUDCA, and TDCA, 25 μM each) were added. The dissociation of the substrates was tracked by UPLC-MS. The assays were performed in biological triplicates. Data are represented as mean ± SEM.

[0036] Figures 4A to 4DIt is shown that compound 7 is an effective and non-toxic inhibitor of BSH in the growth cultures of Gram-positive and Gram-negative gut bacteria. Figure 4A It is shown that compound 7 inhibits BSH activity in live Gram-negative (Bacteroides thetaiotaomicron VPI 5482, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482) as well as Gram-positive (Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32) bacteria. The inhibitor (100 μM of compound 7 or CAPE) and taurine-conjugated bile acid substrates (TβMCA, TCA, TUDCA, and TDCA, 25 μM each) were added to the bacterial cultures at an OD 600 of 0.1. The bacterial cultures were allowed to grow to stationary phase, and the percentage of dissociation was measured by UPLC-MS over 24 h. The assays were performed in biological triplicates. Data are represented as mean ± SEM. Tukey's multiple comparison test was performed after one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.00001. No statistical analysis was performed on B. vulgatus because the standard error of parallel assays was zero. Figure 4B It is shown that compound 7 is not bactericidal. The OD of the bacterial cultures was measured at 24 h 600 . CAPE inhibited the growth of the Gram-positive strains tested. The red downward arrows indicate the percentage decrease compared to the DMSO control. Figure 4C It is shown the dose-response curve and calculated IC 50 values of compound 7 incubated with the growth cultures of Bacteroides thetaiotaomicron (Gram-negative) and Bifidobacterium adolescentis (Gram-positive), demonstrating that compound 7 is an effective broad-spectrum BSH inhibitor. Figure 4D It is shown a representative UPLC-MS trace that shows the inhibitor structure determines the BSH inhibitory activity against the growing Bacteroides thetaiotaomicron cultures. Compounds 1, 7, and 9 were tested at 1 and 10 μM concentrations. For simplicity, one substrate (GUDCA) was added to the bacterial cultures, and its dissociation to UDCA was followed by UPLC-MS. The inhibitor 9 with a cholic acid (C12 = OH) core and an α-FMK warhead showed significantly attenuated activity in inhibiting Bacteroides thetaiotaomicron BSH.

[0037] Figures 5A to 5CShows that compound 7 covalently modifies Bacteroides thetaiotaomicron BSH at the active site cysteine residue. Figures 5A to 5B Shows the mass spectrum, revealing that compound 7 singly labels Bacteroides thetaiotaomicron BSH. Figure 5A Shows the mass spectrum (left) and zero charge mass spectrum (right, superimposed) of BSH treated with DMSO (top, red trace) or 10-fold excess of inhibitor compound 7 (bottom, green trace) for 2 hours. The 388 Da mass shift is consistent with covalent modification of BSH and HF loss. Figure 5B Shows the top-down MS of BSH treated with 10-fold excess of inhibitor compound 7. Ions of type c and z are represented by red and green glyphs, respectively. Ion c3 indicates the modification at the N-terminal Cys2 residue. Figure 5C Shows the X-ray co-crystal structure of compound 7 bound to Bacteroides thetaiotaomicron BSH, confirming that compound 7 is covalently linked to the active site Cys2 rather than Cys67, and the C25 fluorine has been eliminated. C3 of the steroid core is exposed to the solvent, indicating that this site is suitable for modification.

[0038] Figures 6A to 6C Shows that compound 7 exhibits minimal off-target effects. Figure 6A Shows that compound 7 is neither a farnesoid X receptor (FXR) agonist nor an antagonist, as determined by the FXR co-activator recruitment assay. The FXR antagonist activity of compound 7 was evaluated at its EC 50 value (50 nM) in the presence of the known FXR agonist GW4064. n = 4 biological replicates per concentration. Data are represented as mean ± SEM. Figure 6B Shows that compound 7 is neither a G protein-coupled bile acid receptor (GPBAR1, also known as TGR5) agonist nor an antagonist. The endogenous TGR5 agonist activity was measured by incubating Caco-2 cells with different concentrations of compound 7 overnight. The endogenous TGR5 antagonist activity was evaluated in the presence of the 10 μM TGR5 agonist LCA. n ≥ 3 biological replicates per concentration. Data are represented as mean ± SEM. Dunnett's multiple comparison test was performed after one-way ANOVA, ns = not significant. Figure 6C Shows that compound 7 does not show toxicity against Caco-2 cells at concentrations up to 50 μM. n ≥ 3 biological replicates per concentration. Data are represented as mean ± SEM. Dunnett's multiple comparison test was performed after one-way ANOVA, *p < 0.05.

[0039] Figures 7A to 7F Shows that compound 7 inhibits BSH activity ex vivo and in vivo. Figure 7AShows the design for fecal BSH activity assay. Feces freshly collected from conventional mice (1 mg / mL) were resuspended in PBS and incubated with 20 μM of inhibitor (Compound 1, 7, or CAPE) for 30 minutes. Glycochenodeoxycholic acid-d4 (GCDCA-d4, 100 μM) was added as a substrate, and dissociation was determined by UPLC-MS after 18 hours. Figure 7B Shows that Compound 7 effectively inhibits BSH activity in fecal slurry, while CAPE shows minimal inhibitory activity. Consistent with the in vitro results, Compound 1 shows moderate BSH inhibition. The assays were performed in biological triplicates. Data are represented as mean ± SEM. Figures 7C to 7E Shows that treatment of conventional mice with a single dose of Compound 7 led to a reversible inhibition of BSH activity and a shift towards deconjugated bile acids. n = 4 mice per group, Welch's t-test, *p < 0.05, **p < 0.01, ns = not significant. Figure 7C Shows the design of the in vivo BSH inhibition experiment. Male conventional C57BL / 6 mice were gavaged with a single dose of Compound 7 (10 mg / kg) or vehicle control. Feces were collected at 1 day, 1.5 days, 2 days, and 2.5 days after gavage. Bile acid analysis was performed at 1 day after gavage. Figure 7D Shows that BSH activity in the group treated with Compound 7 at 1 day and 1.5 days after gavage was significantly lower compared to the control group, as determined by BSH activity in feces. BSH recovered at 2 days after gavage. BSH activity was determined by resuspending fresh feces from inhibitor- or vehicle-treated groups with substrate (GCDCA-d4, 100 μM), incubating for 25 minutes, and quantifying dissociation by UPLC-MS. Figure 7E Shows the fecal bile acid composition at 1 day after gavage. Deconjugated bile acids, including the secondary bile acid deoxycholic acid (DCA), were reduced in the inhibitor-treated group. Figure 7F Shows that there was no difference in microbial biomass between inhibitor- and vehicle-treated groups at 1 day or 2.5 days after gavage. n = 4 mice per group, Mann-Whitney test.

[0040] Figures 8A to 8D Demonstrates that administration of an intestine-restricted derivative of Compound 7, namely 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK), led to a significant decrease in BSH activity within 1 week when fed in the diet. Figure 8A Shows the structure of 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK). Figure 8B Shows the design of the in vivo BSH inhibition experiment. Male conventional C57Bl / 6 mice were fed ad libitum a normal diet or a diet containing 3S-LCA-FMK (0.03% weight / weight) for 7 days. Feces were collected before dietary change and at days 3, 4, and 7 after dietary change. n = 5 mice per group. Figure 8CIt shows that the BSH activity in the feces of mice fed a diet containing 3S-LCA-FMK is significantly reduced. Figure 8D It shows the concentration of 3S-LCA-FMK, as measured in feces and cecal contents at sacrifice. On day 4, 3S-LCA-FMK was not detected in the circulating plasma, indicating that the compound is gut-restricted.

[0041] Figure 9A It shows that the key reaction in the primary to secondary bile acid conversion is the hydrolysis (cleavage) of the C24 amide bond of the conjugated primary bile acid. Figure 9B It shows that although the BSH protein sequences vary significantly among gut strains, all BSHs have a conserved active site that includes the catalytic cysteine (Cys2). Figure 9C It shows the co-crystal structure of Clostridium perfringens BSH and the substrate taurodeoxycholic acid, which shows that hydrophobic interactions involve the bile acid core and orient the amide towards Cys2, exposing the amino acid to the solvent. Figure 9D It shows the compounds of the present disclosure.

[0042] Figure 10 shows the screening of inhibitors against Bacteroides thetaiotaomicron BSH ( Figure 10A ) and Bifidobacterium longum BSH ( Figure 10B ), which shows the % cleavage of taurocholic acid at 2 and 21 hours. Bacterial strains were incubated with 100 μM of the conjugated bile acid and plated at 21 hours to assess strain viability ( Figure 10C ). Compound 7 is not bactericidal ( Figure 10D ). CAPE reduces the cell viability of the tested Gram-negative strains. The red downward arrow indicates the fold reduction compared to the DMSO control. For ( Figure 10C ) and ( Figure 10D ), Dunnett's multiple comparison test was performed after one-way ANOVA. ( Figure 10E ) Compound 7 inhibits BSH activity in fecal slurry. All assays were performed in biological triplicates, and the data are represented as mean ± SEM.

[0043] Figure 11A It shows the X-ray structure of compound 7 bound to Bacteroides thetaiotaomicron BSH. BSH (cyan) is shown as a ribbon, with the indicated side chains (cyan, with heteroatoms in CPK colors) presented as rods. Figure 11B It shows the co-crystal structure of Bacteroides thetaiotaomicron BSH and compound 7 represented as a ribbon (left, the electron density of the compound is shown as a blue mesh) and a surface (right). The A ring of 7, including the C3 hydroxyl, is exposed to the solvent. Figures a and b were made using PYMOL software generated.

[0044] Figure 12A shows the structure of "clickable" 7, 7-N 3 (12) for hit and off-target studies. Figure 12B shows 7-N 3 shows significant BSH inhibition in normal mouse feces, indicating that the probe retains its function as a BSH inhibitor. Figure 12C shows treating the Bifidobacterium adolescentis L-32 culture with 7-N 3 for 1 hour, followed by cell lysis, click reaction with Fluor 488-alkyne, and visualization by in-gel fluorescence revealing labeling of only one protein of approximately 35 kDa (i.e., the mass of the annotated Bifidobacterium adolescentis BSH). Figure 12D shows the lysate from the Bifidobacterium adolescentis culture treated with 7-N 3 reacting with desthiobiotin-alkyne, resolved by SDS-PAGE, and visualized by silver staining. The arrow indicates the band at the predicted molecular weight of BSH (approximately 35 kDa) in the probe-treated sample. Figure 12E shows treating the Bifidobacterium adolescentis culture with decreasing concentrations of compound 7, followed by treatment with 10 μM 7-N 3 and click reaction with Fluor 488-alkyne, resulting in a dose-dependent increase in fluorescence labeling of the annotated Bifidobacterium adolescentis BSH. Figure 12F shows that compared to control-treated cells, treating NCI-H716 intestinal cells with 7-N 3 for one hour, followed by click reaction with Fluor488-alkyne and visualization by in-gel fluorescence results in no significant protein labeling. For ( Figure 12B , 12C , 12D and 12F), n = 3 biological replicates per condition. For ( Figure 12B ), data are represented as mean ± SEM.

[0045] Figures 13A to 13C shows that treating normal mice with a single dose of compound 7 results in reversible inhibition of BSH activity and a shift to conjugated bile acids. n = 4 mice per group, Student's t-test. Figure 13A shows the design of the in vivo BSH inhibition experiment. Adult male C57BL / 6 mice were gavaged with a single dose of compound 7 (10 mg / kg) or vehicle control. Figure 13B shows measuring BSH activity at half-day increments starting 1 day after gavage. Resuspended fresh feces from the inhibitor- or vehicle-treated groups were incubated with substrate (GCDCA-d4, 100 μM) for 25 minutes, and product formation was quantified by UPLC-MS. n = 4 mice per group, two-tailed Student's t-test. Figure 13CShows the fecal bile acid composition 1 day after gavage. Dissociated bile acids, including the secondary bile acid deoxycholic acid (DCA), were reduced in the inhibitor-treated group. n = 4 mice per group, two-tailed Student's t-test. Figure 13D Shows that 1 day after gavage, there was no difference in bacterial OTUs (operational taxonomic units) between the inhibitor- and vehicle-treated groups. n = 4 mice per group, Tukey's multiple comparison test after one-way ANOVA. Figure 13E Shows the structure of the gut-restricted compound 7 (GR-7, 13). Figure 13F Shows the design of the proof-of-concept in vivo study of GR-7. Adult male C57BL / 6 mice were fed a powdered diet containing 0.09% (w / w) GR-7 or powdered diet alone for 30 h. Fecal pellets were collected 8 h after the dietary change. n = 10 mice per group. Figure 13G Shows that freshly resuspended feces (20 mg / mL) from inhibitor- or control-treated mice were incubated with the substrate (GCDCA-d4, 100 μM) for 25 min, and the formation of products was quantified by UPLC-MS. A significant inhibition of BSH activity was observed in GR-7-treated feces compared to control-treated mice. Student's t-test. n = 10 mice per group, two-tailed Student's t-test. Figure 13H Shows the quantification of GR-7 in tissues and plasma. The inhibitor was detected in feces 8 h after the dietary change and in cecal contents at the time of sacrifice. GR-7 was not detected in the liver or plasma. N.D. = not detected. n = 10 mice per group. All data are expressed as mean ± SEM.

[0046] Figure 14 shows the purification and kinetic characterization of BSH. ( Figure 14A ) SDS-PAGE of the purification of Bacteroides thetaiotaomicron BSH. The experiment was repeated seven times with similar results. ( Figure 14B ) SDS-PAGE of the purification of Bifidobacterium longum BSH. Michaelis-Menten analysis of BSH kinetic data. Bacteroides thetaiotaomicron BSH ( Figure 14C ) and Bifidobacterium longum BSH ( Figure 14D ) rate vs substrate concentration curves.

[0047] Figure 15 shows the identification of compound 7 as an effective broad-spectrum BSH inhibitor. Figure 15A -B shows the inhibitor against Bacteroides thetaiotaomicron BSH ( Figure 15A ) and Bifidobacterium longum BSH ( Figure 15B) Screening, which shows the % dissociation of taurocholic acids over 5 hours. The inhibitor (100 μM) was incubated with 200 nM rBSH for 30 minutes, then the taurine-conjugated bile acid substrates (tauro-β-muricholic acid, TβMCA; taurocholic acid, TCA; tauroursodeoxycholic acid, TUDCA; and taurodeoxycholic acid, TDCA, each 25 μM) were added. The dissociation of the substrates was followed by UPLC-MS. The assays were performed in biological triplicates and all data are represented as mean ± SEM.

[0048] Figure 16 shows the bile acid quantification for reporting purified BSH protein in % dissociation. The products (dissociated bile acids) and the concentration of the unreacted starting material (SM) formed by Bacteroides thetaiotaomicron BSH ( Figure 16A ) and Bifidobacterium longum BSH ( Figure 16B ) at each time point were determined using UPLC-MS. Then the following formula was used to determine the % dissociation for each sample: % dissociation = product concentration / (product concentration + starting material concentration) * 100.

[0049] Figure 17 shows that the compound structure affects the BSH inhibitory activity against growing Bacteroides thetaiotaomicron cultures. ( Figure 17A ) Compounds 8 and 9 are less effective inhibitors of Bacteroides thetaiotaomicron BSH compared to compound 7. The inhibitor (10 μM of compound 7, 8, or 9) and 100 μM TUDCA were added to the Bacteroides thetaiotaomicron culture at OD 600 0.1. ( Figure 17B ) Structural comparison of compounds 7, 8, and 9. Compound 8 lacks the α-FMK warhead and compound 9 has a C12=OH hydroxyl group.

[0050] Figure 18 shows that compound 7 is an effective recombinant BSH inhibitor. The dose-response curve of compound 7 and the calculated IC values. 200 nM recombinant Bacteroides thetaiotaomicron BSH ( Figure 18A , Gram-negative) or Bifidobacterium adolescentis BSH ( Figure 18B , Gram-positive) was pre-incubated with different concentrations of compound 7 for 60 minutes, after which the conjugated bile acid substrates TUDCA and TDCA were added respectively.

[0051] Figure 19A Shows the time required to completely inhibit Bacteroides thetaiotaomicron BSH. 100 μM compound 7 and the conjugated bile acids (tauro-β-muricholic acid, TβMCA; taurocholic acid, TCA; tauroursodeoxycholic acid, TUDCA; and taurodeoxycholic acid, TDCA, each 25 μM) were added simultaneously to 200 nM rBSH without a pre-incubation period. The formation of the dissociated bile acids was measured using a UPLC-MS-based assay and reported as % conversion. Figure 19BIt shows that in the presence of compound 7, no increase in product formation was observed after 15 seconds, indicating that enzyme activity was inhibited.

[0052] Figure 20 shows the quantification of bile acids for reporting bacterial cultures in % dissociation. UPLC-MS was used to determine the concentrations of the products (dissociated bile acids) ( Figure 20A ) and the unreacted starting materials (SM) ( Figure 20B ) formed in each culture. Then the following formula was used to determine the % dissociation for each sample: % dissociation = product concentration / (product concentration + starting material concentration) * 100.

[0053] Figure 21 shows that compound 7 does not alter the bile acid pool when incubated with the Bacteroides thetaiotaomicron BSH KO strain. ( Figure 21A ) A 100 μM pool of taurine-conjugated bile acids (TCA, TβMCA, TUDCA, and TDCA, 25 μM each) and 100 μM inhibitor (compound 7 or CAPE) or DMSO were added to growing Bacteroides thetaiotaomicron. The cultures were incubated for 24 hours and then bile acid analysis was performed using UPLC-MS. No bile acids other than the starting materials (TCA, TβMCA, TUDCA, and TDCA) were detected in any of the cultures. ( Figure 21B ) Colony-forming units (CFUs) were determined by the assay group ( Figure 21A ) after 24 hours. Compound 7 was not found to be bactericidal against BSH-deficient Bacteroides thetaiotaomicron, while CAPE was found to significantly affect the growth of this bacterium.

[0054] Figure 22 shows that compound 7 is an effective BSH inhibitor in growing bacterial cultures. The dose-response curve of compound 7 and the calculated IC values. Logarithmic pre-growth cultures of Bacteroides thetaiotaomicron ( Figure 22A , Gram-negative) and Bifidobacterium adolescentis ( Figure 22B , Gram-positive) were incubated with the conjugated substrates (TUDCA or TDCA) and allowed to grow anaerobically for 48 hours and 24 hours, respectively.

[0055] Figure 23 shows the mass spectrometry, revealing that compound 7 monolabels Bacteroides thetaiotaomicron BSH. ( Figure 23A ) Mass spectrometry (left) and zero-charge mass spectrometry (right) of BSH treated with DMSO (top, red trace) or 10-fold excess of compound 7 (bottom, green trace) for 2 hours. The 388 Da mass shift is consistent with covalent modification of BSH and HF loss. Two independent labeling reactions gave similar results. ( Figure 23B ) Top-down MS / MS of BSH treated with 10-fold excess of compound 7. Ions of type c and z are represented by red and green glyphs, respectively. Ion c3 indicates the modification at the N-terminal Cys2 residue.

[0056] Figure 24 shows the Apo and co-crystal structures of Bacteroides thetaiotaomicron BSH. The X-ray structure of the Bacteroides thetaiotaomicron BSH apoprotein ( Figure 24A ) is superimposed on the X-ray structure of Bacteroides thetaiotaomicron BSH covalently bound to compound 7 ( Figure 24B ). BSH (apo magenta, co-crystal structure cyan) is shown as a ribbon, with the indicated side chains (magenta or cyan respectively, with heteroatoms in CPK colors) presented as rods. Compound 7 (green, with heteroatoms in CPK colors) is presented in stick form. The box (dashed line) indicates the loop (residues 127 - 138) repositioned in the co-crystal structure. The figure was made using PYMOL software (Schroedinger).

[0057] Figure 25 shows that compound 7 is neither an agonist nor an antagonist of FXR or TGR5 and is non-toxic to human cells. ( Figure 25A ) Compound 7 is not a Farnesoid X receptor (FXR) agonist, as determined by the FXR co-activator recruitment assay. n = 4 biological replicates per concentration. ( Figure 25B ) The FXR antagonist activity of compound 7 was evaluated in the presence of the FXR agonist GW4064 at its EC50 value (50 nM, as determined in the corresponding agonist assay). n = 4 biological replicates per concentration. ( Figure 25C ) Compound 7 is not a G protein-coupled bile acid receptor (GPBAR1 / TGR5) agonist. The endogenous TGR5 agonist activity was measured by incubating Caco-2 cells overnight with different concentrations of compound 7. n = 3 biological replicates per concentration, with Dunnett's multiple comparison test after one-way ANOVA. ( Figure 25D ) The endogenous TGR5 antagonist activity was measured by incubating Caco-2 cells overnight with different concentrations of compound 7 in the presence of the TGR5 agonist LCA at 10 μM. n = 3 biological replicates per concentration, with Dunnett's multiple comparison test after one-way ANOVA. ( Figure 25E ) Compound 7 showed no toxicity against Caco-2 or NCI-H716 cells at concentrations up to 50 μM and 100 μM respectively. n = 5 and n = 3 were the number of biological replicates per concentration respectively, with Dunnett's multiple comparison test after one-way ANOVA. All data are represented as mean ± SEM.

[0058] Figure 26It is shown that neither compound 7 nor GR-7 significantly affects epithelial barrier integrity. Incubation of compound 7 or GR-7 with differentiated Caco-2 cells for 6 hours and 12 hours does not impair the integrity of the epithelial monolayer, as measured by the passive transport of 4 kDa FITC-dextran. For the DMSO control, n = 2 biological replicates were performed, and for the inhibitor-treated conditions, n = 3 biological replicates were performed. All data are presented as mean ± SEM.

[0059] Figure 27A It is shown that compound 7-N labels Bifidobacterium adolescentis BSH with minimal off-target reactivity. Figure 27B The fluorescence intensity of the BSH band is shown. Two-tailed Student's t-test. Data are presented as mean ± SEM. Figure 27C It is shown Figure 12E The full SDS-PAGE gel of the experiment described in. The Bifidobacterium adolescentis culture was treated with decreasing concentrations of compound 7 for 1 hour and then with 10 μM compound 7-N for an additional 1 hour. A dose-dependent labeling of BSH was observed as the concentration of compound 7 decreased. The experiment was repeated twice with similar results. Figure 27D It is shown for Figure 12D the silver-stained gel of the experiment described in, performed in biological triplicate. Figure 27E It is shown by Figure 27D LC-MS / MS analysis of in-gel digestion of the indicated band identified BSH-derived tryptic peptides. Amino acids highlighted in the red spectrum are tryptic peptides identified at approximately 1% FDR.

[0060] Figure 28 It is shown that 7-N 3 shows minimal off-target labeling in mammalian cells. Figure 12F The SDS-PAGE gel of the experiment described in was performed in biological triplicate (i.e., NCI-H716 cells were treated with 10 μM 7-N3 for 1 hour, followed by a click reaction with Fluor 488-alkyne).

[0061] Figure 29 shows that compound 7 does not significantly affect the in vivo bacterial community composition or microbial biomass. ( Figure 29A ) By taxonomic-based analysis, the mean relative abundance of the microbiota at the phylum level, n = 4 groups of mice. ( Figure 29B ) There was no difference in CFU / g between inhibitor- and vehicle-treated groups at 0.5, 1, 1.5, 2, or 2.5 days post-gavage. n = 4 mice per group, two-tailed Mann-Whitney test. All data are presented as mean ± SEM.

[0062] Figure 30 shows the activity and in vivo effects of GR-7. ( Figure 30A)GR-7 inhibits the BSH activity in fecal slurry. Feces freshly collected from conventional mice were resuspended in PBS (1 mg / mL) and incubated with 20 μM or 60 μM of GR-7 for 30 minutes. Glycochenodeoxycholic acid-d4 (GCDCA-d4, 100 μM) was added as a substrate, and the formation of the product was determined by UPLC-MS after 18 hours. The assays were performed in biological triplicates.( Figure 30B )16S rDNA copies / g of cecal content 30 hours after dietary change. There was no difference in microbial biomass between the inhibitor- and vehicle-treated groups. Two-tailed Mann-Whitney test. n = 10 mice per group. All data are presented as mean ± SEM.

[0063] Figure 31 It is shown that 3S-LCA-FMK decreased food intake in conventional mice compared to mice given vehicle (n = 8 mice per group).

[0064] Definition

[0065] Chemical definition

[0066] For convenience, the meanings of some terms and phrases used in the specification, examples, and the appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to assist in describing particular embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0067] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999 - 2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are hereby incorporated by reference in their entirety.

[0068] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, CAS version, inside cover of the Handbook of Chemistry and Physics, 75th Edition, and specific functional groups are generally defined as therein described. Additionally, general principles of organic chemistry and specific functional moieties and reactivity are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March’s Advanced Organic Chemistry, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0069] The compounds described herein may contain one or more asymmetric centers and can thus exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions at page 268 (edited by E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure additionally encompasses compounds that are in the form of individual isomers that are substantially free of other isomers, or in the form of mixtures of various isomers.

[0070] The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valence known in the art of chemistry.

[0071] Where substituents are specified by their conventional chemical formulas written from left to right, they also encompass chemically identical substituents arising from writing the structure from right to left, e.g., -CH 2 O- is equivalent to -OCH 2 -.

[0072] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight-chain (i.e., unbranched) or branched carbon chain (or carbon), or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and may include having the specified number of carbon atoms (i.e., C 1 -C 10refers to mono-, di- and polyvalent groups having one to ten carbons). Alkyl groups are uncyclized chains. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy group is an alkyl group attached to the remainder of the molecule via an oxygen linking group (–O–).

[0073] Unless otherwise indicated, the term “alkylene” by itself or as part of another substituent refers to a divalent group derived from an alkyl group, such as, but not limited to, -CH 2 CH 2 CH 2 CH 2 -. Generally, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. Alkylene is an uncyclized chain. “Lower alkyl” or “lower alkylene” are shorter-chain alkyl or alkylene groups, generally having eight or fewer carbon atoms. Unless otherwise indicated, the term “alkenylene” by itself or as part of another substituent refers to a divalent group derived from an alkene.

[0074] Unless otherwise indicated, the term “heteroalkyl” by itself or in combination with another term refers to a stable straight-chain, branched-chain, or combination thereof that includes at least one carbon atom and at least one heteroatom selected from O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Heteroalkyl is an uncyclized chain. One or more of the heteroatoms O, N, P, S, B, As, and Si may be located at any internal position of the heteroalkyl or at the position where the alkyl is attached to the remainder of the molecule. Examples include, but are not limited to: -CH 2 -CH 2 -O-CH 3 、-CH 2 -CH 2 -NH-CH 3 、-CH 2 -CH 2 -N(CH 3 )-CH 3 、-CH 2 -S-CH 2 -CH 3 、-CH 2 -CH 2, -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -CH═CH-O-CH 3 , -Si(CH 3 ) 3 , -CH 2 -CH═N-OCH 3 , -CH═CH-N(CH 3 )-CH 3 , -O-CH 3 , -O-CH 2 -CH 3 and -CN. Up to two or three heteroatoms may be consecutive, such as -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 .

[0075] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent group derived from a heteroalkyl, such as, but not limited to, -CH 2 -CH 2 -S-CH 2 -CH 2 -and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -. For heteroalkylene, the heteroatom(s) may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkylamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the orientation of the linking group does not imply the orientation in which the formula of the linking group is written. For example, the formula -C(O) 2 R′- represents both -C(O) 2 R′- and -R′C(O) 2 -. Heteroalkylene is an uncyclized chain. As noted above, heteroalkyl as used herein includes those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR′, -NR′R″, -OR′, -SR′, and / or -SO 2 R′. In the case where "heteroalkyl" is listed followed by specific heteroalkyls such as -NR′R″, etc., it should be understood that the terms heteroalkyl and -NR′R″ are not redundant or mutually exclusive. Rather, the listing of specific heteroalkyls is for increased clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyls such as -NR′R″, etc.

[0076] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, mean the cyclic forms of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position where the heterocycle is attached to the remainder of the molecule. Cycloalkyl or heteroalkyl is not aromatic. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene", alone or as part of another substituent, mean divalent groups derived from cycloalkyl and heterocycloalkyl, respectively.

[0077] Unless otherwise indicated, the term "halo" or "halogen", alone or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include both mono-haloalkyl and poly-haloalkyl. For example, the term "halo(C 1 -C 4 )alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0078] Unless otherwise indicated, the term "acyl" means -C(O)R, where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0079] Unless otherwise indicated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be monocyclic or fused together (i.e., fused-ring aryl) or a plurality of covalently linked rings (preferably 1 to 3 rings). Fused-ring aryl means a plurality of fused-together rings, wherein at least one fused ring is an aryl ring. The term "heteroaryl" means an aryl (or ring) containing at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and one or more nitrogen atoms are optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl (i.e., a plurality of fused-together rings, wherein at least one fused ring is a heteroaryl ring). 5,6-fused-ring heteroarylene means two fused-together rings, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Similarly, 6,6-fused-ring heteroarylene means two fused-together rings, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And 6,5-fused-ring heteroarylene means two fused-together rings, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. Heteroaryl may be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents of each of the above aryl and heteroaryl ring systems are selected from the following acceptable substituents. "Arylene" and "heteroarylene", alone or as part of another substituent, mean divalent groups derived from aryl and heteroaryl, respectively. A heteroaryl substituent may be -O- bonded to the ring heteroatom nitrogen.

[0080] "Fused polycyclic aryl - heterocycloalkyl" is an aryl fused to a heterocycloalkyl. "Fused polycyclic heteroaryl - heterocycloalkyl" is a heteroaryl fused to a heterocycloalkyl. "Fused polycyclic heterocycloalkyl - cycloalkyl" is a heterocycloalkyl fused to a cycloalkyl. "Fused polycyclic heterocycloalkyl - heterocycloalkyl" is a heterocycloalkyl fused to another heterocycloalkyl. Fused polycyclic aryl - heterocycloalkyl, fused polycyclic heteroaryl - heterocycloalkyl, fused polycyclic heterocycloalkyl - cycloalkyl, or fused polycyclic heterocycloalkyl - heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein. Fused polycyclic aryl - heterocycloalkyl, fused polycyclic heteroaryl - heterocycloalkyl, fused polycyclic heterocycloalkyl - cycloalkyl, or fused polycyclic heterocycloalkyl - heterocycloalkyl may each independently be named according to the size of each fused ring. Thus, for example, a 6,5 aryl - heterocycloalkyl fused ring describes a 6 - membered aryl moiety fused to a 5 - membered heterocycloalkyl. A spiro ring is two or more rings where adjacent rings are connected by a single atom. Each ring within the spiro ring may be the same or different. Each ring within the spiro ring may be substituted or unsubstituted and may have different substituents from the other individual rings within a set of spiro rings. When not part of a spiro ring, the possible substituents for each ring within the spiro ring are the possible substituents for the same ring (e.g., substituents for a cycloalkyl or heterocycloalkyl ring). The spiro ring may be a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heterocycloalkylene, and each ring within the spiro ring group may be any of those listed immediately above, including all rings of one type (e.g., all rings are substituted heterocycloalkylene, where each ring may be the same or different substituted heterocycloalkylene). When referring to a spiro ring system, a heterospiro ring means a spiro ring where at least one ring is a heterocycle and where each ring may be a different ring. When referring to a spiro ring system, a substituted spiro ring means at least one ring is substituted and each substituent may optionally be different.

[0081] As used herein, the term "oxo group" means an oxygen double - bonded to a carbon atom.

[0082] Each of the above terms (e.g., "alkyl", "heteroalkyl", "aryl", and "heteroaryl") includes both substituted and unsubstituted forms of the indicated group.

[0083] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), boron (B), arsenic (As), and silicon (Si).

[0084] As used herein, "substituent" means a group selected from the following moieties:

[0085] (A) oxo group, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO2 、 -SH, -SO 2 Cl, -SO 3 H, -SO 4 H, -SO 2 NH 2 、 -NHNH 2 、 -ONH 2 、 -NHC═(O)NHNH 2 、 -NHC═(O)NH 2 、 -NHSO 2 H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF 3 、 -OCHF 2 、 unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0086] (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl substituted with at least one substituent selected from:

[0087] (i) oxo group, halogen, -CF 3 、 -CN, -OH, -NH 2 、 -COOH, -CONH 2 、 -NO 2 、

[0088] -SH, -SO 2 Cl, -SO 3 H, -SO 4 H, -SO 2 NH 2 、 -NHNH 2 、 -ONH 2 、 -NHC═(O)NHNH 2 、

[0089] -NHC═(O)NH 2 、 -NHSO 2 H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF 3 、

[0090] -OCHF 2 、 unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0091] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl substituted with at least one substituent selected from:

[0092] (a) oxo group, halogen, -CF 3 、-CN, -OH, -NH 2 、-COOH, -CONH 2 、

[0093] -NO 2 、-SH, -SO 2 Cl, -SO 3 H, -SO 4 H, -SO 2 NH 2 、-NHNH 2 、-ONH 2 、

[0094] -NHC═(O)NHNH 2 、-NHC═(O)NH 2 、-NHSO 2 H, -NHC═(O)H, -

[0095] NHC(O)-OH, -NHOH, -OCF 3 、-OCHF 2 、unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0096] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from: oxo group, halogen, -CF 3 、-CN, -OH,

[0097] -NH 2 、-COOH, -CONH 2 、-NO 2 、-SH, -SO 2 Cl, -SO 3 H, -SO 4 H,

[0098] -SO 2 NH 2 、-NHNH 2 、-ONH 2 、-NHC═(O)NHNH 2 、-NHC═(O)NH 2 、

[0099] -NHSO 2 H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF 3 、-OCHF 2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0100] As used herein, the term "isomer" refers to compounds having the same number and kind of atoms and thus the same molecular weight but differing in the structural arrangement or configuration of the atoms.

[0101] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomeric form to another.

[0102] It will be apparent to those skilled in the art that the specific compounds of the invention may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the invention.

[0103] As used herein, the term "silyl ether" refers to a compound containing a silicon atom covalently bonded to an alkoxy group, which generally has the structure R w R x R y Si–O–R z , where R w , R x , R y and R z are independently alkyl or aryl.

[0104] The term "pharmaceutically acceptable salts" is intended to include salts of the active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents present on the compounds described herein. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfide, hydroiodic acid, or phosphorous acid, etc., and those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginine salts, etc., and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see, e.g., Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the invention contain both basic and acidic functional groups, which allow the compounds to be converted into base or acid addition salts.

[0105] As used herein, the term "salt" refers to acid or base salts of the compounds for use in the methods of the invention. Exemplary examples of salts include salts of inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. The term salt also refers to the formation of a salt between two compounds.

[0106] The term "metabolic disorder" refers to any disorder involving an alteration in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or combinations thereof. Metabolic disorders are associated with a deficiency or excess in metabolic pathways, resulting in an imbalance in the metabolism of nucleic acids, proteins, lipids, and / or carbohydrates. Factors affecting metabolism include, but are not limited to, the endocrine (hormonal) control system (e.g., the insulin pathway, enteric endocrine hormones, including GLP-1, PYY, etc.), the nervous control system (e.g., GLP-1 in the brain), etc. Examples of metabolic disorders include, but are not limited to, diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity.

[0107] The term "obesity" refers to an excess of body fat. Obesity can be determined by any measure that is accepted and utilized by those skilled in the art. Currently, the accepted measure of obesity is the body mass index (BMI), which is a measure of body weight (in kilograms) relative to the square of height (in meters). Generally speaking, for adults over 20 years old, a BMI of approximately 18.5 to 24.9 is considered normal, a BMI of approximately 25.0 to 29.9 is considered overweight, a BMI of approximately 30.0 or higher is considered obese, and a BMI of approximately 40 or higher is considered morbidly obese (see, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.). These BMI ranges are based on the impact of body weight on increased disease risk. Some common conditions associated with high BMI and obesity include cardiovascular disease, hypertension (i.e., high blood pressure), osteoarthritis, cancer, and diabetes. Although BMI is related to body fat, the relationship between BMI and actual body fat varies with age and gender. For example, for the same BMI, women are more likely than men to have a higher percentage of body fat. In addition, the BMI thresholds that distinguish normal, overweight, and obese can vary, for example, with factors such as age, gender, sex, health, and body type. In some embodiments, a subject with obesity can be a subject having a body mass index of at least about 25 kg / m 2 prior to administration of a treatment as described herein. In some embodiments, a subject with obesity can be a subject having a body mass index of at least about 30 kg / m 2 prior to administration of a treatment, compound, or agent as described herein.

[0108] As used herein, the terms "inflammation" or "inflamed" or "inflammatory" refer to the activation or recruitment of the immune system or immune cells (e.g., T cells, B cells, macrophages). Tissues with inflammation may become red, white, swollen, hot, painful, exhibit loss of function, or have membranes or mucus. Methods for identifying inflammation are well known in the art. Inflammation typically occurs after injury or microbial infection. In some embodiments, the infection is caused by bacteria selected from: Staphylococcus; Helicobacter pylori; Escherichia coli; Salmonella; Campylobacter; Yersinia enterocolitica; Shingella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus.

[0109] As used herein, the term "inflammatory disease" refers to any disease that affects the immune system. Inflammatory diseases can cause at least one symptom of the disease. Symptoms may include, but are not limited to, diarrhea, vomiting, nausea, stomach discomfort, pain, joint swelling, lethargy, fever, weight loss, weight gain, bleeding, any change in bowel movement or consistency or frequency of stools, or any other symptom associated with the subject's inflammatory disease. In some embodiments, the inflammatory disease is an autoimmune disease.

[0110] In some embodiments in any aspect, the inflammatory disease is selected from: infection; Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis, or any other inflammatory disease known in the art.

[0111] As used herein, the term "gastrointestinal disease" refers to any disease that affects the gastrointestinal tract or intestine. Gastrointestinal diseases can cause at least one symptom of the disease. Symptoms can include, but are not limited to, diarrhea, vomiting, nausea, stomach discomfort, pain, malaise, fever, weight loss, weight gain, bleeding, any change in bowel movement or the consistency or frequency of stools, or any other symptom associated with the subject's gastrointestinal disease. Non-limiting examples of gastrointestinal diseases include gastrointestinal infections, inflammatory bowel disease (IBD), gastrointestinal injury, appendicitis, Crohn's disease (CD), ulcerative colitis (UC), gastritis, enteritis, esophagitis, gastroesophageal reflux disease (GERD), celiac disease, diverticulitis, food intolerance, ulcers, infectious colitis, irritable bowel syndrome, leaky gut syndrome, pancreatitis, diabetes, hepatitis, liver disease, and cancer.

[0112] As used herein, the term "liver disease" refers to any disease that affects the liver.

[0113] Liver diseases can cause at least one symptom of the disease. These symptoms include, but are not limited to, bile acid disorders, fatigue, weight loss, pain, yellowing of the skin and / or eyes, or dark urine. Examples of liver diseases include, but are not limited to, non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis.

[0114] As used herein, the term "cancer" refers to the excessive proliferation of cells that exhibits a loss of normal cell control leading to unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Cancer can be a solid tumor, leukemia, lymphoma, or multiple myeloma. As used herein, the term "tumor" refers to the abnormal growth of cells or tissues, for example, of a malignant or benign type. Non-limiting examples of cancer include digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers.

[0115] As used herein, "subject" means a human or an animal. Typically, the animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species (such as domestic cats), canine species (such as dogs, foxes, wolves), avian species (such as chickens, emus, ostriches), and fish (such as trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient", and "subject" are used interchangeably herein.

[0116] As used herein, the terms "treat", "treatment", "treating", or "ameliorate" refer to a therapeutic treatment, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a disorder associated with a disease. The term "treatment" includes reducing or alleviating at least one side effect or symptom of diabetes. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only an improvement in symptoms or markers, but also a halt or at least a slowing in the progression or worsening of symptoms, as compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, diminishment of the degree of a disease, stabilization (i.e., not worsening) of a disease state, delay or slowing of disease progression, improvement or palliation of a disease state, alleviation (whether partial or total) and / or reduction of mortality, whether detectable or not. The "treatment" of a disease also includes providing alleviation of the symptoms or side effects of the disease (including palliative treatment).

[0117] As used herein, the term "small molecule" refers to a natural (i.e., occurring in nature) or non-natural (i.e., not occurring in nature) organic or inorganic molecule, which may include, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (e.g., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Examples of naturally occurring "small molecules" include, but are not limited to, paclitaxel, dynemicin, and rapamycin. In certain other preferred embodiments, natural product-like small molecules are utilized.

[0118] As used herein, "compound" refers to any chemical, test chemical, drug, new chemical entity (NCE), or other moiety. For example, a compound can be any foreign chemical that is not normally present in a subject such as a mammal (including a human). A compound can also be an endogenous chemical that is normally present and synthesized in a biological system such as a mammal (including a human). For example, a compound, such as a test compound, such as a drug, can reduce the dissociation of primary and secondary bile acids as provided herein.

[0119] As used herein, the term "derivative" means any chemical, conservative substitution, or structural modification of a reagent. Derivatives can improve the characteristics of a reagent or small molecule, such as pharmacodynamics, pharmacokinetics, absorption, distribution, delivery, targeting of specific receptors, or efficacy. For example, for a small molecule, a derivative can consist essentially of from at least one chemical modification to about ten modifications. A derivative can also be the corresponding salt of a reagent. A derivative can be a prodrug of a small molecule as provided herein.

[0120] As used herein, the term "bile acid" refers to steroid acids that act as emulsifiers of fats to aid in digestion and can also play a role in various systemic endocrine hormone-like functions. Bile acids in mammals are synthesized from cholesterol in the liver as primary bile acids and are metabolized by specific mammalian gut microbiota into secondary bile acids. Bile acids are stored in the gallbladder and released into the duodenum upon ingestion of food, where they aid in the absorption of lipids and fat-soluble vitamins. More than 95% of bile acids are reabsorbed in the ileum and recycled to the liver. The remaining approximately 5% enters the colon where most gut bacteria reside. Gut bacterial enzymes then enzymatically modify the primary bile acids, producing a class of molecules called secondary bile acids.

[0121] Bile acids in mammals regulate metabolic pathways by activating the farnesoid X receptor as well as G protein-coupled receptors (GPCRs) such as TGR5. By activating these various signal transduction pathways, bile acids can regulate their own enterohepatic circulation and also regulate triglyceride, cholesterol, energy, and glucose homeostasis. Non-limiting examples of bile acids include cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid, taurochenodeoxycholic acid (TCDA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), muricholic acid, obeticholic acid, and any other bile acids known in the art. The term "bile acid" or "bile salt" can further refer to the salt forms of bile acids, sulfated bile acids, and other metabolites.

[0122] As used herein, "bile salt hydrolase" or "BSH" refers to an enzyme that is widely expressed by mammalian gut bacteria and that converts host-produced primary bile acids into bacterially modified secondary bile acids. Figure 1AExamples are provided where primary and secondary bile acids are deconjugated by BSH and converted to secondary bile acids by bacterial bile acid modifying enzymes. Many amino acid sequences of BSH of various bacterial species are known in the art (e.g., NCBI accession No. accession: ABC26911.1; accession: ABC26910.1; accession: ACL98203.1; accession: AAS98803.1; accession: AKI55714.1; accession: AAP20760.1). Without limitation, BSH can refer to any bacterial BSH enzyme. The key reaction in the conversion of primary to secondary bile acids is the hydrolysis of the C24 amide bond of the conjugated primary bile acid by intestinal bacterial BSH( Figure 1A ).

[0123] As used herein, "suitable control" refers to an otherwise identical cell or population that has not been treated (e.g., a subject that has not been administered the reagents provided herein, or has only been administered a subset of the reagents provided herein, as compared to non-control cells). As used herein, the term "pharmaceutical composition" can include any material or substance that, when combined with an active ingredient (e.g., Compound 7 or a derivative thereof), allows that ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, emulsions such as oil / water emulsions, and various types of wetting agents. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0124] As used herein, a "reagent" is a chemical molecule of synthetic or biological origin. In the context of the present invention, a reagent is typically a molecule that can be used in a pharmaceutical composition.

[0125] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject reagent from one organ or part of the body to another. The term "pharmaceutically acceptable carrier" does not include tissue culture media. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, e.g., the carrier does not reduce the effect of the reagent on the treatment. In other words, the carrier is pharmaceutically inert. The terms "physiologically tolerable carrier" and "biocompatible delivery carrier" are used interchangeably. Non-limiting examples of pharmaceutical carriers include particulate or polymeric vehicles such as nanoparticles, microparticles, polymeric microspheres, or polymer-drug conjugates.

[0126] As used herein, the term "restricting delivery of a composition to the gastrointestinal tract" refers to a formulation that permits or facilitates delivery of a reagent or pharmaceutical composition described herein in a viable form to the colon, large intestine, or small intestine. Enteric coatings or micron- or nanoparticle formulations can facilitate such delivery, as can, for example, buffering agents or other protective formulations.

[0127] The term "effective amount" may be used interchangeably with the terms "therapeutically effective amount" or "sufficient amount" and refers to the amount of at least one BSH inhibitor (e.g., any one of formulas (I)-(XVIII) or a derivative thereof) in a pharmaceutical composition, the dosage and time period of which are necessary to achieve a desired therapeutic outcome (e.g., "alleviate," reduce, or halt at least one symptom of diabetes, obesity, or an inflammatory disease). For example, an effective amount using the methods disclosed herein would be considered to be an amount sufficient to reduce one or more symptoms of diabetes, obesity, or an inflammatory disease by at least 10%. As used herein, an effective amount also includes an amount sufficient to prevent or delay the development of such symptoms, alter the course of a symptomatic disease (e.g., but not limited to slowing the progression of the symptoms of the disease), or reverse the symptoms of the disease in a subject suffering from diabetes, prediabetes, hyperglycemia, obesity, or an inflammatory disease. Thus, as used herein, the terms "effective amount" or "therapeutically effective amount" refer to the amount of a therapeutic agent (e.g., a compound of formulas (I)-(XVIII) or a derivative thereof) in a pharmaceutical composition that alleviates at least one symptom of a disease. In other words, a "therapeutically effective amount" of a BSH inhibitor as disclosed herein is the amount of an agonist that exerts a beneficial effect on the symptoms of a disease (e.g., an inflammatory disease, a gastrointestinal disease, cancer, obesity, etc.). The dosage administered to an individual as a single dose or multiple doses will vary depending on a variety of factors, including the pharmacokinetic properties of the inhibitor, the route of administration, the condition and characteristics of the subject (gender, age, weight, health, body type), the degree of the symptoms, concurrent therapy, the frequency of treatment, and the desired effect. A therapeutically effective amount is also an amount where the therapeutic beneficial effect exceeds any toxic or detrimental effect of the therapeutic agent. The effective amount in each individual case can be determined empirically by one of ordinary skill in the art using methods established in the art without undue experimentation. Generally speaking, the phrases "therapeutically effective" and "effective for treatment, prevention, or inhibition" are intended to define an agonist as disclosed herein that will achieve the goal of reducing the severity of diabetes, cancer, gastrointestinal disease, obesity, or an inflammatory disease or one of its associated symptoms.

[0128] As used herein, the term "co-administering" and the like are intended to encompass administering selected therapeutic agents to a single patient and are intended to include treatment regimens in which the reagents are administered by the same or different routes of administration or at the same or different times.

[0129] As used herein, the term "unit dosage form" refers to a dosage suitable for single administration. By way of example, a unit dosage form may be a quantity of therapeutic agent disposed in a delivery device such as a syringe or an intravenous drip bag. In one embodiment in any aspect, the unit dosage form is administered as a single administration. In another embodiment, more than one unit dosage form may be administered simultaneously.

[0130] The terms "administer" and "subject to" are used interchangeably in the context of treating a disease or disorder.

[0131] In jurisdictions that prohibit the patenting of methods practiced on the human body, the meaning of "administering" a composition to a human subject shall be limited to a controlled substance that prescribes that the human subject self-administer by any technique (e.g., orally, inhaled, topically applied, injected, inserted, etc.). The broadest reasonable interpretation consistent with the laws or regulations defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods practiced on the human body, "administering" a composition includes both methods practiced on the human body and the foregoing activities.

[0132] As used herein, the term "administer" means placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site to produce a desired effect. The compounds or compositions described herein may be administered by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0133] As used herein, the phrases "parenteral administration" and "parenterally administered" mean but are not limited to modes of administration other than enteral and topical administration, typically by injection, and include but are not limited to intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection, infusion, and other injection or infusion techniques. Without limitation, oral administration may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, mouthwashes, powders, etc.

[0134] As used herein, the term "modulate" means including increasing or decreasing the effect of a given parameter, as defined by those terms herein.

[0135] As used herein, the term "contact" when used in reference to a cell or an organ encompasses both introducing or administering a reagent, surface, hormone, etc. to a cell, tissue, or organ in a manner that permits physical contact of the cell with the reagent, surface, hormone, etc., and introducing an element (such as a genetic construct or vector) that permits the expression of a reagent (such as miRNA, polypeptide, or other expression product) in the cell. It should be understood that a cell genetically modified to express a reagent is in "contact" with the reagent, such as the progeny of the cell expressing the reagent.

[0136] The term "statistically significant" or "significant" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

[0137] As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and their corresponding components, which are essential for the method or composition but are open to including unspecified elements, whether essential or not.

[0138] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional characteristics of the embodiment of the present invention. Detailed Description

[0139] Human - associated bacteria play crucial roles in health and disease. Microbial dysbiosis is associated with a broad range of disease states. Studies in germ - free mice colonized with a single strain, multiple strains, or defined bacterial communities have revealed the ability of gut bacteria to influence host processes, including metabolism, immune function, and neural responses. Compounds that selectively alter specific bacterial metabolite and protein levels can be used to evaluate how bacterial products affect host physiology in a fully - developed animal with a complex microbial community and as therapeutic agents for treating diseases such as metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases, cancer (e.g., liver cancer), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0140] The compositions and methods provided herein relate in part to the discovery of several compounds that inhibit bile salt hydrolase (BSH) and modulate the dissociation of primary and secondary bile acids in a subject.

[0141] The compounds provided herein are selective, effectively inhibit BSH in a broad spectrum of bacteria, have no off - target effects in the host, can be restricted to the gut, and modulate bile acids present in a host subject.

[0142] Compound

[0143] In one aspect, the present disclosure provides a compound of formula (I):

[0144]

[0145] Wherein:

[0146] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0147] m is 1, 2, 3 or 4;

[0148] X is an electrophilic group;

[0149] R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 18 , N(R 18 ), 2 , SR 18 , halogen, CN, -CHO, -CO 2 H, -CO 2 R 18 , -NO 2 , -ONO 2 , -SO 2 Cl, -SO 3 - , -OSO 3 - , -NR 18 SO 3 - , -PO 3 2- , -OPO 3 2- , -OSO 2 R 18 , -SO 2 , N(R 18 ), 2 , -OSO 2 , N(R 18 ), 2 , -NR18 SO 2 R 18 、 -SO 2 N(R 18 ) 2 、 -NHNH 2 、 -ONH 2 、 or -NHC(O)NHNH 2 wherein each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0150] or a pharmaceutically acceptable salt thereof.

[0151] In certain embodiments, the compound of formula (I) is of formula (I’):

[0152]

[0153] wherein:

[0154] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0155] m is 1, 2, 3 or 4;

[0156] X is an electrophilic group;

[0157] R 1 、 R 2 、 R 3 、 R 4 、 R 6 、 R 7 、 R 11 、 R 12 、 R 15 、 R 16 and R 17 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 18 、 N(R 18 ) 2 、 SR 18 、 halogen, CN, -CHO, -CO 2 H、 -CO 2 R 18 、 -NO 2 、 -ONO 2 、 -SO 2 Cl、 -SO 3 H、 -OSO 3 H、 -NR18 SO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 R 18 , -SO 2 N(R 18 ) 2 , -OSO 2 N(R 18 ) 2 , -NR 18 SO 2 R 18 , -SO 2 N(R 18 ) 2 , -NHNH 2 , -ONH 2 , or -NHC(O)NHNH 2 , wherein each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0158] or a pharmaceutically acceptable salt thereof.

[0159] In certain embodiments, the compound of formula (I) is of formula (I-a):

[0160]

[0161] wherein:

[0162] each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.

[0163] In certain embodiments, the compound of formula (I-a) is of formula (I-a’):

[0164]

[0165] wherein:

[0166] each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.

[0167] In certain embodiments, the compound of formula (I) is of formula (I-b):

[0168]

[0169] Wherein:

[0170] Each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.

[0171] In certain embodiments, the compound of formula (I-b) is of formula (I-b’):

[0172]

[0173] Wherein:

[0174] Each R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a pharmaceutically acceptable salt thereof.

[0175] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-c):

[0176]

[0177] or a pharmaceutically acceptable salt thereof.

[0178] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-d):

[0179] or a pharmaceutically acceptable salt thereof.

[0180] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-e):

[0181] or a pharmaceutically acceptable salt thereof.

[0182] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-f):

[0183] or a pharmaceutically acceptable salt thereof.

[0184] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-g):

[0185] or a pharmaceutically acceptable salt thereof.

[0186] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-h):

[0187]

[0188] or a pharmaceutically acceptable salt thereof.

[0189] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-i):

[0190]

[0191] or a pharmaceutically acceptable salt thereof.

[0192] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-c'):

[0193]

[0194] or a pharmaceutically acceptable salt thereof,

[0195] wherein:

[0196] R 3a 、R 7a and R 12a are independently selected from -OR 18 、-SO 3 R 18 、-OSO 3 R 18 、-PO 3 (R 18 ) 2 、-OPO 3 (R 18 ) 2 、-OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a 、R 7a and R 12a are independently selected from -OR 18 、-SO 3 H、-OSO3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , wherein each R 18 is independently H, or a substituted or unsubstituted alkyl group. In certain embodiments, R 3a , R 7a and R 12a are independently selected from -OR 18 , -SO 3 H and -OSO 3 H, wherein R 18 is H, or a substituted or unsubstituted alkyl group. In certain embodiments, R 3a , R 7a and R 12a are independently selected from -OH and -OSO 3 H.

[0197] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-d’):

[0198]

[0199] or a pharmaceutically acceptable salt thereof,

[0200] wherein:

[0201] R 3a and R 12a are independently selected from -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 (R 18 ) 2 , -OPO 3 (R 18 ) 2 , -OSO 2 R 18 and -SO 2 N(R 18 ) 2 , wherein each R 18 is independently H, or a substituted or unsubstituted alkyl group. In certain embodiments, R 3a and R 12a are independently selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO3 H 2 、 -OPO 3 H 2 、 -OSO 2 H and -SO 2 NH 2 wherein R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 12a are independently selected from -OH and -OSO 3 H. In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-d"):

[0202]

[0203] or a pharmaceutically acceptable salt thereof.

[0204] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-e'):

[0205]

[0206] or a pharmaceutically acceptable salt thereof,

[0207] wherein:

[0208] R 3a and R 7a are independently selected from -OR 18 、 -SO 3 R 18 、 -OSO 3 R 18 、 -PO 3 (R 18 ) 2 、 -OPO 3 (R 18 ) 2 、 -OSO 2 R 18 and -SO 2 N(R 18 ) 2 where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 7a are independently selected from -OR 18 、 -SO 3 H、 -OSO 3 H、 -PO 3 H 2 、 -OPO 3 H 2 、 -OSO2 H and -SO 2 NH 2 , wherein R 18 is independently H, or a substituted or unsubstituted alkyl group. In certain embodiments, R 3a and R 7a are independently selected from -OH and -OSO 3 H. In certain embodiments, R 3a is -OSO 3 H, and R 7a is selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , wherein R 18 is independently H, or a substituted or unsubstituted alkyl group. In certain embodiments, R 3a is -OH, and R 7a is selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , wherein R 18 is independently H, or a substituted or unsubstituted alkyl group.

[0209] In certain embodiments, the compound of formula (I-e) is of formula (I-e”):

[0210]

[0211] or a pharmaceutically acceptable salt thereof.

[0212] In certain embodiments, the compound of formula (I-e”) is the following formula:

[0213]

[0214] or a pharmaceutically acceptable salt thereof.

[0215] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-f’):

[0216]

[0217] or a pharmaceutically acceptable salt thereof,

[0218] wherein:

[0219] R 3a is selected from -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 (R 18 ) 2 , -OPO 3 (R 18 ) 2 , -OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a is selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a is -OSO 3 H. In certain embodiments, R 3a is -OH.

[0220] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-f"):

[0221]

[0222] or a pharmaceutically acceptable salt thereof.

[0223] In certain embodiments, the compound of formula (I-f') is of formula (I-f'''):

[0224]

[0225] or a pharmaceutically acceptable salt thereof.

[0226] In certain embodiments, the compound of formula (I-f''') is of the following formula:

[0227]

[0228] or a pharmaceutically acceptable salt thereof.

[0229] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-g'):

[0230]

[0231] or a pharmaceutically acceptable salt thereof,

[0232] wherein:

[0233] R 3a 、R 6a and R 7a are independently selected from -OR 18 、-SO 3 R 18 、-OSO 3 R 18 、-PO 3 (R 18 ) 2 、-OPO 3 (R 18 ) 2 、-OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a 、R 6a and R 7a are independently selected from -OR 18 、-SO 3 H、-OSO 3 H、-PO 3 H 2 、-OPO 3 H 2 、-OSO 2 H and -SO 2 NH 2 , where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a 、R 6a and R 7a are independently selected from -OH and -OSO 3 H.

[0234] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-g"):

[0235]

[0236] or a pharmaceutically acceptable salt thereof.

[0237] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-h'):

[0238]

[0239] or a pharmaceutically acceptable salt thereof,

[0240] wherein:

[0241] R 7a and R 12a are independently selected from -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 (R 18 ) 2 , -OPO 3 (R 18 ) 2 , -OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 7a and R 12a are independently selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 7a and R 12a are independently selected from -OH and -OSO 3 H.

[0242] In certain embodiments, the compound of formula (I) or formula (I') is of formula (I-h"):

[0243]

[0244] or a pharmaceutically acceptable salt thereof.

[0245] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-i’):

[0246]

[0247] or a pharmaceutically acceptable salt thereof,

[0248] wherein:

[0249] R 3a and R 6a are independently selected from -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 (R 18 ) 2 , -OPO 3 (R 18 ) 2 , -OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 6a are independently selected from -OR 18 , -SO 3 H, -OSO 3 H, -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 H and -SO 2 NH 2 , where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 6a are independently selected from -OH and -OSO 3 H.

[0250] In certain embodiments, the compound of formula (I) or formula (I’) is of formula (I-i”):

[0251]

[0252] or a pharmaceutically acceptable salt thereof.

[0253] In certain embodiments, a compound of any one of formulas (I-c’), (I-d’), (I-e’), (I-f’), (I-g’), (I-h’), or (I-i’) may contain substituent R 3a , R 6a , R 7a , or R 12a . In certain embodiments, R 3a , R 6a , R 7a and R 12a are independently selected from -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 (R 18 ) 2 , -OPO 3 (R 18 ) 2 , -OSO 2 R 18 and -SO 2 N(R 18 ) 2 , where each R 18 is independently H, or a substituted or unsubstituted alkyl. In certain embodiments, at least one instance of R 3a , R 6a , R 7a , or R 12a is independently -OR 18 . In certain embodiments, at least one instance of R 3a , R 6a , R 7a , or R 12a is independently -SO 3 R 18 . In certain embodiments, at least one instance of R 3a , R 6a , R 7a , or R 12a is independently -OSO 3 R 18 . In certain embodiments, at least one instance of R 3a , R 6a , R 7a , or R 12a is independently -PO 3 (R 18 ) 2 . In certain embodiments, R3a , R 6a , R 7a , or R 12a at least one example of is independently -OPO 3 (R 18 ) 2 . In certain embodiments, R 3a , R 6a , R 7a , or R 12a at least one example of is independently -OSO 2 R 18 . In certain embodiments, R 3a , R 6a , R 7a , or R 12a at least one example of is independently -SO 2 N(R 18 ) 2 . In certain embodiments, R 3a is -OH, and R 6a , R 7a and R 12a are independently selected from -OH and -OSO 3 H. In certain embodiments, R 3a is -OSO 3 H, and R 6a , R 7a and R 12a are independently selected from -OH and -OSO 3 H.

[0254] In some embodiments of the various aspects disclosed herein, the compound of formula (I) can be a compound of any one of formulas (II)-(XV):

[0255]

[0256]

[0257] In the compounds of formulas (I)-(XV), X is an electrophilic group. The terms "electrophile" and "electrophilic" refer to a functional group that is susceptible to nucleophilic attack, i.e., is prone to react with an introduced nucleophilic group (e.g., thiol, amine). Generally speaking, an electrophilic group is a group of atoms in which one or more are electron-deficient. Usually, electrophilic groups include electron-withdrawing groups. Examples of electron-withdrawing groups include, but are not limited to, halogen substituents, nitro groups, cyano groups, ester groups, aldehyde groups, ketone groups, sulfone groups, or amide groups. One or more electron-deficient atoms are called electrophilic centers, and representative examples thereof include carbonyl, thiocarbonyl, oxophosphoryl, and thiophosphoryl groups. Exemplary electrophilic groups include, but are not limited to, acyl halides, isothiocyanates, isocyanates, epoxy groups, and acid anhydride groups.

[0258] In some embodiments of the various aspects disclosed herein, X is a thiol-reactive electrophilic group. As used herein, the term "thiol-reactive electrophilic group" is any group that is susceptible to nucleophilic attack by the lone pair of electrons on the sulfur atom of a thiol group or a thiolate anion. Examples of thiol-reactive electrophilic groups include groups having a good leaving group. For example, α-halocarbonyl, isothiocyanate, isocyanate, alkyl groups attached with halide or alkoxy, and electron-deficient vinyl groups. In some embodiments, X is an α-halocarbonyl or an isothiocyanate group.

[0259] In some embodiments of the various aspects disclosed herein, X is -C(O)R 19 , -NCS, -NHC(O)R 19 , -CH═C(CN)CO 2 R 20 , or -CN, where R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, and R 20 is alkyl.

[0260] In certain embodiments, X is -C(O)R 19 , where R 19 is alkyl, haloalkyl, alkenyl, or alkynyl. In certain embodiments, X is -C(O)R 19 , where R 19 is -CH 2 F. In certain embodiments, X is -NCS. In certain embodiments, X is -NHC(O)R 19 , where R 19 is alkyl (e.g., Me, Et, Pr), haloalkyl (e.g., CH 2 F). In certain embodiments, X is -CH═C(CN)CO 2 R 20 , where R 20 is alkyl. In certain embodiments, X is -CN.

[0261] In certain embodiments, X is an electrophilic group selected from .

[0262] In certain embodiments, X is In certain embodiments, X is In certain embodiments, X is In certain embodiments, X is In certain embodiments, X is In certain embodiments, X is In certain embodiments, X is

[0263] In some embodiments, X is -C(O)R 19 or -NCS, where R 19 is haloalkyl. For example, X is -C(O)CH 2 F or -NCS.

[0264] In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently H. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted alkyl. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted heteroalkyl. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted cycloalkyl. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R11 , R 12 , R 15 , R 16 and R 17 are independently a substituted or unsubstituted heterocycloalkyl. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently a substituted or unsubstituted aryl. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently a substituted or unsubstituted heteroaryl. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently OR 18 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently N(R 18 ) 2 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R15 , R 16 and R 17 are independently SR 18 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently halogen. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently CN. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -CHO. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -CO 2 H. In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -CO 2 R18 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -NO 2 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -ONO 2 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -SO 2 Cl. In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -SO 3 - 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -OSO 3- 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -NR 18 SO 3 - 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -PO 3 2- 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -OPO 3 2- 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15 、R 16 and R 17 are independently -OSO 2 R 18 。In certain embodiments, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 11 、R 12 、R 15, R 16 and R 17 are independently -SO 2 N(R 18 ) 2 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -OSO 2 N(R 18 ) 2 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -NR 18 SO 2 R 18 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -SO 2 N(R 18 ) 2 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -NHNH 2 . In certain embodiments, R 1 , R 2 , R 3, R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -ONH 2 . In certain embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently -NHC(O)NHNH 2 .

[0265] In certain embodiments, R 18 is H. In certain embodiments, R 18 is a substituted or unsubstituted alkyl. In certain embodiments, R 18 is a substituted or unsubstituted heteroalkyl. In certain embodiments, R 18 is a substituted or unsubstituted cycloalkyl. In certain embodiments, R 18 is a substituted or unsubstituted heterocycloalkyl. In certain embodiments, R 18 is a substituted or unsubstituted aryl. In certain embodiments, R 18 is a substituted or unsubstituted heteroaryl.

[0266] In the compounds of formula (I), at least one of R 1 , R 2 , R 4 , R 6 , R 11 , R 15 and R 16 may be H. For example, one, two, three, four, five, six, or all seven of R 1 , R 2 , R 4 , R 6 , R 11 , R 15 and R 16 may be H. In some embodiments of the various aspects disclosed herein, R 1 , R 2 , R 4 , R 6 , R 11 , R 15and R 16 are all H.

[0267] In some compounds of formula (I), R 3 , R 7 and R 12 at least one of which may be -OR 18 . For example, one, two, or all three of R 3 , R 7 and R 12 may be -OR 18 . Thus, in some embodiments of the various aspects disclosed herein, R 3 is -OR 18 . In some embodiments of the various aspects disclosed herein, R 7 is -OR 18 . In some embodiments of the various aspects disclosed herein, R 12 is -OR 18 . In some embodiments, R 3 and R 7 are -OR 18 . In some embodiments, R 3 and R 12 are -OR 18 . In some embodiments, R 7 and R 12 are -OR 18 . In some embodiments, R 3 , R 7 and R 12 may all be -OR 18 .

[0268] In some embodiments of the various aspects disclosed herein, at least one of R 3 and R 7 is -OR 18 , and R 12 is H or OR 18 . For example, at least one of R 3 and R 7 is -OH, and R 12 is H or -OH.

[0269] In some additional embodiments of the various aspects disclosed herein, R 3 and R 7 are -OR 18 , and R 12 is H or -OR 18 . For example, R 3 and R 7 are -OH, and R 12 is H or -OH.

[0270] In some compounds of formula (I), R 3 , R 6 , R 7 and R 12 at least one of which may be -OSO 3 - , -NR 18 SO 3 - or -OPO 3 2- . In some other embodiments of these, R 3 , R 6 , R 7 and R 12 at least one of which is -OSO 3 - . In some specific embodiments, R 3 is -OSO 3 - .

[0271] Exemplary R 18 groups include but are not limited to H and C 1 -C 6 alkyl. In some embodiments of the various aspects disclosed herein, R 18 is H.

[0272] In the compounds of formula (I), R 17 may be C 1 -C 6 alkyl. For example, R 17 may be methyl, ethyl, propyl, isopropyl, butyl, pentyl. In some embodiments of the various aspects disclosed herein, R 17 is methyl.

[0273] In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, n is 7. In certain embodiments, n is 8. In certain embodiments, n is 9. In certain embodiments, n is 10.

[0274] In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4.

[0275] In the compounds of formula (I), n may be 1 or 2. In some exemplary compounds of formula (I)-(XVIII), n is 2.

[0276] In the compounds of formula (I), m can be 1, 2 or 3. In some exemplary compounds of formula (I)-(XVIII), m is 1.

[0277] In some embodiments of the various aspects described herein, the compound of formula (I) is of formula (XVI):

[0278]

[0279] wherein R 19 is haloalkyl; R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 , R 17 , n and m are as defined for formula (I). In some exemplary compounds of formula (XVI), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 is H; R 3 and R 7 are OH; R 12 is H or -OH; R 17 is methyl; and R 19 is haloalkyl, such as -CH 2 F. In some other exemplary compounds of formula (XVI), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 are H; R 3 is -OSO 3 - ; R 7 is OH; R 12 is H or -OH; R 17 is methyl; and R 19 is haloalkyl, such as -CH 2 F.

[0280] In some embodiments of the various aspects described herein, the compound of formula (I) is of formula (XVII):

[0281]

[0282] wherein R 19 is a haloalkyl; R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 , R 17 , n and m are as defined for formula (I). In some exemplary compounds of formula (XVII), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 is H; R 3 and R 7 are OH; R 12 is H or OH; R 17 is methyl; and R 19 is a haloalkyl. In some other exemplary compounds of formula (XVII), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 is H; R 3 is -OSO 3 - ; R 7 is OH; R 12 is H or -OH; R 17 is methyl; and R 19 is a haloalkyl, such as -CH 2 F.

[0283] In some embodiments of the various aspects described herein, the compound of formula (I) is of formula (XVIII):

[0284]

[0285] wherein R 19 is a haloalkyl; R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R11 , R 12 , R 15 , R 16 , R 17 , n and m are as defined for formula (I). In some exemplary compounds of formula (XVIII), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 is H; R 3 and R 7 are OH; R 12 is H or OH; R 17 is methyl; and R 19 is haloalkyl, such as -CH 2 F. In some other exemplary compounds of formula (XVIII), m is 1 or 2; n is 1 or 2; R 1 , R 2 , R 4 , R 6 , R 11 , R 15 , R 16 , R 3 is -OSO 3 - ; R 7 is OH; R 12 is H or OH; R 17 is methyl; and R 19 is haloalkyl, such as -CH 2 F.

[0286] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-a):

[0287]

[0288] Wherein:

[0289] R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

[0290] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 18 is H and R 19 is haloalkyl (such as -CH 2 F).

[0291] In certain embodiments, the compound of formula (XVIII-a) is of formula (XVIII-a’):

[0292]

[0293] Wherein:

[0294] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH 2 F).

[0295] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-b):

[0296]

[0297] Wherein:

[0298] R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

[0299] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 18 is H and R 19 is haloalkyl (e.g., -CH 2 F).

[0300] In certain embodiments, the compound of formula (XVIII-b) is of formula (XVIII-b’):

[0301]

[0302] Wherein:

[0303] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH 2 F).

[0304] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-c):

[0305]

[0306] Wherein:

[0307] R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

[0308] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 18 is H and R 19 is haloalkyl (e.g., -CH 2 F).

[0309] In certain embodiments, the compound of formula (XVIII-c) is of formula (XVIII-c’):

[0310]

[0311] wherein:

[0312] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH 2 F).

[0313] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-d):

[0314]

[0315] wherein:

[0316] R 18 is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

[0317] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 18 is H and R 19 is haloalkyl (e.g., -CH 2 F).

[0318] In certain embodiments, the compound of formula (XVIII-d) is of formula (XVIII-d’):

[0319]

[0320] Wherein:

[0321] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH 2 F).

[0322] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F, -NCS, -C(O)CH=CH 2 , -C(O)C≡CH, -NHC(O)CH=CH 2 , -CN, -CH=C(CN)CO 2 Et, or -C(O)CH 3 ; R 1 , R 2 , R 4 , R 16 , R 11 , R 15 and R 16 are H; R 3 is -OH or -OSO 3 - ; R 7 is -OH; and R 12 is H or -OH.

[0323] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F, -NCS, -C(O)CH=CH 2 , -C(O)C≡CH, -NHC(O)CH=CH 2 , -CN, -CH=C(CN)CO 2 Et, or -C(O)CH 3 ; R 1 , R 2 , R 4 , R 16 , R 11 , R 15 and R 16 are H; R 3 is -OH or -OSO 3 H; R 7 is -OH; and R 12 is H or -OH.

[0324] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F or -NCS; R 1 , R2 , R 4 , R 16 , R 11 , R 15 and R 16 is H; R 3 is -OH or -OSO 3 - ; R 7 is -OH; and R 12 is H or -OH.

[0325] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F or -NCS; R 1 , R 2 , R 4 , R 16 , R 11 , R 15 , R 16 and R 3 is -OH or -OSO 3 H; R 7 is -OH; and R 12 is H or -OH.

[0326] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F; R 1 , R 2 , R 4 , R 16 , R 11 , R 15 , R 16 and R 3 is -OH or -OSO 3 - ; R 7 is -OH; and R 12 is H or -OH.

[0327] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F; R 1 , R 2 , R 4 , R 16 , R 11 , R 15 , R 16 and R 3 is -OH or -OSO 3 H; R 7 is -OH; and R 12 is H or -OH.

[0328] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F; R 1 、R 2 、R 4 、R 16 、R 11 、R 15 and R 16 are H; R 3 is -OH and R 7 is OH; and R 12 is H or -OH.

[0329] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F; R 1 、R 2 、R 4 、R 16 、R 11 、R 15 and R 16 are H; R 3 is -OSO 3 - ; and R 7 is -OH; and R 12 is H.

[0330] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH 2 F; R 1 、R 2 、R 4 、R 16 、R 11 、R 15 and R 16 are H; R 3 is -OSO 3 H; and R 7 is -OH; and R 12 is H.

[0331] In embodiments of the various aspects disclosed herein, the compounds of formula (I) do not modulate the activity of TGR5. In other words, the compounds of formula (I) are neither agonists nor antagonists of TGR5.

[0332] Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heteroalkyl, cycloalkenyl and heteroalkenyl) can be one or more of a variety of groups selected from, but not limited to, the following: -OR′, ═O, ═NR′, ═N-OR′, -NR′R″, -SR′, -halogen, -SiR′R″R′″, -OC(O)R′, -C(O)R′, -CO 2 R′, -CONR′R″, -OC(O)NR′R″, -NR″C(O)R′, -NR′-C(O)NR″R′″, -NR″C(O) 2 R′, -NR-C(NR′R″R′″)═NR″″, -NR-C(NR′R″)═NR′″, -S(O)R′, -S(O) 2 R′, -S(O) 2 NR′R″, -NRSO 2 R′, -NR′NR″R′″, -ONR′R″, -NR′C═(O)NR″NR′″R″″, -CN, -NO 2 , -NR′SO 2 R″, -NR′C═(O)R″, -NR′C(O)-OR″, -NR′OR″, the number of which ranges from 0 to (2m′ + 1), where m′ is the total number of carbon atoms in such groups. R′, R′, R″, R′″ and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy, or aralkyl. When the compounds of the present invention include more than one R group, for example, when there are more than one of these groups, each R group is independently selected as each of the R′, R″, R′″ and R″″ groups. When R′ and R″ are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., —CF 3 and —CH 2 CF 3 ) and acyl (e.g., —C(O)CH 3 , —C(O)CF 3 , —C(O)CH 2 OCH 3 , etc.).

[0333] Substituents similar to those described for alkyl groups and used for aryl and heteroaryl groups are variable and are selected from, for example: –OR′, –NR′R″, –SR′, –halogen, –SiR′R″R′″, –OC(O)R′, –C(O)R′, –CO 2 R′, –CONR′R″, –OC(O)NR′R″, –NR″C(O)R′, –NR′–C(O)NR″R′″, –NR″C(O) 2 R′, –NR–C(NR′R″R′″)=NR″″, –NR–C(NR″R″)–NR′″, –S(O)R′, –S(O) 2 R′, –S(O) 2 NR′R″, –NRSO 2 R′, –NR′NR″R′″, –ONR′R″, –NR′C=(O)NR″NR′″R″″, –CN, –NO 2 , –R′, –N 3 , –CH(Ph) 2 , fluorinated (C 1 -C 4 ) alkoxy and fluorinated (C 1 -C 4 ) alkyl, –NR′SO 2 R″, –NR′C=(O)R″, –NR′C(O)–OR″, –NR′OR″, the number of which ranges from zero to the total number of open valences on the aromatic ring system; and wherein R′, R″, R′″ and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When the compounds of the present invention include more than one R group, for example, when there are more than one of these groups, each R group is independently selected as each of the R′, R″, R′″ and R″″ groups.

[0334] Substituents for a ring (such as cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) can be described as substituents on the ring rather than on a specific atom of the ring (commonly referred to as floating substituents). In such cases, the substituent can be attached to any ring atom (subject to valence rules), and in the case of fused or spiro rings, a substituent depicted as being associated with a member of the fused or spiro ring (a floating substituent on a single ring) can be a substituent on any of the fused or spiro rings (a floating substituent on multiple rings). When a substituent is attached to a ring but not to a specific atom (a floating substituent) and the subscript of the substituent is an integer greater than one, multiple substituents can be on the same atom, the same ring, different atoms, different fused rings, different spiro rings, and each substituent can be optionally different. In cases where the point of attachment of the ring to the rest of the molecule is not limited to a single atom (a floating substituent), the point of attachment can be any atom of the ring, and in the case of fused or spiro rings, any atom of any of the fused or spiro rings, subject to valence rules. When a ring, fused ring, or spiro ring contains one or more ring heteroatoms and the ring, fused ring, or spiro ring is shown to have one or more floating substituents (including but not limited to the point of attachment to the rest of the molecule), the floating substituent can be bonded to the heteroatom. When a ring heteroatom is shown bonded to one or more hydrogens (e.g., a ring nitrogen having two bonds to ring atoms and a third bond to a hydrogen) in a structure or formula having a floating substituent, when the heteroatom is bonded to a floating substituent, the substituent will be understood to replace the hydrogen, subject to valence rules.

[0335] Two or more substituents can optionally be joined to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl. Such so-called ring-forming substituents are typically but not necessarily found attached to a cyclic base structure. In some embodiments in any aspect, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure form a fused ring structure. In another embodiment in any aspect, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure form a spiro ring structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0336] Two substituents on adjacent atoms of an aryl or heteroaryl ring can optionally form a ring of the formula -T-C(O)-(CRR′) q -U-, where T and U are independently -NR-, -O-, -CRR′-, or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring can optionally be replaced by the formula -A-(CH 2 ) rReplacement of the substituent of -B-, wherein A and B are independently -CRR′-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR′-, or a single bond, and r is an integer from 1 to 4. One of the single bonds of the newly formed ring can optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring can optionally be replaced by a substituent of the formula -(CRR′) s -X′-(C″R″R′″) d -, wherein r and d are independently integers from 0 to 3, and X′ is -O-, -NR′-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR′-. The substituents R′, R′, R″ and R′″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0337] In certain embodiments, the compound of formula (I) is of the following formula:

[0338]

[0339]

[0340] or a pharmaceutically acceptable salt thereof.

[0341] In certain embodiments, the compound of formula (I) is of the following formula:

[0342]

[0343] or a pharmaceutically acceptable salt thereof.

[0344] In certain embodiments, the compound of formula (I) is of the following formula: or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound of formula (I) is of the following formula: or a pharmaceutically acceptable salt thereof.

[0345] In certain embodiments, the compound of formula (I) is not of the following formula:

[0346]

[0347] In certain embodiments, the compound of formula (I) is not of the following formula:

[0348]

[0349] In some embodiments, each substituted group in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene in the compounds herein is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-restricted substituent. In other embodiments, at least one or all of these groups are substituted with at least one lower-level substituent.

[0350] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl can be substituted or unsubstituted C 1 -C 20 alkyl, each substituted or unsubstituted heteroalkyl is substituted or unsubstituted 2- to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is substituted or unsubstituted C 3 -C 8 cycloalkyl, and / or each substituted or unsubstituted heterocycloalkyl is substituted or unsubstituted 3- to 8-membered heterocycloalkyl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is substituted or unsubstituted C 1 -C 20 alkylene, each substituted or unsubstituted heteroalkylene is substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is substituted or unsubstituted C 3 -C 8 cycloalkylene, and / or each substituted or unsubstituted heterocycloalkylene is substituted or unsubstituted 3- to 8-membered heterocycloalkylene.

[0351] In some embodiments, each substituted or unsubstituted alkyl is substituted or unsubstituted C 1 -C 8 alkyl, each substituted or unsubstituted heteroalkyl is substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is substituted or unsubstituted C 3 -C 7 cycloalkyl, and / or each substituted or unsubstituted heterocycloalkyl is substituted or unsubstituted 3- to 7-membered heterocycloalkyl. In some embodiments, each substituted or unsubstituted alkylene is substituted or unsubstituted C 1 -C 8 alkylene, each substituted or unsubstituted heteroalkylene is substituted or unsubstituted 2- to 8-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is substituted or unsubstituted C 3 -C 7a subcycloalkyl group, and / or each substituted or unsubstituted heterocycloalkylidene group is a substituted or unsubstituted 3- to 7-membered heterocycloalkylidene group.

[0352] Certain compounds of the present invention have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms (which may be defined as (R)- or (S)- according to absolute stereochemistry, or (D)- or (L)- for amino acids), and each isomer is encompassed within the scope of the present invention. Compounds of the present invention do not include those that are known in the art to be too unstable to synthesize and / or isolate. The present invention is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain an olefinic bond or other geometrically asymmetric center, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.

[0353] Accordingly, the compounds of the present invention may exist as salts, such as salts with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art.

[0354] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs in certain physical properties, such as solubility in polar solvents, from the various salt forms.

[0355] In addition to the salt forms, the present invention also provides prodrug forms of the compounds. Prodrugs of the compounds described herein are those compounds that undergo chemical changes under physiological conditions readily to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an ex vivo environment. For example, a prodrug can be slowly converted to the compound of the present invention when placed in a transdermal patch reservoir together with a suitable enzyme or chemical reagent.

[0356] Certain compounds of the present invention may exist in non-solvated forms as well as solvated forms (including hydrates). Generally, the solvated forms are equivalent to the non-solvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended uses of the present invention and are intended to be within the scope of the present invention.

[0357] Unless otherwise indicated, the structures shown herein are also intended to include all stereochemical forms of the structure; that is, the R and S configurations of each asymmetric center. Thus, single stereoisomers as well as enantiomeric and diastereomeric mixtures of the compounds of the invention are within the scope of the invention.

[0358] Unless otherwise indicated, the structures shown herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structure of the invention but in which hydrogen is replaced by deuterium or tritium or carbon is replaced by 13 C- or 14 C-enriched carbon are within the scope of the invention.

[0359] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more atoms that make up such compounds. For example, the compounds may be radiolabeled with a radioactive isotope such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variants of the compounds of the invention, whether radioactive or not, are encompassed within the scope of the invention.

[0360] Pharmaceutical Compositions, Kits, and Administration

[0361] In yet another aspect, provided herein is a pharmaceutical composition that comprises a compound of formula (I)-(XVIII) and a pharmaceutically acceptable carrier or excipient.

[0362] In some embodiments in any aspect, the reagents or compounds provided herein are formulated as pharmaceutical compositions. In another embodiment in any aspect, the pharmaceutical composition is formulated for the treatment of diseases (such as metabolic disorders (such as diabetes, obesity), gastrointestinal diseases (such as gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or for example inflammatory diseases (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver diseases, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis)).

[0363] In another aspect of any embodiment, the present disclosure provides a composition comprising a reagent that inhibits bile salt hydrolase (BSH) in a subject.

[0364] In another embodiment in any aspect, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0365] The present disclosure provides a pharmaceutical composition comprising a compound of formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises a compound of formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0366] In some embodiments, the pharmaceutical composition is in liquid or solid dosage form. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the compounds of any one of formulas (I)-(XVIII), the liquid dosage form may also contain, for example, inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of dehydrated sorbitol, and mixtures thereof. In addition to the inert diluent, the oral composition may also contain adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0367] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound of any one of formulas (I)-(XVIII) is admixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or calcium phosphate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) adsorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0368] Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycols. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be compositions that are optionally formulated to release one or more active ingredients in a delayed manner, only or preferentially in a particular part of the intestine. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycols.

[0369] Compounds of any of formulas (I)-(XVIII) may also be in microencapsulated form with one or more of the excipients described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings well known in the art of pharmaceutical formulations. In such solid dosage forms, the compound of any of formulas (I)-(XVIII) can be admixed with at least one inert diluent such as sucrose, lactose, and starch. As is normal practice, such dosage forms may also contain additional substances in addition to the inert diluent, for example, tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. They may optionally contain emulsifying agents and may also be compositions that release one or more active ingredients in a delayed manner, only or preferentially, in a specific part of the intestine. Examples of embedding compositions that can be used include polymeric materials and waxes.

[0370] In some embodiments, the carrier or excipient limits the delivery of the composition to the gastrointestinal tract. In some embodiments, the compositions provided herein are restricted to the gastrointestinal tract by increasing sulfate groups or polar groups to the compound.

[0371] In some embodiments, the carrier or excipient is an enteric coating or an enteric-coated drug delivery device. As used herein, the term "enteric coating" or "enteric-coated delivery device" refers to any drug delivery method that can be orally administered but does not degrade or activate until the device reaches the intestine. Such methods can utilize coatings or capsules that degrade in a pH-dependent manner, for example, thereby allowing protection of the delivery device as well as the reagent to be administered or transplanted throughout the gastrointestinal tract until the device reaches the alkaline pH of the intestine (e.g., the cecum or colon).

[0372] An enteric coating can control the location of release of the reagent in the digestive system. Thus, an enteric coating can be used such that the pharmaceutical composition does not dissolve and release the reagent in the stomach, but travels to the intestine where it dissolves and releases the reagent in an environment most conducive to inhibiting BSH (e.g., targeting bacteria located in the cecum, ileum, large intestine, or colon). The enteric coating can be stable at low pH (such as in the stomach) and dissolve at higher pH (such as in the intestine). Materials that can be used for the enteric coating include, for example, alginic acid, cellulose acetate phthalate, plastics, waxes, shellac, and fatty acids (e.g., stearic acid, palmitic acid). Enteric coatings are described, for example, in U.S. Patent Nos. 5,225,202, 5,733,575, 6,139,875, 6,420,473, 6,455,052, and 6,569,457, all of which are incorporated herein by reference in their entirety. The enteric coating can be an aqueous enteric coating. Examples of polymers that can be used for the enteric coating include, for example, shellac (trade names EmCoat 120N, Marcoat 125); cellulose acetate phthalate (trade names AQUACOAT TM , AQUACOAT ECD TM , SEPIFILM TM , KLUCEL TM , and METOLOSE TM ); polyvinyl acetate phthalate (trade name SURETERIC TM ); and methacrylic acid (trade names EUDRAGIT TM , EUDRAGIT L 100-55 TM , obtained from Evonik Industries, Germany).

[0373] Another example of a method known in the art for confining a pharmaceutical composition to the intestine includes enteric magnesium micromotors (EMgM). EMgM is described in the art, for example, in Li et al., ACS NANO, (2016).

[0374] Pharmaceutical compositions include formulations suitable for oral administration, which can be provided as discrete units such as tablets, capsules, cachets, syrups, elixirs, ready-to-eat foods, microemulsions, solutions, suspensions, lozenges, or gel-coated ampoules, each containing a predetermined amount of the active compound; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or water-in-oil or oil-in-water emulsions.

[0375] Accordingly, formulations suitable for rectal administration can be used, including gels, creams, lotions, aqueous or oily suspensions, dispersible powders or granules, emulsions, soluble solid materials, enemas, etc. The formulation is preferably provided as a unit dose suppository, which contains the active ingredient in one or more solid carriers (such as cocoa butter) that form the suppository base. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycol, alcohols, and combinations thereof. Alternatively, a colon lavage solution with the rapid recolonization deployment agent of the present disclosure can be formulated for colon or rectal administration.

[0376] In certain embodiments, the compound or pharmaceutical composition is a solid. In certain embodiments, the compound or pharmaceutical composition is a powder. In certain embodiments, the compound or pharmaceutical composition can be dissolved in a liquid to prepare a solution. In certain embodiments, the compound or pharmaceutical composition is dissolved in water to prepare an aqueous solution. In certain embodiments, the pharmaceutical composition is a liquid for parenteral injection. In certain embodiments, the pharmaceutical composition is a liquid for oral administration (e.g., ingestion). In certain embodiments, the pharmaceutical composition is a liquid for intravenous injection (e.g., an aqueous solution). In certain embodiments, the pharmaceutical composition is a liquid for subcutaneous injection (e.g., an aqueous solution).

[0377] After being formulated with the desired dose and appropriate pharmaceutically acceptable excipients, depending on the disease or condition being treated, the pharmaceutical compositions of the present disclosure can be administered orally, parenterally, intracisternally, intraperitoneally, topically, buccally, etc. to humans and other animals.

[0378] In certain embodiments, a pharmaceutical composition comprising a compound of formula (I)-(XVIII) is administered orally or parenterally at a dose level sufficient to deliver from about 0.001 mg / kg to about 200 mg / kg of each pharmaceutical composition, in one or more doses over one day or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose is from about 0.001 mg / kg to about 200 mg / kg per day, from about 0.001 mg / kg to about 100 mg / kg per day, from about 0.01 mg / kg to about 100 mg / kg per day, from about 0.01 mg / kg to about 50 mg / kg per day, preferably from about 0.1 mg / kg to about 40 mg / kg per day, preferably from about 0.5 mg / kg to about 30 mg / kg per day, from about 0.01 mg / kg to about 10 mg / kg per day, from about 0.1 mg / kg to about 10 mg / kg per day, subject body weight change, once or more than once a day, to obtain the desired therapeutic and / or prophylactic effect. In certain embodiments, the compounds described herein may be at a dose level sufficient to deliver from about 0.001 mg / kg to about 200 mg / kg per day, from about 0.001 mg / kg to about 100 mg / kg per day, from about 0.01 mg / kg to about 100 mg / kg per day, from about 0.01 mg / kg to about 50 mg / kg per day, preferably from about 0.1 mg / kg to about 40 mg / kg per day, preferably from about 0.5 mg / kg to about 30 mg / kg per day, from about 0.01 mg / kg to about 10 mg / kg per day, from about 0.1 mg / kg to about 10 mg / kg per day, and more preferably from about 1 mg / kg to about 25 mg / kg of subject body weight, once or more than once a day, to obtain the desired therapeutic and / or prophylactic effect. The desired dose may be delivered three times a day, twice a day, once a day, every other day, every three days, weekly, bi-weekly, tri-weekly, or monthly. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations). In certain embodiments, the dose administered of the compositions described herein is below the dose at which the compound or agent causes a non-specific effect.

[0379] In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.001 mg to about 1000 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.01 mg to about 200 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.01 mg to about 100 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.01 mg to about 50 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.01 mg to about 10 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered in a dose of from about 0.1 mg to about 10 mg per unit dose.

[0380] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. Generally, such preparation methods include the steps of combining a composition comprising a compound of (I)-(XVIII) with a carrier and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose unit.

[0381] The pharmaceutical composition can be prepared, packaged, and / or sold as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is usually equal to the dose of the active ingredient to be administered to a subject, and / or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

[0382] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route of administration of the composition. By way of example, the composition can contain from 0.1% to 100% (w / w) of the active ingredient.

[0383] Pharmaceutically acceptable excipients for manufacturing the provided pharmaceutical compositions include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and aromatic agents can also be present in the composition.

[0384] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and mixtures thereof.

[0385] Exemplary granulating agents and / or dispersants include potato starch, corn starch, tapioca starch, sodium carboxymethyl starch, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium carboxyethyl starch), carboxymethyl cellulose, croscarmellose sodium (croscarmellose), methyl cellulose, pregelatinized starch (Starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0386] Exemplary surfactants and / or emulsifiers include natural emulsifiers (such as gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (such as bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long-chain amino acid derivatives, high molecular weight alcohols (such as stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (such as carboxypolymethylene, polyacrylic acid, acrylic polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (such as sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (such as polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80)), polyethylene oxide esters (such as polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyethylene oxide stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (such as Cremophor TM ), polyoxyethylene ethers (such as polyoxyethylene lauryl ether (Brij 30)), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, Poloxamer-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium dioctyl sulfosuccinate, and / or mixtures thereof.

[0387] Exemplary adhesives include starches (such as corn starch and starch paste), gelatin, sugars (such as sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (such as gum arabic, sodium alginate, Irish moss extract, panwar gum, Indian gum, isapol hull mucilage, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum), and pine polysaccharides), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, alcohol, and / or mixtures thereof.

[0388] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0389] Exemplary antioxidants include α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, thioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0390] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (such as sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (such as citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0391] Exemplary antifungal preservatives include butyl p-hydroxybenzoate, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0392] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenethyl alcohol.

[0393] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, β-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0394] Other preservatives include tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium bisulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.

[0395] Exemplary buffers include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconolactate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate (calciumhydroxide phosphate), potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0396] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, sodium lauryl sulfate, sodium laureth sulfate, and mixtures thereof.

[0397] Exemplary natural oils include almond, apricot, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba wax, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango seed, melissa officinalis, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, seabuckthorn, sesame, shea butter, siloxane, soybean, sunflower, tea tree, thistle, tsukemoto, vetiver, walnut, and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0398] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active agent, the liquid dosage forms may also contain, for example, inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, oral compositions may also contain adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and fragrances. In certain embodiments for parenteral administration, the agents of the present invention are mixed with solubilizing agents, such as CREMOPHOR (polyethoxylated castor oil), alcohols, oils, modified oils, diols, polysorbates, cyclodextrins, polymers, and combinations thereof.

[0399] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3 - butanediol. Acceptable vehicles and solvents that may be employed are water, Ringer's solution U.S.P. and isotonic sodium chloride solution. In addition, a sterile fixed oil is conventionally used as a solvent or suspending medium. For this purpose, any mild fixed oil may be employed, including synthetic mono - or diglycerides of fatty acids. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0400] The injectable preparation may be sterilized, for example, by filtration through a bacteria - retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition which may be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0401] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is admixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or calcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) adsorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0402] Solid compositions of a similar type may also be employed as fillers in soft and hard - filled gelatin capsules using excipients such as lactose or sucrose syrup and high - molecular - weight polyethylene glycols. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other coatings well - known in the art of pharmaceutical formulations. They may optionally contain opacifying agents and may also be compositions that optionally release one or more active ingredients only, or preferentially, in a delayed manner in a particular part of the intestine. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard - filled gelatin capsules using excipients such as lactose or sucrose syrup and high - molecular - weight polyethylene glycols.

[0403] The active agent may also be in microencapsulated form with one or more of the excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active agent can be admixed with at least one inert diluent such as sucrose, lactose, or starch. As in normal practice, such dosage forms may also contain additional substances in addition to the inert diluent, for example, tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. They may optionally contain emulsifying agents and may also be compositions that optionally release one or more active ingredients in a delayed manner only in or preferentially in a particular part of the intestine. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0404] Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, gels, applicators, oil-in-water or water-in-oil emulsions such as creams, ointments, or pastes; or solutions or suspensions such as drops. Formulations for topical administration to the skin surface can be prepared by dispersing the drug in a dermatologically acceptable carrier such as a lotion, cream, ointment, or soap. The available carriers are capable of forming a film or layer on the skin to localize the administration and impede removal. For topical administration to the surface of internal tissues, the agent can be dispersed in a liquid tissue adhesive or other substances known to enhance adsorption to the tissue surface. For example, the use of hydroxypropyl cellulose or fibrinogen / thrombin solutions can be beneficial. Alternatively, tissue coating solutions such as those containing pectin preparations can be used. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the additional advantage of providing controlled delivery of the agent to the body. Such dosage forms can be prepared by dissolving or dispensing the agent in a suitable medium. Penetration enhancers can also be used to increase the flux of the agent through the skin. The rate can be controlled by providing a rate controlling membrane or by dispersing the agent in a polymeric matrix or gel.

[0405] Additionally, the form of the carrier for topical formulations can be a hydroalcoholic system (such as liquids and gels), an anhydrous oil or silicone-based system, or an emulsion system, including but not limited to oil-in-water, water-in-oil, water-in-oil-in-water, and silicone-in-water-in-oil emulsions. The emulsions can cover a wide range of consistencies, including thin lotions (which can also be suitable for spray or aerosol delivery), milky lotions, light creams, heavy creams, etc. The emulsions can also include microemulsion systems. Other suitable topical carriers include anhydrous solids and semi-solids (such as gels and sticks); and water-based mousse systems.

[0406] The present disclosure also encompasses kits (e.g., pharmaceutical packages). The provided kits may include the pharmaceutical compositions or compounds described herein and a container (e.g., vial, ampoule, bottle, syringe, and / or dispenser package, or other suitable container). In some embodiments, the provided kits may also optionally include a second container that contains a pharmaceutical excipient for diluting or suspending the pharmaceutical compositions or compounds described herein. In some embodiments, the pharmaceutical compositions or compounds described herein provided in the first and second containers are combined to form a unit dosage form.

[0407] Accordingly, in one aspect, there is provided a kit that includes a first container containing a compound or pharmaceutical composition of formula (I)-(XVIII) described herein. In certain embodiments, the kit can be used to treat diseases (such as metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases (e.g., gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), inflammatory diseases (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis)) in a subject in need thereof. In certain embodiments, the kit can be used to prevent diseases (such as metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases (e.g., gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), inflammatory diseases (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis)) in a subject in need thereof.In certain embodiments, the kit can be used to reduce the risk of developing a disease (such as a metabolic disorder (such as diabetes, obesity), a gastrointestinal disease, cancer, an inflammatory disease (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, and gastroesophageal reflux disease (GERD)), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof.

[0408] In certain embodiments, the kits described herein further include instructions for using the kits. The kits described herein may further include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and the instructions provide for treating a disease (such as a metabolic disorder (such as diabetes, obesity), an inflammatory disease (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, and gastroesophageal reflux disease (GERD)), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof. In certain embodiments, the kit and the instructions provide for preventing a disease (such as a metabolic disorder (such as diabetes, obesity), a gastrointestinal disease (such as a gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as a digestive system cancer; liver malignancy; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancer), or an inflammatory disease (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis)) in a subject in need thereof. In certain embodiments, the kit and the instructions provide for reducing the risk of developing a disease (such as a metabolic disorder (such as diabetes, obesity), a gastrointestinal disease, cancer (such as liver cancer), or an inflammatory disease (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis)) in a subject in need thereof. The kits described herein may include one or more of the additional pharmaceutical compounds described herein in separate compositions.

[0409] Method of treatment

[0410] In one aspect, the present disclosure provides a method of modulating bile acids in a subject. In another aspect, the present disclosure provides a method of inhibiting bile acid dissociation in a subject. In yet another aspect, the present disclosure provides a method of promoting bile acid conjugation in a subject.

[0411] In one aspect of any of the embodiments, the present disclosure provides a method of modulating bile acids in a subject, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a compound of any one of Formulas I-XVIII provided herein, a derivative thereof, or a pharmaceutical composition.

[0412] In some embodiments of any aspect, the agent is an inhibitor of BSH. In some embodiments of any aspect, the agent is an inhibitor of bacterial BSH present in a host subject.

[0413] In another embodiment of any aspect, the agent or inhibitor is a compound of Formulas (I)-(XVIII) or a derivative thereof; Compounds 1-9 or a derivative thereof; riboflavin; or caffeic acid phenethyl ester (CAPE). Compounds 1-9 are also shown in Figure 2D in.

[0414] In another embodiment of any aspect, the inhibitor is selected from small molecules, antibodies, peptides, genome editing systems, antisense oligonucleotides, shRNA, and siRNA.

[0415] In some embodiments of any aspect, the agent that inhibits BSH is RNAi, siRNA, or shRNA. As used herein, the term “RNAi” or “siRNA” or “shRNA” refers to interfering RNA or RNA interference. RNAi refers to a mode of selective post-transcriptional gene silencing by disrupting a specific mRNA by a molecule that binds to the mRNA and inhibits mRNA processing (e.g., inhibits mRNA translation or causes mRNA degradation). As used herein, the term “RNAi” refers to any type of interfering RNA, including but not limited to siRNA, shRNA, endogenous microRNA, and artificial microRNA. For example, it includes sequences previously identified as siRNA, regardless of the downstream processing mechanism of the RNA.

[0416] In some embodiments in any aspect, the reagent that inhibits BSH is an antisense oligonucleotide. As used herein, "antisense oligonucleotide" refers to a synthetic nucleic acid sequence that is complementary to a DNA or mRNA sequence, such as the sequence of a microRNA. Antisense oligonucleotides are typically designed to block the expression of a DNA or RNA target by binding to the target and stopping expression at the transcriptional, translational, or splicing level. The antisense oligonucleotides as described herein are complementary nucleic acid sequences designed to hybridize to a gene under cellular conditions. Thus, such oligonucleotides are selected that are sufficiently complementary to the target, i.e., hybridize well and have sufficient specificity under the circumstances of the cellular environment to give the desired effect. For example, antisense oligonucleotides that directly or indirectly inhibit the level or activity of BSH may include at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or more bases that are complementary to a portion of the coding sequence of bacterial BSH. In addition, antisense oligonucleotides can target transcription factors that regulate the expression of bacterial BSH.

[0417] In some embodiments, the reagent that inhibits BSH is an antibody. As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. Antibody reagents can comprise an antibody or a polypeptide containing the antigen-binding domain of an antibody. In some embodiments in any aspect, the antibody reagent can comprise a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). Alternatively, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; the full text of which is incorporated herein by reference)) as well as intact antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as their subtypes and combinations). Antibodies can be from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates) and primatized antibodies. Antibodies also include broadly neutralizing antibodies, midibodies, nanobodies, humanized antibodies, chimeric antibodies, and the like.

[0418] In other embodiments, the agent that inhibits BSH is a polypeptide. As used herein, the term "polypeptide" is intended to cover both the singular "polypeptide" and the plural "polypeptides", and includes any chain or chains of two or more amino acids. Thus, as used herein, the terms including but not limited to "peptide", "dipeptide", "tripeptide", "protein", "enzyme", "amino acid chain", and "continuous amino acid sequence" are all covered within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term also includes polypeptides that have undergone one or more post-translational modifications, including for example but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or modification by the inclusion of one or more non-naturally occurring amino acids. There are conventional nomenclatures for polynucleotide and polypeptide structures. For example, single-letter and three-letter abbreviations are widely used to describe amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine (C; Cys), glutamine (Q; Gln), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val), and lysine (K; Lys). The amino acid residues provided herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form may replace any L-amino acid residue as long as the desired properties of the polypeptide are retained.

[0419] In another embodiment in any aspect, a bacterial cell genome is used to inhibit BSH using any genome editing system, including but not limited to zinc finger nucleases, TALENs, meganucleases, and CRISPR / Cas systems. In some embodiments in any aspect, the genome editing system used to introduce a nucleic acid encoding one or more guide RNAs into the genome of a cell is not a CRISPR / Cas system; this can prevent unwanted cell death in cells that retain a small amount of Cas enzyme / protein. It is also contemplated herein that the Cas enzyme or sgRNA is each expressed under the control of a different inducible promoter, thereby allowing their respective transient expression to prevent such interference. The gene editing system can directly or indirectly regulate the level or activity or expression of BSH.

[0420] In one aspect of any embodiment, provided herein is a method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound provided herein.

[0421] In some embodiments in any aspect, the agent is an inhibitor of bile salt hydrolase (BSH). In another embodiment in any aspect, the agent is a compound of any one of Formulas I - XVIII or 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK). In another embodiment in any aspect, the agent is a derivative of any one of Formulas I - XVIII or 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK). In another embodiment in any aspect, the agent is a bile acid or a derivative thereof. In another embodiment in any aspect, the agent is chenodeoxycholic acid (CDCA) or a derivative thereof.

[0422] In some embodiments, inhibition of BSH results in a decrease in secondary bile acids. In other embodiments, inhibition of BSH promotes the conjugation of bile acids. In another embodiment, inhibition of BSH reduces the dissociation of bile acids. The activity of BSH can be determined by the presence or absence of dissociated bile acids.

[0423] In some embodiments in any aspect, the activity or level of BSH is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to an appropriate control.

[0424] Imbalances in bile acid homeostasis can play a causal role in the pathophysiology of diseases including hypercholesterolemia, obesity, diabetes, cancer, gastrointestinal diseases, and gallstone formation, further highlighting the biological importance of these metabolites.

[0425] In some embodiments in any aspect, the subject is at risk of having or has a gastrointestinal disease (e.g., gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer).

[0426] In some embodiments in any aspect, the disease is a gastrointestinal disease. In certain embodiments, the gastrointestinal disease is a gastrointestinal infection. The gastrointestinal infection is an infection caused by bacteria selected from the following: Staphylococcus; Helicobacter pylori; Escherichia coli; Salmonella; Campylobacter; Yersinia enterocolitica; Shigella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus. In certain embodiments, the gastrointestinal disease is inflammatory bowel disease (IBD). In certain embodiments, the gastrointestinal disease is appendicitis. In certain embodiments, the gastrointestinal disease is Crohn's disease (CD). In certain embodiments, the gastrointestinal disease is ulcerative colitis (UC). In certain embodiments, the gastrointestinal disease is gastritis. In certain embodiments, the gastrointestinal disease is enteritis. In certain embodiments, the gastrointestinal disease is esophagitis. In certain embodiments, the gastrointestinal disease is pancreatitis. In certain embodiments, the gastrointestinal disease is diabetes. In certain embodiments, the gastrointestinal disease is hepatitis. In certain embodiments, the gastrointestinal disease is liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis). In certain embodiments, the gastrointestinal disease is gastroesophageal reflux disease (GERD). In certain embodiments, the gastrointestinal disease is celiac disease. In certain embodiments, the gastrointestinal disease is diverticulitis. In certain embodiments, the gastrointestinal disease is food intolerance. In certain embodiments, the gastrointestinal disease is ulcer. In certain embodiments, the gastrointestinal disease is infectious colitis. In certain embodiments, the gastrointestinal disease is irritable bowel syndrome. In certain embodiments, the gastrointestinal disease is leaky gut syndrome. In certain embodiments, the gastrointestinal disease is cancer.

[0427] In another embodiment in any aspect, the gastrointestinal disease is liver disease. In certain embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the liver disease is non-alcoholic steatohepatitis (NASH). In certain embodiments, the liver disease is hepatitis A. In certain embodiments, the liver disease is hepatitis B. In certain embodiments, the liver disease is hepatitis C. In certain embodiments, the liver disease is autoimmune hepatitis. In certain embodiments, the liver disease is cirrhosis.

[0428] In another embodiment in any aspect, the subject is at risk of having or has obesity. As used herein, the term "obesity" refers to an excess of body fat.

[0429] In some embodiments in any aspect, a subject with obesity may have at least about 25 kg / m before administration of a treatment as described herein 2Subjects with a body mass index. In some embodiments, subjects with obesity may have at least about 30 kg / m² before administration of a treatment, compound, or agent as described herein 2 Subjects with a body mass index.

[0430] In another embodiment in any aspect, the subject is at risk of having or has an inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0431] In one aspect, the present disclosure provides a method for treating diabetes in a subject.

[0432] In some embodiments, the diabetes is type I diabetes, type II diabetes, neonatal diabetes, maturity-onset diabetes of the young, or gestational diabetes.

[0433] In some embodiments, the diabetes is caused by obesity. In one aspect, the present disclosure provides a method for treating obesity in a subject.

[0434] In certain embodiments, the disease is cancer. In certain embodiments, the cancer is a digestive system cancer. In certain embodiments, the cancer is a liver malignancy. In certain embodiments, the cancer is liver cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is esophageal cancer. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is hepatocellular carcinoma. In certain embodiments, the cancer is kidney cancer or renal carcinoma. In certain embodiments, the cancer is oral cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is rectal cancer. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is basal cell carcinoma. In certain embodiments, the cancer is cholangiocarcinoma. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is cervical cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is uterine cancer. In certain embodiments, the cancer is urinary tract cancer.

[0435] In some embodiments, the inflammatory disease is selected from: Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis. In some embodiments, the inflammatory disease is Crohn's disease. In some embodiments, the inflammatory disease is inflammatory bowel disease. In some embodiments, the inflammatory disease is ulcerative colitis. In some embodiments, the inflammatory disease is pancreatitis, hepatitis. In some embodiments, the inflammatory disease is appendicitis. In some embodiments, the inflammatory disease is gastritis. In some embodiments, the inflammatory disease is diverticulitis. In some embodiments, the inflammatory disease is celiac disease. In some embodiments, the inflammatory disease is food intolerance. In some embodiments, the inflammatory disease is enteritis. In some embodiments, the inflammatory disease is ulcers. In some embodiments, the inflammatory disease is gastroesophageal reflux disease (GERD). In some embodiments, the inflammatory disease is psoriatic arthritis. In some embodiments, the inflammatory disease is psoriasis. In some embodiments, the inflammatory disease is rheumatoid arthritis.

[0436] In certain embodiments, the subject being treated is an animal. The animal can be of either sex and can be at any stage of development. In certain embodiments, the subject is a mammal. In certain embodiments, the subject being treated is a human. In certain embodiments, the subject is a domesticated animal such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal.

[0437] In another embodiment in any aspect, the subject is at risk of having or has cancer. The conversion of primary to secondary bile acids can lead to a reduction in tumor suppressors in the liver. This mechanism is expected to extend to other types of cancer. See, e.g., Ma et al. Science (2018), which is incorporated herein by reference in its entirety.

[0438] The methods and compositions provided herein can also be used to treat or prevent prediabetes in a subject. The subject may also have or be at risk of developing diabetes or prediabetic conditions. The cause of diabetes can be due to gene mutations, genetic diabetes, obesity, lifestyle, or idiopathic.

[0439] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such a compound is preferably within the circulating concentration range that includes ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of use or administration utilized.

[0440] The effective dosage can initially be estimated by cell culture assays. The dosage can be formulated in animals. Generally, the composition is administered such that the compounds disclosed herein are used or administered at a dosage of 1 μg / kg to 1000 mg / kg; 1 μg / kg to 500 mg / kg; 1 μg / kg to 150 mg / kg, 1 μg / kg to 100 mg / kg, 1 μg / kg to 50 mg / kg, 1 μg / kg to 20 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 50 mg / kg, 100 μg / kg to 20 mg / kg, 100 μg / kg to 10 mg / kg, 100 μg / kg to 1 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, or 10 mg / kg to 20 mg / kg. It should be understood that the ranges given herein include all intermediate ranges, e.g., the range 1 mg / kg to 10 mg / kg includes 1 mg / kg to 2 mg / kg, 1 mg / kg to 3 mg / kg, 1 mg / kg to 4 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 6 mg / kg, 1 mg / kg to 7 mg / kg, 1 mg / kg to 8 mg / kg, 1 mg / kg to 9 mg / kg, 2 mg / kg to 10 mg / kg, 3 mg / kg to 10 mg / kg, 4 mg / kg to 10 mg / kg, 5 mg / kg to 10 mg / kg, 6 mg / kg to 10 mg / kg, 7 mg / kg to 10 mg / kg, 8 mg / kg to 10 mg / kg, 9 mg / kg to 10 mg / kg, etc. Dosages of about 0.1 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, or 0.5 mg / kg to about 3 mg / kg (as a bolus or continuous infusion) are also contemplated. It should also be understood that the intermediate ranges of those given above are also within the scope of the present disclosure, e.g., within the range of 1 mg / kg to 10 mg / kg, e.g., use or dosage ranges such as 2 mg / kg to 8 mg / kg, 3 mg / kg to 7 mg / kg, 4 mg / kg to 6 mg / kg, etc.

[0441] The compounds described herein can be administered in a single dose, or can be divided into multiple smaller doses and administered at intervals of time. It should be understood that the precise dosage and duration of treatment will be a function of the location of the parenteral administration composition, the vehicle, and other variables that can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that the concentration and dosage values can also vary with the age of the individual being treated. It should also be understood that for any particular subject, the specific dosing regimen may need to be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the formulation. Accordingly, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.

[0442] In one embodiment in any aspect, the reagent, compound, or composition is administered continuously (e.g., at a constant level over a period of time). Continuous administration of the reagent or compound can be achieved, for example, by an epidermal patch, a sustained release formulation, or an in vivo syringe.

[0443] The compound can be administered as a single bolus or multiple boluses, a continuous infusion, or a combination thereof. For example, the compound can be initially administered as a single bolus and then as a continuous infusion after the bolus. The infusion rate can be any desired rate. Some contemplated infusion rates include 1 μg / kg / min to 100 mg / kg / min, or 1 μg / kg / hour to 1000 mg / kg / hour. The infusion rate can include 0.2 to 1.5 mg / kg / min, or more specifically 0.25 to 1 mg / kg / min, or even more specifically 0.25 to 0.5 mg / kg / min. It should be understood that the infusion rate can be determined based on the dose required to maintain an effective plasma concentration and the elimination rate of the compound such that the compound is administered via infusion at a rate sufficient to safely maintain an adequate effective plasma concentration of the compound in the bloodstream.

[0444] The dosage of the reagent or compound as described herein can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effect. With regard to the duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when the treatment is providing efficacy and to determine whether to administer additional reagent, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so large as to cause adverse side effects such as cytokine release syndrome. Generally speaking, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of ordinary skill in the art. In the event of any complications, the dosage can also be adjusted by the individual physician.

[0445] In one embodiment in any aspect, the reagents, compounds, or compositions described herein are used as a monotherapy. In another embodiment in any aspect, the reagents or compounds described herein can be used in combination with other reagents and therapies known to be used for diabetes. As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the course of the subject's affliction with a disorder, e.g., delivering two or more treatments after the subject has been diagnosed with a disorder (such as diabetes) and before the disorder has been cured or eliminated or the treatment has been stopped for other reasons. In some embodiments, the delivery of one treatment still occurs at the start of the delivery of the second treatment, such that there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery".

[0446] In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to combination administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment compared to that observed when the second treatment is administered in the absence of the first treatment, or the second treatment reduces symptoms to a greater extent, or a similar situation is observed in the case of the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters related to the disorder is greater than that observed when delivering one treatment in the absence of the other treatment. The effects of the two treatments can be partially additive, fully additive, or more than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. The compounds and reagents described herein and at least one additional therapy can be administered simultaneously in the same or separate compositions or sequentially. For sequential administration, the reagent described herein can be administered first and the additional reagent second, or the order of administration can be reversed. The reagent and / or other therapeutic agents, procedures, or modalities can be administered during the active disorder period or during a remission or less active disease period. The reagent can be administered before, simultaneously with, after, or during remission of the disorder.

[0447] Currently, therapeutic agents used for treating or preventing gastrointestinal diseases, inflammatory diseases, liver diseases, and metabolic disorders (such as obesity) include, but are not limited to, insulin therapy, sulfonylureas (such as glibenclamide), meglitinides (such as nateglinide), SGLT2 inhibitors (such as canaglifozin), bile acid sequestrants (such as colesevelam), dopamine-2 agonists (such as bromocriptine), biguanides (such as metformin), DPP-4 inhibitors (such as alogliptin, linagliptin, etc.), α-glucosidase inhibitors (such as acarbose and miglitol), thiazolidinediones (such as rosiglitazone), antibiotics (such as aminosalicylic acid, norfloxacin, penicillin, cephalosporin), antiviral agents (such as zanamivir, oseltamivir), vaccines, corticosteroids (such as hydrocortisone, prednisone, prednisolone, budesonide), analgesics (such as paracetamol, ibuprofen), non-steroidal anti-inflammatory drugs (such as mesalazine), anti-inflammatory drugs (such as sulfasalazine), immunosuppressants (such as infliximab, azathioprine, adalimumab, mercaptopurine), dietary supplements (such as iron), surgical procedures (such as colostomy, ileostomy, colectomy, proctocolectomy, gastric bypass), ursodeoxycholic acid (UDCA, also known as ursodiol, INN, NAN, AAN, or USAN), cholestyramine, stanozolol, naltrexone, rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinyl esters, vitamin A, liver dialysis, or liver transplantation, intravenous infusions, enemas, and other therapeutic agents known in the art.

[0448] In addition to the therapeutic agents for the above diseases, chemotherapeutic agents may also be administered. Non-limiting examples of therapeutic agents for cancer (e.g., liver cancer) include nucleoside analogs (e.g., tegafur), folic acid antagonists, anthracyclines, podophyllotoxin, taxanes, alkaloids, alkylating agents, platinum compounds, antibodies, retinoids, histone deacetylase inhibitors, arsenic trioxide, kinase inhibitors (e.g., sorafenib), surgery, or any other chemotherapeutic agent known in the art. Those skilled in the art can readily identify the chemotherapeutic agents to be used (see, e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86, Harrison’s Principles of Internal Medicine, 14th ed.; Perry et al., Chemotherapy, Chapter 17, Abeloff, Clinical Oncology 2nd ed 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds.): The Cancer Chemotherapy Handbook, 4th ed St. Louis, Mosby-Year Book, 1993).

[0449] In addition to the therapeutic agents for the above diseases, chemotherapeutic agents can also be administered. Non-limiting examples of therapeutic agents for cancer (such as liver cancer) include nucleoside analogs (such as tegafur), folic acid antagonists, anthracyclines, podophyllotoxin, taxanes, alkaloids, alkylating agents, platinum compounds, antibodies, retinoids, histone deacetylase inhibitors, arsenic trioxide, kinase inhibitors (such as sorafenib), surgery, or any other chemotherapeutic agent known in the art. Those skilled in the art can readily identify the chemotherapeutic agents to be used (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86, Harrison's Principles of Internal Medicine, 14th Edition; Perry et al., Chemotherapy, Chapter 17, Abeloff, Clinical Oncology 2nd Edition 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds.): Oncology Pocket Guide to Chemotherapy, 2nd Edition St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds.): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

[0450] When administered in combination, the reagent or composition and the additional reagent (such as a second or third reagent) or all can be administered in an amount or dose that is higher, lower, or the same as the amount or dose of each reagent used alone (for example, as a single therapy). In certain embodiments, the amount or dose of the reagent, the additional reagent (such as a second or third reagent), or all is lower than the amount or dose of each reagent used alone (for example, at least 20%, at least 30%, at least 40%, or at least 50%). In other embodiments, the amount or dose of the reagent, the additional reagent (such as a second or third reagent), or all that results in the desired effect (such as treating diabetes) is lower than the amount or dose of each reagent alone required to achieve the same therapeutic effect (for example, at least 20%, at least 30%, at least 40%, or at least 50% lower).

[0451] Administration

[0452] In some embodiments in any aspect, the reagent is administered by direct injection, subcutaneous injection, intramuscular injection, oral administration, or nasal administration. In some embodiments, administration of the reagent or pharmaceutical composition provided herein reduces the glucose level in the serum of the subject.

[0453] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In certain preferred embodiments, the composition is administered orally. In some embodiments, the reagents or compositions provided herein are directly injected into the portal vein. For example, injection into the portal vein can limit systemic side effects of the reagent or pharmaceutical composition. In some embodiments, the compositions provided herein are implanted into the portal vein for sustained release. In some embodiments, the composition is administered via an injection port.

[0454] Because parenteral dosage forms are generally administered by bypassing the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready for dissolution or suspension in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled release parenteral dosage forms, and emulsions.

[0455] Suitable vehicles for providing the parenteral dosage forms of the present disclosure are known to those of skill in the art. Examples include, but are not limited to, sterile water; water for injection USP; saline solutions; glucose solutions; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0456] In some embodiments in any aspect, reagents or pharmaceutical compositions are described herein that are administered to a subject in a controlled or sustained release manner. Ideally, optimally designed controlled release preparations for use in medical treatment are characterized by curing or controlling a condition with the least amount of drug substance in the shortest amount of time. Advantages of controlled release formulations include: 1) extended activity of the drug; 2) reduced frequency of dosing; 3) increased patient compliance; 4) lower total drug dosage; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved therapeutic efficacy; 9) reduced potentiation or loss of drug activity; and 10) improved rate of control of a disease or condition (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled release formulations can be used to control the onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels of compounds of formula (I). In particular, controlled or extended release dosage forms or formulations can be used to ensure maximum effectiveness of a reagent while minimizing potential side effects and safety issues that can occur due to insufficient drug dosage (i.e., below the minimum therapeutic level) and exceeding the toxic level of the drug.

[0457] A variety of known controlled or extended release dosage forms, formulations, and devices are suitable for use with any of the reagents described herein. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185, each of which is incorporated herein by reference in its entirety. These dosage forms can be used to provide slow or controlled release of one or more active ingredients, using, for example, different ratios of hydroxypropyl methylcellulose, other polymeric matrices, gels, osmotic membranes, osmotic systems (such as (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or combinations thereof to provide the desired release characteristics. Additionally, ion exchange materials can be used to prepare fixed adsorbed salt forms of the disclosed compounds, thus achieving controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to A568 and AP143 (Rohm & Haas, Spring House, Pa. USA).

[0458] Efficacy

[0459] The efficacy of the reagents described herein, such as for treating diseases, can be determined by a skilled practitioner. However, if one or more of the signs or symptoms of diabetes, obesity, gastrointestinal diseases, cancer, or inflammatory diseases are altered in a beneficial manner, other clinically acceptable symptoms are improved or even ameliorated, or a desired response of, for example, at least 10% is induced after treatment according to the methods described herein, the treatment is considered to be "effective treatment" as the term is used herein. Efficacy can be evaluated, for example, by measuring markers, metrics, symptoms, and / or incidence of the condition being treated according to the methods described herein or any other suitable measurable parameter (such as glucose level or glucose tolerance). Efficacy can also be measured by the absence of deterioration in an individual, as evaluated by the need for hospitalization or medical intervention (i.e., progression of symptoms). Methods for measuring these metrics are known to those of skill in the art and / or are described herein.

[0460] Efficacy can be evaluated in animal models of the conditions described herein, e.g., a mouse model or a suitable animal model of the disease provided herein, as appropriate. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant change in a marker is observed, such as a decreased blood glucose level in a diabetes model.

[0461] It should be understood that the present disclosure is not limited to the specific methods, protocols, reagents, etc. provided herein and can thus vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure, which is defined only by the claims.

[0462] In certain embodiments, provided herein are methods of treating a metabolic disorder (e.g., diabetes, obesity), a gastrointestinal disease (e.g., gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., digestive system cancers; liver malignancy; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or an inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof.

[0463] In certain embodiments, provided herein are methods of preventing a metabolic disorder (e.g., diabetes, obesity), a gastrointestinal disease (e.g., gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., digestive system cancers; liver malignancy; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or an inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof.

[0464] The present disclosure also provides a compound of formula (I)-(XVIII) or a pharmaceutically acceptable salt thereof for treating metabolic disorders (such as diabetes, obesity), gastrointestinal diseases (such as gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or inflammatory diseases (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis and rheumatoid arthritis).

[0465] The present disclosure also provides a compound of formula (I)-(XVIII) or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating metabolic disorders (such as diabetes, obesity), gastrointestinal diseases (such as gastrointestinal infections; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; gastric cancer; hepatocellular carcinoma; kidney cancer or renal carcinoma; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; gastric cancer; basal cell carcinoma, cholangiocarcinoma; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancers), or inflammatory diseases (such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis and rheumatoid arthritis).

[0466] In certain embodiments, the disease is a metabolic disorder. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the diabetes is type I diabetes. In certain embodiments, the diabetes is type II diabetes. In certain embodiments, the metabolic disorder is obesity.

[0467] In certain embodiments, the disease is an inflammatory disease. In certain embodiments, the inflammatory disease is Crohn's disease. In certain embodiments, the inflammatory disease is inflammatory bowel disease. In certain embodiments, the inflammatory disease is ulcerative colitis. In certain embodiments, the inflammatory disease is pancreatitis, hepatitis. In certain embodiments, the inflammatory disease is appendicitis. In certain embodiments, the inflammatory disease is gastritis, diverticulitis. In certain embodiments, the inflammatory disease is celiac disease. In certain embodiments, the inflammatory disease is food intolerance. In certain embodiments, the inflammatory disease is enteritis, ulcer, gastroesophageal reflux disease (GERD). In certain embodiments, the inflammatory disease is psoriatic arthritis. In certain embodiments, the inflammatory disease is psoriasis. In certain embodiments, the inflammatory disease is rheumatoid arthritis.

[0468] In certain embodiments, the methods of the present disclosure include administering to a subject an effective amount of a compound of formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount.

[0469] The specific methods described herein may include administering a combination of one or more additional agents with the compounds described herein. The one or more additional agents may be administered at the same time as the compound of formula (I)-(XVIII), or at a different time than the compound of formula (I)-(XVII). For example, the compound of formula (I)-(XVIII) and any one or more additional agents may be on the same dosing schedule or different dosing schedules. All or some of the doses of the compound of formula (I)-(XVIII) may be administered before all or some of the doses of the additional agent, after all or some of the doses of the additional agent, within the dosing schedule of the additional agent, or a combination thereof. For different additional agents, the timing of administration of the compound of formula (I)-(XVIII) and the additional agent may be different.

[0470] Examples

[0471] Assay protocol

[0472] Bacterial culture. All bacterial strains were grown in Cullen-Haiser Gut (CHG) medium, which consists of Brain Heart Infusion medium (Bacto TM BHI, BD), supplemented with 1% BBL vitamin K 1- Hemin solution (BD), 1% trace mineral solution (ATCC), 1% trace vitamin solution (ATCC), 5% fetal bovine serum (Hyclone), 1 g / L cellobiose, 1 g / L maltose and 1 g / L fructose) or BHI + (Bacto TM BHI, BD, supplemented with 5 mg / L hemin and 2.5 μL / L vitamin K 1 ) were cultured at 37 °C. All strains were grown under anaerobic conditions in an anaerobic chamber (Coy Lab Products Airlock) with a gas mixture of 5% hydrogen, 20% carbon dioxide and nitrogen. Escherichia coli was grown aerobically at 37 °C in LB medium supplemented with ampicillin for selection against the pET21b plasmid.

[0473] UPLC-MS analysis. Bile acid analysis was performed by UPLC-MS using a published method. 16 A correction factor for extraction efficiency was used and determined by extracting known concentrations of relevant bile acids from buffer or bacterial medium and comparing with a standard curve. The detection limits for individual bile acids were determined using commercially available standards / synthetic compounds dissolved in 1:1 MeOH / water and were as follows: βMCA, 0.03 picomoles / μL; TβMCA, 0.01 picomoles / μL; CA, 0.04 picomoles / μL; TCA, 0.01 picomoles / μL; UDCA, 0.04 picomoles / μL; TUDCA, 0.01 picomoles / μL; DCA, 0.04 picomoles / μL; TDCA, 0.05 picomoles / μL; GCDCA-d4, 0.1 picomoles / μL; CDCA-d4, 0.1 picomoles / μL; 7-oxo-CA, 0.5 picomoles / μL; 7, 1.0 picomoles / μL; GR-7, 0.05 picomoles / μL.

[0474] Protein expression and purification.

[0475] Bacteroides thetaiotaomicron rBSH. The gene encoding BT_2086 (without leader sequence) of Escherichia coli was codon-optimized and cloned into a vector containing a C-terminal His 6In the pET-21b(+) vector of the label (primers are shown in Table 2). Then, under ampicillin selection, the expression plasmid was transformed into BL21(DE3)pLysS Escherichia coli (New England Biolabs) cells. The overnight culture grown in LB medium containing ampicillin (50 μg / mL) was diluted 1:1000 with fresh LB medium containing ampicillin and grown at 37 °C. Expression was induced by adding 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) at an OD of 0.6 - 0.7 and further incubated overnight at 18 °C. Cells were pelleted by centrifugation at 7,000 g for 20 minutes at 4 °C. The pelleted cells were then resuspended in PBS buffer (with 5% glycerol) containing 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The resuspended cells were sonicated and pelleted by centrifugation at 16,000 g for 20 minutes at 4 °C. The supernatant was then mixed with preformed Ni-NTA at 4 °C for 45 minutes. Nickel-bound proteins were eluted with PBS buffer containing gradually increasing concentrations of imidazole (with 0.25 mM TCEP and 5% glycerol). The purity of the collected fractions was tested by SDS-PAGE. The pure fractions were pooled and concentrated, and then dialyzed using a storage buffer (PBS at pH 7.5 with 0.25 mM TCEP and 5% glycerol). 600 For crystallization purposes, the protein was further purified on a BioRad FPLC using an S200 size exclusion column (obtained from GE) in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 7.5 containing 300 mM NaCl, 0.25 mM TCEP, and 5% glycerol.

[0476] Recombinant BSH from Bifidobacterium longum was expressed and purified as above from Bifidobacterium longum SBT2928, except that 0.25 mM IPTG was used for protein expression and 1 mM TCEP was used for protein purification.

[0477] Enzyme kinetics. The enzyme was characterized using a modified BSH activity assay

[0478] 26 ​To 144.8 μL of PBS buffer (containing 10 mM TCEP and 5% glycerol), 35.2 μL of rBSH was added to obtain final concentrations of 6.2 μM and 7.0 μM for Bacteroides thetaiotaomicron BSH and Bifidobacterium longum BSH, respectively. The solution was preheated to 37 °C in a water bath. 20 μL of DMSO containing conjugated bile acids at an appropriate concentration was preheated to 37 °C in a water bath and added to the above solution. At each time interval, 15 μL of the mixture was quenched with 15 μL of 15% trichloroacetic acid. The turbid solution was centrifuged at 4,200 g for 15 minutes. 10 μL of the supernatant was added to 190 μL of ninhydrin mixture (15 mL of 0.5 M sodium citrate (pH 5.5) containing 1% [weight / volume] ninhydrin, 36 mL of glycerol, and 6 mL of 0.5 M sodium citrate buffer (pH 5.5)), and the mixture was heated to 100 °C in a BioRad thermal cycler for 18 minutes. The obtained solution was cooled at 4 °C for 20 minutes, and the absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices).

[0479] Inhibitor screening using rBSH. 200 nM rBSH was incubated with 100 μM inhibitor in 3 mL of PBS buffer (pH 7.5) containing 0.25 mM TCEP and 5% glycerol at 37 °C for 30 minutes. The bile acid pool (100 μM) was added to the above solution and incubated at 37 °C. At time point intervals, 1 mL of the above buffer solution was acidified to pH = 1 with 6 M HCl and extracted twice with 1 mL of ethyl acetate. Then the combined organic layers were dried using a Biotage TurboVap LV. The dried extract was resuspended in 1:1 methanol:water and transferred to a mass spectrometry vial. The samples were analyzed according to the method described in "UPLC-MS analysis". The obtained bile acid concentrations were used to determine % dissociation.

[0480] Formula for calculating % dissociation.

[0481] % dissociation = concentration of detected dissociated bile acids / (concentration of detected dissociated bile acids + concentration of detected conjugated bile acids) * 100.

[0482] Kinetic study of compound 7. The assay was run in PBS buffer (containing 0.25 mM TCEP and 5% glycerol), and all reactants were incubated at 37 °C before the start time of the reaction. Bacteroides thetaiotaomicron BSH (200 nM) was added to a pool of 100 μM bile acids and a pool of 100 μM 7. At the designated time points, 500 μL aliquots were removed and snap-frozen in liquid nitrogen. After thawing, the solution was acidified to pH = 1 with 6 M HCl and then processed as described in "Inhibitor screening using rBSH". The procedure was repeated with 8.2 mM TUDCA.

[0483] Determination of the IC 50 value of compound 7 against the recombinant protein. 200 nM rBSH was incubated with increasing concentrations of 7 in 1 mL PBS buffer (pH 7.5) containing 0.25 mM TCEP and 5% glycerol at 37 °C for 1 hour. 100 μM bile acids (TUDCA for Bacteroides thetaiotaomicron BSH and TDCA for Bifidobacterium longum BSH) were added to the above solution and incubated at 37 °C for 2 hours. The solution was acidified to pH = 1 with 6 M HCl and then processed as described in "Inhibitor screening using rBSH".

[0484] Inhibitor screening in bacteria. Bacterial cultures were diluted to an OD + of 0.1 with 4 mL BHI containing 100 μM taurine-conjugated bile acid pool and 100 μM inhibitor 600 . These cultures were then grown anaerobically at 37 °C. After 21 hours, serial dilutions were plated on BHI + agar to determine cell viability (CFU / mL). 1 mL of the whole bacterial culture was acidified to pH = 1 with 6 M HCl, then 2 mL of ethyl acetate was added and vortexed. The culture was centrifuged at 2,500 g for 5 minutes in a centrifuge to obtain better separation. Then the organic layer was removed, and the aqueous layer was extracted again with 2 mL of ethyl acetate. The dried organic extract was resuspended in 1:1 methanol:water and transferred to a mass spectrometry vial and analyzed as described in "UPLC-MS analysis". The bile acid concentrations obtained were used to determine the % dissociation.

[0485] Determination of the IC 50 value of compound 7 in bacterial cultures. Note that due to the slow growth of Bifidobacterium longum, Bifidobacterium adolescentis was used for the study in growing bacteria. Overnight cultures of Bacteroides thetaiotaomicron and Bifidobacterium adolescentis were diluted to an OD 600, the fresh CHG medium contained 100 μM TUDCA or TDCA and increasing concentrations of inhibitor 7, respectively. In the inhibitor screening in bacterial assays, the dissociation of TUDCA and TDCA by Bacteroides thetaiotaomicron and Bifidobacterium adolescentis reached the maximum extent of any conjugated substrate, and thus these substrates were used to determine the IC 50 values. The cultures were then grown anaerobically at 37 °C for 24 h (Bifidobacterium adolescentis) or 48 h (Bacteroides thetaiotaomicron). Bacteroides thetaiotaomicron requires a longer incubation time because for this bacterium, significant BSH activity was observed only during the stationary phase. The cultures were extracted and analyzed according to the method described in "Inhibitor Screening in Bacteria".

[0486] Screening of inhibitors in conventional mouse feces. A modified form of the published method was used to quantify BSH activity in fecal pellets 45 . Fecal pellets (approximately 10 - 20 mg) were disrupted into fine particles in buffer (10% PBS, 90% sodium acetate, pH 5.2) to obtain a concentration of 1 mg / mL. The inhibitor at the designated concentration was added to the fecal slurry, and the mixture was incubated at 37 °C for 30 min. 100 μM glycodeoxycholic acid-d4 (GCDCA-d4) was added to the mixture and incubated at 37 °C for 18 h. The tubes were then frozen in dry ice for 5 min and, after thawing, diluted with an equal volume of methanol. The slurry was centrifuged at 12,500 g for 10 min. The supernatant was transferred to a clean eppendorf tube and centrifuged again. The supernatant was transferred to an MS vial and the samples were analyzed according to the method described in "UPLC-MS Analysis". The product concentrations detected from these assays were reported directly.

[0487] Crystallization, data collection, and structure determination. Crystals of BSH and BSH complexed with 7 were grown by hanging-drop cultivation in 24-well format at room temperature. After 3 days, BSH crystals (5.0 mg / mL) were grown by microseeding in 42% tacimate 100 mM Tris pH 7.4. After 5 days, BSH-7 complex (5.0 mg / mL) crystals were grown in 21% PEG 3350 and 100 mM trisodium citrate dihydrate pH 5.0. The crystals were cryoprotected by supplementing the mother liquor with 10% 2-methyl-2,4-pentanediol (v / v). At the Advanced Photon Source NE-CAT beamline 24ID-C, data collection was performed at 100 K using the wavelength of. The diffraction images were processed and scaled using XDS. To obtain the phases for the apo BSH structure, 3HBC was used as a search model and Phaser was used in Phenix 46Molecular replacement was performed. Iterative modeling and reciprocal lattice space refinement were performed in COOT and phenix.refine, respectively. The BSH-7 structure was phased using molecular replacement with the apo BSH as the search model. Iterative modeling and refinement of the atomic B-factors of the BSH-7 ensemble were carried out, and the applied twinning law of k h–l was used. Composite omit density maps were used to evaluate the model quality of the two structures. In the final cycle of model building, NCS restraints were removed. The final model quality was evaluated using MolProbity 48 . For 6UFY, 97% of the residues are in the favored regions of the Ramachandran plot, 3% are in the allowed regions, and none are in the outlier regions; for 6UH4, 89.3% of the residues are in the favored regions, 10.3% are in the allowed regions, and 0.4% are outliers. All crystallographic data processing, refinement, and analysis software are curated and supported by the SBGrid Consortium 49 . Figures were prepared using Pymol .

[0488] Mass spectrometry for identification of residues labeled on BSH. The BSH protein was incubated with DMSO or 10-fold molar excess of inhibitor 7 at room temperature for 2 h. The reaction was then analyzed by LC-MS using a Shimadzu LC and autosampler system (Shimadzu, Marlborough, MA) interfaced with an LTQ ion trap mass spectrometer (ThermoFisher Scientific, San Jose, CA).

[0489] To determine the modification sites, the compound 7-modified protein was analyzed as described above, except that the LC system was interfaced with an Orbitrap Lumos mass spectrometer (ThermoFisher Scientific). The mass spectrometer was programmed to perform consecutive cycles consisting of 1 MS scan (m / z 300 - 2000, profile mode, electron multiplier detection), followed by an ETD MS / MS scan of the +41 charge state precursor of the compound 7-modified protein (ETD reagent target = 200 ms, image current detection at 60K resolution, target value = 2E6, ETD reaction time = 100 or 200 ms). Ion assignment was performed using mzStudio software 50 .

[0490] Effect of 7 on FXR. The effect of 7 on FXR was tested using the LanthaScreen TR-FRET Coactivator assay (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The known FXR agonist GW4064G (Sigma, G5172) was used as a positive control (agonist assay) or at its EC50 (50.3 nM, measured in this assay) was added (antagonist assay). After incubation at room temperature for 1 hour, the 520 / 495 TR-FRET ratio was measured using a PerkinElmer Envision fluorescence plate reader with the following filter settings: excitation at 340 nm, emission at 495 nm, and emission at 520 nm. A 100 μsec delay was used, followed by a 200 μsec integration time to collect the time-resolved signal.

[0491] Cell cultures. Caco-2 cells and NCI-H716 cells were obtained from the American Type Culture Collection (Manassas, VA). Caco-2 cells were maintained in Minimal Essential Medium (MEM) supplemented with GlutaMAX and Earle's salts, while NCI-H76 cells were maintained in Roswell Park Memorial Institute (RPMI) medium (Gibco, Life Technologies, UK). All cell media were supplemented with 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin (GenClone). Cells were grown in a 5% CO 2 atmosphere at 37 °C in "complete" medium supplemented with FBS and antibiotics.

[0492] Plasmids and transient transfection. For the luciferase reporter assay, vectors expressing human reporter gene constructs were used. The pGL4.29[luc2P / CRE / Hygro] plasmid (Promega Corporation) was transiently transfected into Caco-2 cells at a concentration of 2 μg / ml of medium each, respectively, for studying TGR5 activation. The pGL4.74[hRluc / CMV] plasmid (Promega Corporation) was used as a transfection efficiency control at a concentration of 0.05 μg / ml of medium. All plasmids were transfected using Opti-MEM (Gibco) and Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. Plasmid transfection was performed in antibiotic-free MEM medium containing 10% FBS. After incubation overnight, 7α and / or bile acids were added in complete medium. 7α and / or bile acids were diluted with DMSO and the concentration of DMSO was kept constant. 10 μM of LCA was added together with 7α to study TGR5 antagonism and incubated overnight. Cells were harvested the next day for luciferase assay.

[0493] Luciferase reporter assay. Luminescence was measured using the Dual-Luciferase Reporter assay system (Promega Corporation) according to the manufacturer's instructions. Cells were gently washed with PBS and lysed in PLB obtained from the kit. Luminescence was measured using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA) at the ICCB-Longwood Screening Facility of HMS. Luminescence was normalized to Renilla luciferase activity and the relative luminescence percentage was calculated compared to the DMSO control.

[0494] Cell viability assay. Caco-2 and NCI-H716 cells were treated with the indicated compounds diluted in DMSO in complete MEM and RPMI media, respectively. The concentration of DMSO was kept constant and used as a negative control. Cells were incubated with the compounds overnight at 37 °C in a 5% CO 2 atmosphere. The next day, cells were treated with HBSS (GenClone) containing 0.25% trypsin for 10 minutes at 37 °C. Cell viability was measured in a Countess II automated cell counter (Invitrogen). The relative viability percentage was calculated compared to the DMSO control.

[0495] Epithelial permeability assay. Undifferentiated Caco-2 cells were seeded at 200,000 cells / transwell in 24-well plate transwells (0.4 uM pore size, Costar). Media were changed on days 4, 8, 12, 16, and 18 to differentiate Caco-2 cells in vitro 51 . On day 21, fully differentiated and polarized cells were used for the FITC-dextran permeability assay. Briefly, the indicated concentrations of 7 and GR-7 in PBS were added to the apical chamber of transwells containing differentiated Caco-2 cells and incubated for 6 or 12 hours. The apical chamber of the transwells contained 100 ul volume of PBS and the compound or DMSO control, while the basolateral chamber contained 500 uL of PBS. Caco-2 epithelial integrity was determined by measuring the passive diffusion of 4 kDa FITC-dextran (Sigma Aldrich) added to the apical chamber at a concentration of 5 uM. At the ICCB-Longwood Screening Facility of HMS, using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA), diffusion from the apical to the basolateral side was measured by fluorescence readings in PBS on the basolateral side of the transwell system. Fluorescence readings were normalized to the DMSO control.

[0496] Using 7-N 3 Target validation and off-target analysis in Bifidobacterium adolescentis. 7-N was performed using Bifidobacterium adolescentis (Gram-positive) and Bacteroides thetaiotaomicron (Gram-negative). 3 Experimental study. Due to the strong total fluorescence signal detected by in-gel fluorescence, we chose to use Bifidobacterium adolescentis. The Bifidobacterium adolescentis culture was diluted to an OD of 0.1 with 6 mL of fresh CHG medium containing a 100 μM pool of taurine-conjugated bile acids. 600 . The culture was grown anaerobically at 37 °C for 21 h. Then 10 μM 7-N 3 (10 mM stock solution, in DMSO) or 6 μL of DMSO (to the control tube) was added to the culture and incubated anaerobically at 37 °C for 1 h. The culture was centrifuged at 2,500 g for 15 min at 4 °C. The medium was decanted, and the cells were resuspended in PBS containing 1 mM TCEP and 1 mM PMSF and centrifuged at 4,200 rpm for 15 min at 4 °C. The buffer was decanted, the cells were suspended in 300 μL of fresh buffer, and transferred to a homogenization tube with ceramic beads (Precellys lysing kit toughmicro-organism lysing VK05 tube). The suspension was then homogenized (5000 speed 90 s * 2, 6500 speed 60 s) and centrifuged at 15,000 at 4 °C for 20 min. The supernatant was removed, and the protein concentration in the lysate was quantified by Bradford assay. Then, the lysate was subjected to a click reaction according to "Click chemistry for in-gel fluorescence imaging" for fluorescence imaging and "Click chemistry for MS / MS of bacterial lysates" for quantitative and identification based on mass spectrophotometer.

[0497] Dose-dependent labeling of BSH in Bifidobacterium adolescentis via competition of 7 and 7-N 3 The Bifidobacterium adolescentis culture was diluted to an OD of 0.1 with 6 mL of fresh CHG containing a 100 μM pool of taurine-conjugated bile acids 600 . The culture was allowed to grow anaerobically at 37 °C for 21 h. Decreasing concentrations of 7 were added to different tubes, and the culture was incubated anaerobically at 37 °C for 1 h. Then 10 μM 7-N 3 was added to the culture and incubated anaerobically at 37 °C for an additional 1 h. The culture was further processed according to the methods reported in "Target validation and off-target analysis of 7-N in Bifidobacterium adolescentis" and "Click chemistry for in-gel fluorescence imaging". 3

[0498] Using 7-N 3 ​Off-target analysis in mammalian cells. The human epithelial cell line NCI-H716 was used to study the interaction with mammalian proteins. 10 μM 7-N 3 (10 mM stock solution, in DMSO) or 1 μL DMSO (for control) was added to approximately 8×10 6 cells in 1 mL DPBS (HiMedia), and incubated for 1 hour. The cells were collected in a 15 mL Falcon tube and washed twice in 15 mL DBPS by centrifugation at 500 g for 5 minutes. A third wash was performed by centrifugation in a DPBS solution containing 1 mM cOmplete TM protease inhibitor mixture (Roche, Switzerland). The cells were resuspended in 250 μL of DPBS containing 1 mM cOmplete TM protease inhibitor mixture and sonicated for 2 seconds at 50% amplitude, followed by 30 seconds on ice, for a total of 3 cycles. The lysate was centrifuged at 15,000 g for 15 minutes at 4 °C. The supernatant was removed and the protein concentration was measured by Bradford assay. Then, the lysate was subjected to a click reaction according to "Click Chemistry for In-Gel Fluorescence Imaging" for in-gel fluorescence and "Click Chemistry for MS / MS of Mammalian Lysates" for quantitative and identification based on mass spectrophotometry.

[0499] Click Chemistry for In-Gel Fluorescence Imaging. The click reaction was performed on a 25 μL scale. The lysate pretreated with 10 μM compound 7-N 3 (normalized to 1.5 mg / mL for both bacteria and mammalian cells) was incubated with 100 μM fluor 488-alkyne (10 mM stock solution, in DMSO), 100 μM CuBr (5 mM stock solution, in DMSO), and 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (5 mM stock solution, 4:1 t-BuOH:DMSO) together in the dark at 37 °C for 1 hour. 10 μL of 2x Laemmli buffer (containing 5% β-mercaptoethanol) was added to the reaction, and the tube was heated at 95 °C for 10 minutes. Then, 15 μL of the protein sample was resolved by 10% SDS-PAGE. The ladder was diluted 100-fold and 10 μL was loaded. The gel was decolorized for 30 minutes using 40% methanol, 50% acetic acid, 10% water, and visualized using a Bio-Rad ChemiDoc MP imaging system. The gel was stained in Coomassie blue for 20 minutes and decolorized for 2 hours before imaging.

[0500] Click Chemistry for MS / MS of Bacterial Lysates. The click reaction was performed on a 100 μL scale. The lysate pretreated with 10 μM 7-N 3The pretreated lysate (normalized to 1.3 mg / mL) was incubated with 100 μM desthiobiotin-PEG4-alkyne (10 mM stock solution, in DMSO), 1 mM CuBr (50 mM stock solution, in DMSO), and 1 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (50 mM stock solution, in 4:1 t-BuOH:DMSO) at 37 °C for 1 h. Samples were then processed for further analysis according to "Proteomic analysis of click-labeled proteins".

[0501] Click chemistry for MS / MS of mammalian lysates. The click reaction was performed on a 100 μL scale. The 10 μM 7-N 3 The pretreated lysate (1.5 mg / mL for mammalian cells) was incubated with 100 μM desthiobiotin-PEG4-alkyne (10 mM stock solution, in DMSO), 100 μM CuBr (5 mM stock solution, in DMSO), and 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (5 mM stock solution, in 4:1 t-BuOH:DMSO) at 37 °C for 1 h. Samples were then processed for further analysis according to "Proteomic analysis of click-labeled proteins".

[0502] Proteomic analysis of click-labeled proteins. The pull-down precipitation and bead digestion of desthiobiotinylated proteins were performed similar to the previously described protocol 52 After resuspending tryptic peptides in 5% acetonitrile containing 0.1% formic acid, the peptides were analyzed by nanoflow LC-MS / MS as described 53 . Using multiplierz 54 The raw data were converted to.mgf and searched against the forward reversed databases of human or Bifidobacterium adolescentis proteins (uniprot) using Mascot 2.6.2. The search results were downloaded from Mascot, converted to xls, and filtered to 1% FDR using the multiplierz script. The normalized spectral abundance factors were obtained as described 55 . Filtering the data of proteins with more than 5 spectral counts (average of biological triplicates) for the 7-N 3 -treated samples. In a separate experiment, the click-labeled bacterial lysate proteins were subjected to avidin enrichment and washed as described above. The proteins were then eluted with LDS loading buffer, subjected to SDS-PAGE and silver staining. The indicated bands were excised, subjected to in-gel digestion, and the extracted peptides were analyzed by nanoflow LC-MS / MS as described 53 .

[0503] Animal studies. C57BL / 6 mice obtained from the Jackson Laboratory were maintained under a strict 12-hour / 12-hour light / dark cycle and at a constant temperature (21 ± 1 °C) and humidity (55–65%). All experiments were conducted on male mice at 8–9 weeks of age.

[0504] Gavage of 7. Based on the efficacy of 7 in in vitro assays at 10 μM to 100 μM, our target concentration of 7 in vivo was approximately 50 μM: (0.00005 M) × (approx. 10 mL volume / gastrointestinal tract of 1 mouse) × (1 mmol of compound 7 / 408 mg) = 0.2 mg / mouse × (1 mouse / approx. 0.02 kg) = 10 mg / kg.

[0505] Mice were maintained on a standard diet (LabDiet, catalog number 5053) throughout the duration of the experiment. The mice were divided into two groups of four mice each and gavaged with 200 μL of corn oil containing 5% DMSO (vehicle group) or with 200 μL of corn oil containing 7 at a concentration of 1.25 mg / mL (experimental group). To collect fecal pellets, each mouse was transferred to a temporary cardboard cage for a few minutes until it defecated.

[0506] Feeding a GR-7-containing diet for one day. Mice were fed a powdered standard diet (LabDiet, catalog number 5053) throughout the duration of the experiment. The mice were divided into two groups of ten mice each and maintained on the powdered diet (control group) or fed a powdered diet containing 0.09% (w / w) GR-7 (experimental group). Feces from these mice were collected at 8 hours as described above. Thirty hours after obtaining the powdered diet with or without GR-7, the mice were euthanized with carbon dioxide. Blood samples were collected by cardiac puncture and placed in EDTA-coated tubes on ice. Then, the liver and cecal contents were collected from each mouse, snap-frozen in liquid nitrogen, and stored at -80 °C until further analysis. The blood samples were then centrifuged at 2500 g for 15 minutes at 4 °C. The resulting supernatant (plasma) was collected and stored at -80 °C until analysis.

[0507] BSH activity in feces. A modified form of a published method was used to quantify BSH activity in fecal pellets 45 . Fecal pellets (approx. 10–20 mg) were suspended in a buffer (10% PBS, 90% sodium acetate, pH 5.2) containing 100 μM (GCDCA-d4) to obtain a concentration of 20 mg / mL. The fecal pellets were broken into fine particles, and the mixture was incubated at 37 °C for 25 minutes. Samples were processed and analyzed as described in "Screening for inhibitors in normal mouse feces". The product concentration detected from these assays was reported directly.

[0508] Quantification of bile acids in tissues and plasma. Bile acids were extracted from tissues and plasma collected from mouse experiments using a previously published method 16 .

[0509] Determination of microbial biomass by plating. Frozen fecal pellets were used to determine colony-forming units (CFU / g). Feces were suspended in PBS buffer in an anaerobic chamber. Serial dilutions were plated on CHG agar plates (see "Bacterial culture") and incubated at 37 °C

[0510] Isolation and 16S rRNA gene sequencing analysis of fecal bacterial microbiota. Mouse fecal microbiota DNA was isolated using the ZymoBIOMICS 96 DNA kit (ZymoBIOMICS TM ) according to the manufacturer's instructions. The variable region 4 of the 16S rRNA gene was amplified using primers: forward 5'-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3'

[0511] Reverse 5’-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3’. According to the manufacturer's instructions, the PCR products were quantified using the Quant-IT dsDNA High-Sensitivity Assay (Invitrogen). The success of PCR amplification was examined using gel electrophoresis. The concentration of the PCR products was measured by the Quan-IT dsDNA High-Sensitivity Assay. Approximately 120 ng of DNA from each PCR product was pooled together to generate an aggregated library for downstream processing. According to the manufacturer's instructions, on a target size selection platform (pippin prep 1.5% agarose cassette, obtained from Sage Sciences), PCR DNA amplicons of 300 - 500 bp were selected from the aggregated library. The size of the DNA amplicons was characterized on an Agilent Technologies 2100 Bioanalyzer trace. The DNA concentration of the aggregated library was measured by the Quant-IT dsDNA High-Sensitivity Assay. The DNA in the library was denatured with NaOH and diluted to 7.5 pM with the HT buffer provided in the Illumina kit. 600 ul of the denatured and diluted library containing 20% spike-in phiX (120 ul, 7.5 pM of phiX) was loaded onto a MiSeq V2 reagent cartridge (Illumina), and paired-end 250 bp reads sequencing was performed using the custom primers described above. After running the MiSeq, demultiplexed fastq files were generated using the default parameters by the Illumina MiSeq Control Software and quality control was performed through the pipeline of the Massachusetts Host-Microbiome Center. Then, the resulting FASTQ sequences were quality filtered and analyzed according to QIIME_mothur_DADA2 56-59 Operational taxonomic units (OTUs) with 97% sequence similarity were picked. The phylogenetic relatedness of each OTU was aligned with the Greengenes reference database and 99% ID

[0512] Quantification of bacterial 16S rDNA copy number. Bacterial DNA was isolated from mouse cecal contents using the AllPrep Bact. DNA / RNA / Protein kit (QIAGEN). The 16S rDNA was then amplified using the following primer pair at 10 μM: forward 5’-AGAGTTTGATCCTGGCTCAG-3’, reverse 5’-CTGCTGCCTYCCGTA-3’. Amplification was performed on a QuantStudio 7Flex real-time PCR system using the LightCycler480 SYBR Green I Master according to the provided qPCR protocol. The cycle threshold of each sample was compared to a standard curve obtained from serial dilutions of Bacteroides thetaiotaomicron genomic DNA 60 。

[0513] Example 1. Development of broad-spectrum covalent inhibitors of gut bacterial bile salt hydrolases

[0514] Development of broad-spectrum covalent inhibitors of gut bacterial bile salt hydrolases

[0515] Described herein is the development of broad-spectrum covalent inhibitors of gut bacterial BSH. Using a rational design strategy, a small library of potential BSH inhibitors was generated. By testing these compounds against purified BSH protein and growing gut bacterial cultures, a lead inhibitor bearing an α-fluoromethyl ketone warhead was identified. Another BSH inhibitor, caffeic acid phenethyl ester (CAPE), was determined to inhibit the growth of Gram-negative gut bacteria but lacked the same broad-spectrum activity as the other BSH inhibitors described herein. Mass spectrometry and X-ray crystallography confirmed covalent single-labeling of the protein by the inhibitor at the catalytic cysteine residue. Strikingly, the lead inhibitor completely abolished BSH activity in conventional mouse feces. Conventional mice gavaged with a single dose of the lead inhibitor showed loss of BSH activity in feces and a reduction in deconjugated bile acids. Collectively, these studies demonstrate the potential of covalent BSH inhibitors as chemical tools to modulate bile acid composition in vivo

[0516] Introduction

[0517] Human-associated bacteria play crucial roles in health and disease. Microbial dysbiosis has been associated with a broad range of disease states, including inflammatory bowel disease 1 、cancer 2 、autism 3 and obesity 4 。However, little is known about the ways in which bacterial agents affect the human host at the molecular level. Studies in germ-free mice colonized with single strains, multiple strains, or defined bacterial communities have revealed that gut bacteria influence host processes, including metabolism 5 、immune function 6,7and neural responses 8 ) The ability. Although germ-free mice are available tools, compared to conventional animals, they show physiological differences, including altered food processing for energy acquisition 9 , defects in immune cell balance (especially in the gut) 10,11 , and altered stress response behavior 12 . These differences may complicate determining whether effects observed in germ-free animals can be extrapolated to conventional animals and humans. Chemical tools that selectively alter specific bacterial metabolite and protein levels may allow researchers to study how these bacterial products affect host physiology in fully developed animals with complex microbial communities. Using small molecules as chemical tools may also provide therapeutic opportunities. Indeed, small molecule inhibitors of gut bacterial β-glucuronidase have been shown to reduce dose-limiting diarrhea caused by the colon cancer chemotherapeutic agent CPT-11 in mice 13 . In recent work, small molecule inhibitors of the gut bacterial enzyme cutCc have been shown to reduce the levels of the prothrombotic metabolite trimethylamine N-oxide (TMAO) in vivo 14 . These studies demonstrate the ability of non-bactericidal agents targeting specific bacterial enzymes to favorably alter host physiology.

[0518] Bacteria in the gastrointestinal tract are inundated with molecules from the host, including both dietary compounds and host metabolites. The bacteria then chemically modify these compounds to produce new metabolite types, which can then act as signaling molecules between the bacteria and the host 15 . An important example of a class of host-produced bacterially modified signaling molecules is bile acids 16 . Primary bile acids are produced from cholesterol in the liver and conjugated with taurine or glycine to produce primary conjugated bile acids ( Figure 1A ). These molecules are then stored in the gallbladder and released into the duodenum upon food intake, where they aid in the absorption of lipids and fat-soluble vitamins. More than 95% of bile acids are reabsorbed in the ileum and recycled to the liver. The remaining approximately 5% enters the colon where most gut bacteria reside. The gut bacteria then enzymatically modify these primary bile acids to produce a class of molecules called secondary bile acids ( Figure 1A ). Approximately 50 secondary bile acids are detected in human feces. Due to the higher concentration of bile acids released into the small intestine, the resulting concentration of these molecules in the lower intestine remains in the low millimolar range 17 . As a result, even minor abundances of secondary bile acids are present at physiologically relevant concentrations.

[0519] Although bile acids were initially studied for their detergent properties, it was later recognized that these compounds can act as signaling molecules by binding to host receptors, including nuclear hormone receptors (NhR) and G protein-coupled receptors (GPCR)Figure 1B )。By acting as these receptors, including agonists or antagonists of the farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5), muscarinic receptors 2 and 3, and sphingosine 1-phosphate receptor 2, primary and secondary bile acids affect host processes 18-22 。Specifically, by binding to host receptors, bile acids regulate host metabolism, including energy expenditure and glucose and lipid homeostasis 18,23 , as well as host immune responses, including innate and adaptive immunity 24,25 。In addition, bile acids tightly regulate their own biosynthesis through a negative feedback loop controlled by FXR 23 。Ultimately, imbalances in bile acid homeostasis are thought to play a causal role in the pathophysiology of diseases including hypercholesterolemia, obesity, diabetes, cancer, and gallstone formation 18,26,27 , further highlighting the biological importance of these metabolites

[0520] Importantly, primary and secondary bile acids alone have different binding affinities for host receptors, indicating that the specific composition of the bile acid pool in vivo determines downstream signaling events in the host 18,28 。The key reaction in the conversion of primary to secondary bile acids is the hydrolysis of the C24 amide bond of conjugated primary bile acids ( Figure 1A )。This reaction is carried out by the intestinal bacterial bile salt hydrolase (BSH) 16 。BSH (EC 3.5.1.24) is widely distributed among human intestinal bacteria. Recent studies have identified BSH in intestinal species from 117 genera and 12 phyla, including two dominant intestinal phyla, Bacteroidetes and Firmicutes, as well as Actinobacteria and Proteobacteria 29 。In addition, the study identified BSH in the human microbiota of 11 different populations from 6 continents, including indigenous populations in Tanzania. These results suggest that BSH activity is a conserved function of the human intestinal metagenome. Thus, broad-spectrum non-toxic small molecule inhibitors of intestinal bacterial BSH can limit BSH activity in a variety of Gram-negative and Gram-positive strains without significantly affecting the growth of these bacteria. In addition, the use of such inhibitors in vivo can lead to a conversion of the bile acid pool towards conjugated bile acids and away from deconjugated and secondary bile acids ( Figure 1A )。The compounds described herein can be used to study how secondary bile acids produced by bacteria affect physiology in a fully colonized host

[0521] This article describes the development of broad-spectrum covalent inhibitors of bacterial BSH and their determination using rational design methods. Importantly, the compounds described herein can significantly inhibit BSH activity in conventional mouse feces, indicating their activity as broad-spectrum inhibitors of BSH.

[0522] Experimental results

[0523] Rational design and synthesis of covalent small molecule inhibitors of bile salt hydrolase

[0524] To achieve the goal of generating effective and long-lasting BSH inhibitors, covalent inhibitors of these intestinal bacterial enzymes have been developed and described herein. Covalent inhibitors have gained wide interest in the field of drug discovery because they can inactivate their protein targets with a high degree of potency and selectivity even in the presence of large concentrations of natural substrates. 30 . The substrate of BSH, conjugated bile acids, is present in the colon at high concentrations (1 - 10 mM). 17 , indicating that covalent inhibition can be an effective strategy for targeting these enzymes. In addition, recently developed bacterial cutC inhibitors are irreversible and block the production of trimethylamine in vivo and show minimal off-target effects. 14 . This work demonstrates that covalent inhibitors of bacterial enzymes can be effective in the intestine, thus further validating the method of the present invention.

[0525] Although there are significant differences in the BSH protein sequences among intestinal strains, all BSHs have a conserved active site consisting of five amino acids: cysteine 2 (Cys2), arginine 18 (Arg18), aspartic acid 21 (Asp21), asparagine 175 (Asn175), and arginine 228 (Arg228). 16,29 . Cys2 performs a nucleophilic attack on the substrate carbonyl group, resulting in amide bond cleavage ( Figure 2A ). By designing compounds that target this highly conserved Cys residue, broad-spectrum BSH inhibitors have been developed. Structural data and biochemical information of the Gram-positive species Clostridium perfringens have contributed to the design plan. The co-crystal structure of Clostridium perfringens BSH and the substrate taurodeoxycholic acid (TDCA) shows that the amino acids are solvent-exposed when hydrophobic interactions hold the bile acid core in place and orient the amide bond towards the conserved cysteine ( Figure 2B ). 31 . In addition, purified Clostridium perfringens BSH tolerates a large degree of variability in amino acid side chains, including long-chain conjugates. 32 . These results suggest that the D-ring side chain of bile acids is a possible site for introducing electrophilic groups into inhibitors.

[0526] Next, a small library of potential inhibitors was designed: it contains a bile acid core motif that selectively targets BSH and a side-chain electrophilic warhead that irreversibly binds the inhibitor to the enzyme ( Figure 2C ). Although previous literature suggested that BSH hydrolyzes the amide bond cleavage of all conjugated bile acids regardless of the steroid core 16,26 , it was recently determined that species from the abundant Gram-negative phylum Bacteroidetes cleave C12=H rather than C12=OH primary bile acids ( Figure 1A ). 33 . Since the goal was to develop BSH inhibitors that target both Gram-negative and Gram-positive strains, the steroid moiety of the human primary bile acid chenodeoxycholic acid (CDCA, C12=H) was used as the scaffold for the inhibitors described herein ( Figure 2C ).

[0527] For the electrophilic capture group, warheads that have been successfully adopted in the development of selective and effective protease and kinase inhibitors were selected 34,35 , including isothiocyanate (1) 36-38 , cyanoacrylate (2) 39,40 , α,β-unsaturated systems (3 and 4) 41 , acrylamide (5) 42 and nitrile (6) 43,44 . Inhibitors with an α-fluoromethyl ketone warhead (FMK) (7) were selected in the library. Covalent inhibitors with this warhead have shown high potency and selectivity 45-47 . Contrary to the more electrophilic α-iodo, α-bromo- and α-chloromethyl ketone warheads, the weak leaving group ability of fluorine makes the FMK warhead less reactive and thus more selective 45,47,48 . Therefore, FMK-based inhibitors have been shown to cause minimal off-target effects 45,49 .

[0528] All compounds in the library were obtained from the commercially available bile acid chenodeoxycholic acid (CDCA, 12) in 3-9 steps (Scheme 1). Using a modified one-pot Curtius rearrangement, isothiocyanate (1) and acrylamide (5) were synthesized from CDCA in 3 steps to install a C23-substituted primary amine (Scheme S1) 50 . The synthesis of cyanoacrylate (2), α,β-unsaturated systems (3 and 4) and nitrile (6) compounds was carried out rapidly in 2-3 steps from bis-methoxymethyl ether (MOM)-protected C24-aldehyde CDCA via Grignard addition or condensation reactions (Scheme S1). To obtain compound 7, bis-MOM-protected CDCA (13) was coupled with benzyl fluoromalonic acid magnesium to provide the β-keto-α-fluorobenzyl ester product 14 in 66% yield 51 . Hydrogenation, followed by deprotection, gave the target compound 7.

[0529]

[0530] Scheme 1: Synthesis of Compound 7, which contains an α-fluoromethyl ketone warhead on the chenodeoxycholic acid core. Abbreviations: SOCl 2 , thionyl chloride; DIPEA, N,N-diisopropylethylamine; MOMCl, methyl chloromethyl ether; THF, tetrahydrofuran; CDI, 1,1'-carbonyldiimidazole; Pd / C, palladium on carbon; R = methoxymethyl ether.

[0531] Biochemical Characterization of BSH

[0532] For the ongoing inhibitors 1-9, the next goal is to evaluate the biochemical activity of these compounds against both Gram-negative and Gram-positive BSH. Specifically, these compounds are tested against the selective Bacteroides BSH because the more limited substrate range of this enzyme may make it more difficult to target. So far, biochemical characterization has been mainly limited to BSH from Gram-positive bacteria 16 , 26 ,52 , including Lactobacillus 53 , Bifidobacterium 54 , Clostridium 31 and Enterococcus 55 BSH. Among Gram-negative bacteria, only BSH from Bacteroides vulgatus and Bacteroides fragilis has been biochemically characterized, and the corresponding genes have not been identified 56,57 . In addition, these strains do not have selective BSH selectivity 33 . Recently, BT2086 was identified as the gene responsible for selective BSH activity in the intestinal bacterium Bacteroides thetaiotaomicron VPI-5482 (B. theta) 33 . To test the compounds against this selective BSH, the enzyme encoded by BT2086 was heterologously expressed and purified and molecularly cloned. Since this enzyme had not been characterized previously, kinetic parameters for its hydrolysis of conjugated primary and secondary bile acids (primary, taurocholic acid, TCA, and taurochenodeoxycholic acid, TCDCA; secondary, tauroursodeoxycholic acid, TUDCA, and taurodeoxycholic acid, TDCA) were established using a ninhydrin-based assay 58 . Taurine-conjugated substrates were chosen because taurine conjugates are present in both mice and humans, while sugar-bile acids are essentially absent in mice 28 . Consistent with previous results from B. thetaiotaomicron cultures, purified B. thetaiotaomicron BSH showed a preference for TDCA cleavage over TCA cleavage (Table 1) 33These results suggest that the enzyme selectivity observed in the whole cell culture of B. thetaiotaomicron is due to the intrinsic biochemical properties of BSH, rather than differences in transport or substrate accessibility to the enzyme.

[0533] To test the potency of inhibitors against Gram-positive BSH, the known BSH from Bifidobacterium longum SBT2928 was cloned and expressed 54 , and the kinetic parameters of this enzyme were determined using the same set of taurine-conjugated bile acid substrates (Table 1). Notably, the K m values for all recognized substrates were in the low millimolar range, approximating the concentrations of these bile acids in the intestine. The K m values established here for B. longum were higher than those previously reported 54 . This difference may be the result of the conditions under which the assays were performed, i.e., the physiological pH (7.5) in this work versus the pH (6) optimized for activity in previous studies. In summary, both enzymes showed kinetic parameters comparable to those of previously characterized BSH 53,54,56 .

[0534] Table 1: Kinetic characterization of BSH from Gram-negative B. thetaiotaomicron (B.theta) and Gram-positive B. longum (B.longum).

[0535]

[0536] a Characterization was performed using the ninhydrin reagent, and the experiments were carried out in PBS buffer at pH 7.5 and 37 °C. b The conjugated primary and secondary bile acids used as substrates were taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA).

[0537] Biochemical evaluation identified the α-FMK compound 7 as a lead inhibitor

[0538] Next, the ability of compounds in the library to inhibit BSH from B. thetaiotaomicron and B. longum was evaluated. Two additional compounds, namely riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11), were included in the assay ( Figure 2E ). These molecules have previously been identified as BSH inhibitors by high-throughput screening against BSH from Lactobacillus salivarius chicken intestinal isolates 59。To determine the BSH inhibitory activity of these compounds, Bacteroides thetaiotaomicron BSH was incubated with each inhibitor (100 μM) for 30 minutes, and then an equimolar amount of four conjugated bile acids (TβMCA, TCA, TUDCA, and TDCA, 100 μM in total) was added.

[0539] The conversion of conjugated to unconjugated bile acids was monitored by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) over a total of 21 hours (Figure 3). Among the synthesized inhibitors, isothiocyanate (1) showed moderate inhibition during the experiment. The other compounds containing Michael acceptor warheads (inhibitors 2–6) did not inhibit dissociation ( Figure 3A ). In contrast, incubation with the α-fluoromethyl ketone-based inhibitor 7 led to almost complete inhibition of Bacteroides thetaiotaomicron BSH activity for 21 hours (>98%, Figure 3A ). To verify that the inhibitory activity of compound 7 was due to the presence of fluorine as a leaving group, a methyl ketone analogue lacking a fluorine atom (8) was synthesized 49 . This analogue did not show BSH inhibition, indicating that the α-fluorine group was essential for activity. The previously identified BSH inhibitor riboflavin did not show any inhibitory activity, while CAPE only provided moderate inhibition of Bacteroides thetaiotaomicron BSH.

[0540] Next, the activities of the two most effective inhibitors against Bacteroides thetaiotaomicron BSH were evaluated. Compound 1 and 7, as well as CAPE, were tested against BSH from the Gram-positive species Bifidobacterium longum ( Figure 3B ). These compounds showed the same specific effectiveness against Bifidobacterium longum BSH as observed against Bacteroides thetaiotaomicron BSH. Compound 7 was the most effective inhibitor at the 2-hour, 5-hour, and 21-hour time points, compound 1 showed moderate inhibition, and CAPE was ineffective in inhibiting the dissociation of Bifidobacterium longum BSH at all time points. These data indicate that compound 7 is an effective inhibitor of purified BSH proteins from Gram-negative and Gram-positive bacterial strains. In addition, since the activities of CAPE and riboflavin against genera other than Lactobacillus were not determined 59 , these results suggest that these molecules may not be effective broad-spectrum inhibitors.

[0541] Compound 7 inhibits BSH activity in the growth cultures of intestinal bacteria

[0542] Given that compound 7 showed activity against purified BSH from Bacteroides thetaiotaomicron and Bifidobacterium longum, the potency of the inhibitor in growing bacterial cultures was evaluated. To test the range of BSH inhibition, three Gram-negative and three Gram-positive strains of human intestinal bacteria known to have BSH activity were tested in this screen (Gram-negative: Bacteroides thetaiotaomicron, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482; Gram-positive: Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32) 16,33 .

[0543] Bacterial cultures were diluted in the early logarithmic growth phase, and a mixture of the inhibitor (100 μM) and conjugated bile acids (100 μM final concentration; TCA, TβMCA, TDCA, and TUDCA) was added simultaneously. Dissociation was monitored by UPLC-MS over 24 h. Strikingly, while all six bacterial strains dissociated bile acids in the presence of the vehicle control, little detectable dissociation was observed in any of the growing cultures in the presence of compound 7. These results indicate that compound 7 shows potent BSH inhibition against both Gram-negative and Gram-positive bacteria( Figure 4A ). Compound 7 did not significantly affect the growth of any of the tested strains( Figure 4B ), indicating that the observed BSH inhibition was not due to bacteriostatic activity. To quantify the potency of compound 7, the IC 50 values of the inhibitor against the Gram-negative strain Bacteroides thetaiotaomicron and the Gram-positive strain Bifidobacterium adolescentis were determined to be 913 nM and 227 nM, respectively( Figure 4C ). Collectively, these results indicate that compound 7 is a potent broad-spectrum inhibitor of BSH.

[0544] In contrast, no inhibition of dissociation was observed in five of the six bacterial strains growing in the presence of CAPE (100 μM) over the course of 21 h( Figure 4A ). CAPE was found to inhibit dissociation in Lactobacillus plantarum, results consistent with the hypothesis that the compound inhibits Lactobacillus BSH but is not a broad-spectrum BSH inhibitor. Additionally, in contrast to inhibitor 7, CAPE inhibited the growth of all three tested Gram-negative bacterial strains( Figure 4B ). These results suggest that the primary effect of CAPE on Gram-negative bacteria is not to inhibit BSH activity but to inhibit growth.

[0545] To evaluate the hypothesis that C12=OH compounds are not effective inhibitors, broad-spectrum inhibitors, because they do not inhibit Bacteroides thetaiotaomicron BSH activity, such inhibitors cholate were synthesized: in which the α-fluoromethyl ketone warhead from the most effective inhibitor compound 7 was appended to the C12=OH bile acid core (compound 9, Figure 2D ). Subsequently, a growth culture of Bacteroides thetaiotaomicron was incubated with compound 9 (1 μM or 10 μM) and the conjugated bile acid substrate (GUDCA, 100 μM), and dissociation was monitored using UPLC-MS. While incubation with 10 μM of compound 7 led to almost complete inhibition of dissociation, significant dissociation was observed in the presence of the same concentration of compound 9 ( Figure 4D ). These results support the hypothesis that the bile acid core structure, particularly the C12 substitution, affects the ability of the probe to act as a broad-spectrum inhibitor. In addition, these results suggest that the α-fluoromethyl ketone warhead is not broadly reactive but requires proper positioning within the active site, which can be further tested using mass spectrometry and crystallography studies.

[0546] Compound 7 covalently binds to the catalytic cysteine residue of BSH

[0547] In establishing the potency of compound 7, the mechanism of its inhibition was investigated. To confirm that compound 7 is a covalent inhibitor and that it modifies Cys2 (the catalytic cysteine residue), mass spectrometry experiments were performed. Bacteroides thetaiotaomicron BSH contains two cysteine residues, namely Cys2 and Cys67. Analysis of the apo crystal structure of the enzyme revealed that both cysteine residues point towards the active site, indicating that either residue could be a potential binding site for compound 7 (PDB 3HBC). It was found that reincubation of Bacteroides thetaiotaomicron BSH with compound 7 led to a complete mass shift of the protein by 388 mass units. This mass shift was consistent with the addition of a single equivalent of the inhibitor to the protein ( Figure 5A ). Although no labeled peptides were identified upon digestion with trypsin or Lys-C, a top-down approach revealed that Cys2 was the modified residue, as indicated by the c3 ion ( Figure 5B ).

[0548] To understand the spatial arrangement of the inhibitor within the binding pocket and to guide further inhibitor design, the co-crystal structure of Bacteroides thetaiotaomicron BSH covalently bound to compound 7 was determined at resolution. Consistent with the mass spectrometry data, the co-crystal structure revealed that Cys2 binds to the C25-methylene of the bile acid structure and the fluorine atom has been eliminated. Collectively, these data indicate that compound 7 selectively labels Bacteroides thetaiotaomicron BSH at the nucleophilic cysteine residue in the protein active site. In addition, the co-crystal structure reveals that the C3-hydroxy group is solvent-exposed, suggesting that this site is amenable to further modification.

[0549] Compound 7 shows the least off-target effect

[0550] Although covalent inhibitors have been shown to be highly effective, it has been proposed that the non-specific reactivity of these compounds may lead to problems of acute toxicity 30 。The inhibitors described herein are designed to contain a bile acid core to increase the selectivity of these compounds for BSH. However, bile acids are known ligands for host nuclear hormone receptors (NhR) and G protein-coupled receptors (GPCR) 18 。The lead inhibitors can then bind to these receptors and induce off-target effects in the host. Specifically, the binding of specific bile acids to FXR and GPBAR1 / TGR5 affects core host metabolic and immune processes 18 。To determine whether compound 7 can act as a ligand for FXR, an in vitro co-activator recruitment assay was performed( Figure 6A ) 28 。This assay measures the ability of the compound to enhance the binding of the recombinant FXR ligand-binding domain (LBD) to the co-activator peptide (SRC2-2), as measured by an increase in the time-resolved fluorescence resonance energy transfer (TR-FRET) signal. Although the known FXR agonist GW4064 shows a significant dose-dependent increase in the binding of SRC2-2 to FXR (EC 50 = 50 nM), the binding of SRC2-2 to FXR does not increase in the presence of compound 7, indicating that the inhibitor does not activate FXR. In the presence of GW4064 at its EC 50 concentration, compound 7 does not show a dose-dependent curve, indicating that compound 7 does not have FXR antagonist activity at physiologically relevant concentrations. Next, the effect of compound 7 on TGR5 activation was evaluated in a human intestinal cell line (Caco-2). Compound 7 does not activate TGR5 within the tested concentration range. In addition, in the presence of the known TGR5 agonist LCA (10 μM), compound 7 does not antagonize TGR5( Figure 6B )。These results indicate that inhibitor 7 does not induce off-target effects by binding to either of these key host receptors.

[0551] In addition to their effects on host receptors, bile acids are known to be toxic to cells due to their detergent properties 16,60 。Since the expected in vivo region of inhibitor 7 is the lower intestine, the toxicity of the compound against human intestinal cells (Caco-2) was tested. No resulting toxicity was observed when these cells were incubated with up to 50 μM of compound 7( Figure 6C )。Since the IC 50Values range from 227 nM to 913 nM, and these results suggest that effective in vivo doses should be achievable at concentrations that do not cause cytotoxicity to intestinal cells. Collectively, these results indicate that inhibitor 7 is non-toxic and selective for bacterial BSH relative to potential host targets.

[0552] Compound 7 inhibits BSH activity in feces from conventional mice

[0553] Although the experimental results demonstrated the potency of inhibitor 7 against the growth cultures of six different gut bacterial strains, there are hundreds of bacterial species in the human gut 61 . Previous literature has reported significant BSH activity in mouse feces 62 . To further strengthen the discovery that compound 7 is a broad-spectrum BSH inhibitor, the activity of compound 7 in resuspended feces from conventional (i.e., fully colonized) mice was tested. Compounds 1, 7, and CAPE (20 μM) were added to fecal suspensions in buffer. After 30 minutes, the deuterated substrate GCDCA-d4 was added, and dissociation was determined by quantifying the formation of CDCA-d4 using UPLC-MS after 18 hours ( Figure 7A ). Strikingly, it was observed that while incubation with compound 1 led to reduced dissociation, incubation with compound 7 completely inhibited BSH activity in feces ( Figure 7B ). Consistent with the in vitro results, CAPE did not provide inhibition of BSH in feces from conventional mice. These results further demonstrate that the lead inhibitor compound 7 is an effective broad-spectrum inhibitor of gut bacterial BSH activity.

[0554] A single dose of compound 7 inhibits BSH activity in conventional mice

[0555] Having established the in vitro potency of compound 7, the activity of this inhibitor was evaluated in conventional mice. C57Bl / 6 mice were gavaged with a single dose of compound 7 (10 mg / kg) or vehicle control, and BSH activity was monitored at half-day increments until 2.5 days post-gavage ( Figure 7C ). Without being bound by a particular theory, it was expected that if compound 7 was active in vivo, an initial decrease in BSH activity would be observed, followed by a recovery of BSH activity. This expected effect was observed.

[0556] One and 1.5 days post-gavage, a significant decrease in BSH activity in feces was noted, while at subsequent time points (2 and 2.5 days post-gavage), a recovery of activity was observed ( Figure 7D ). Based on the initial hypothesis ( Figure 1A), and not bound by a particular theory, changes in the bile acid pool upon BSH inhibition were expected to be observed. A significant decrease in conjugated bile acids and an increase in unconjugated bile acids were observed 1 day after gavage. Notably, a decrease in the unconjugated secondary bile acid deoxycholic acid (DCA) was observed at this time point ( Figure 7E ).

[0557] Bacterial culture results indicated that compound 7 did not significantly inhibit bacterial growth. Consistent with this result, no significant decrease in bacterial biomass was observed at any time point after the initial gavage ( Figure 7F ). Collectively, these results suggest that compound 7 in vivo inhibits intestinal bacterial BSH activity in the murine gastrointestinal tract without significantly inhibiting the overall growth of the intestinal bacterial community.

[0558] Derivative 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK) of compound 7 was generated to restrict the delivery of the BSH inhibitor to the intestine ( Figure 8A ). Male conventional C57Bl / 6 mice were fed ad libitum a normal diet or a diet containing 3S-LCA-FMK (0.03% weight / weight) for 7 days. Feces were collected before the dietary change and on days 3, 4, and 7 after the dietary change. n = 5 mice per group ( Figure 8B ). It was found that the BSH activity in the feces of mice fed the diet containing 3S-LCA-FMK was significantly reduced, and on day 4, 3S-LCA-FMK was not detected in the circulating plasma ( Figures 8C - 8D ). Collectively, these results confirmed that the 3S-LCA-FMK compound is intestine-restricted and maintains the inhibition of bile acid deconjugation in an animal model. It was also shown that 3S-LCA-FMK reduced food intake in conventional mice compared to mice given the vehicle (n = 8 mice per group). Mice given 3S-LCA-FMK showed inhibited BSH activity and a significant reduction in food consumption ( Figure 31 ).

[0559] Summary

[0560] The development of such chemical tools, potent selective broad-spectrum inhibitors of intestinal bacterial BSH, is described herein. The lead inhibitor compound 7 was identified, which effectively inhibits the growth cultures of purified BSH protein, Gram-negative and Gram-positive human intestinal strains containing BSH, and the deconjugation of resuspended conventional mouse feces. It was also shown that administration of a single dose of compound 7 to conventional mice reduced BSH activity and predictably shifted the in vivo bile acid pool. Importantly, compound 7 did not significantly affect the growth of these bacteria.

[0561] These results suggest that compound 7 or its derivatives can be used as tools to study the biological effects of primary and secondary bile acids in fully colonized animals. For example, previous studies have shown that bacterial BSH activity affects host metabolism. However, there are conflicting reports on how altering BSH activity in vivo affects host metabolic responses.

[0562] One study found that increasing BSH activity in conventional mice by introducing Escherichia coli engineered to express Lactobacillus salivarius BSH resulted in reduced weight gain and lower serum and liver lipid levels 63 . Introducing exogenous bacterial strains overexpressing proteins from different bacterial sources into the gut is a significant perturbation of the natural ecosystem, however, and the interpretation of how BSH functions in natural systems becomes complicated. Another study found that treating conventional mice with the antioxidant compound TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) resulted in reduced Lactobacillus BSH activity and reduced weight gain 62 . However, TEMPOL did not show a direct role as a BSH inhibitor, and it may exert metabolic effects via mechanisms independent of BSH.

[0563] Furthermore, in recent work, it was shown that compared to the wild-type strain of Bacteroides thetaiotaomicron, deletion of the BSH-encoding gene from the Gram-negative gut commensal Bacteroides thetaiotaomicron resulted in reduced weight gain, lower liver and blood lipid levels, and a reduced respiratory exchange ratio in mice colonized with this bacterium 33 . However, these experiments were performed in mono-colonized germ-free mice and did not reveal how the restricted activity of all BSH affects the metabolism of conventional animals. Without being bound by a particular theory, it is hypothesized that the reduced weight gain phenotype in Bacteroides thetaiotaomicron BSH knockout (KO)-colonized mice is due to reduced food intake. Administering a chemical inhibitor such as compound 7 to mice in a metabolic cage can determine the origin of the metabolic effects of inhibiting individual BSH in mono-colonized mice and all BSH in conventional mice.

[0564] In addition to facilitating studies of the effects of bile acids on host metabolism, selective BSH inhibitors are also capable of studying how primary and secondary bile acids affect host immune responses, particularly in the context of liver cancer. Recent studies have proposed a causal relationship between bacterial bile acid metabolism (specifically the conversion of primary to secondary bile acids) and a reduction in the tumor-suppressive environment in the liver 64。By bile acid feeding, antibiotic treatment, and colonization of mice with bile acid-metabolizing bacteria, these researchers provided support for a model in which secondary bile acids reverse the beneficial NKT cell accumulation and the liver tumor growth inhibition promoted by primary bile acids. Use of a BSH inhibitor in a mouse model of liver cancer can further test this hypothesis by shifting the endogenous in vivo bile acid pool towards primary bile acids without significantly disturbing the enterohepatic system and the microbiota. If such a shift in the bile acid pool limits liver tumor growth, bacterial BSH inhibitors could be developed as novel cancer therapeutics.

[0565] Finally, in the development of BSH inhibitors, two molecules, riboflavin and CAPE, previously identified as inhibitors of BSH from a Lactobacillus salivarius chicken intestinal isolate by high-throughput screening were also evaluated. 59 。In contrast to compound 7, riboflavin and CAPE did not show significant inhibitory activity against any Gram-negative strains and only one of three Gram-positive intestinal bacterial strains, which were also from the genus Lactobacillus. Additionally, while compound 7 (20 μM) almost completely inhibited BSH activity in resuspended mouse feces, CAPE at 20 μM or 100 μM concentrations did not significantly reduce deconjugation in this assay. CAPE significantly inhibited the growth of the tested Gram-negative intestinal bacterial strains. The use of CAPE to inhibit BSH in mice has been reported, and the shift towards a more FXR-antagonistic bile acid pool was thus investigated to see how it affects host metabolism, particularly hepatic gluconeogenesis. 65 。Given these results, especially the finding that CAPE has antibiotic properties, the conclusions of previous in vivo results obtained with CAPE should be re-examined or at least viewed with caution. 66 。The use of selective BSH inhibitors such as compound 7 allows for the evaluation of bacterial bile acid metabolism and its effects on host physiology.

[0566] Covalent inhibitors are able to inactivate their protein targets with a high degree of potency and selectivity even in the presence of large concentrations of natural substrates. 11 。The substrate of BSH, the conjugated bile acids, are present in the colon at high concentrations (1 - 10 mM). 4 。Furthermore, recent work has shown that irreversible inhibitors of bacterial enzymes can be effective in the gut. 12 。

[0567] Although the BSH protein sequences vary significantly among intestinal strains, all BSHs have a conserved active site that includes the catalytic cysteine (Cys2) (Figure 9b). 1,10。Therefore, compounds targeting this conserved residue can be effective pan-BSH inhibitors. The co-crystal structure of Clostridium perfringens BSH and the substrate taurodeoxycholic acid shows that hydrophobic interactions involve the bile acid core and direct the amide towards Cys2, exposing the amino acid to the solvent (Figure 9c) 13 。In addition, Clostridium perfringens BSH tolerates a greater degree of variability in amino acid side chains, including long-chain conjugates 14 。

[0568] A small library of potential inhibitors containing both a bile acid core motif and a side-chain electrophilic warhead was developed (Figure 9d). Without being bound by any particular theory, previous literature indicates that conjugated amino acid identity can largely drive BSH specificity 1 while the sterol core configuration also affects BSH reactivity 15 。In addition, some Bacteroidetes species cleave C12=H but not C12=OH primary bile acids (Figure 9a) 16 。

[0569] Several electrophilic trapping groups were selected 17 including isothiocyanate (1) 18 ,cyanoacrylate (2) 19 ,α,β-unsaturated systems (3 and 4) 20 ,acrylamide (5) 21 and nitrile (6) 22 。An inhibitor with an α-fluoromethyl ketone warhead (FMK) (7) was also synthesized. In contrast to the more electrophilic α-iodo, α-bromo-, and α-chloromethyl ketone warheads, the weak leaving group ability of fluorine makes the FMK warhead less reactive and thus more selective 23,24 。FMK-based inhibitors showed minimal off-target effects 23,25 。

[0570] Example 2. Biochemical characterization of BSH

[0571] Then, using selective Bacteroides BSH for inhibitor optimization, the activities of inhibitors 1-9 against Gram-negative and Gram-positive BSH were evaluated. Thus, the selective BSH (BT_2086) was heterologously expressed and purified (Table 2 and Figure 14) 16 。

[0572] Table 2: Primers for BSH gene amplification.

[0573]

[0574] Kinetic parameters were determined using a ninhydrin-based assay 26The purified BSH of Bacteroides thetaiotaomicron showed a preference for taurodeoxycholic acid (TUDCA) dissociation, without dissociating taurocholic acid (TCA) (Table 3 and Figure 14). 16 。

[0575] Table 3: Kinetic parameters of BSH from Bacteroides thetaiotaomicron (B.theta) and Bifidobacterium longum (B.longum).

[0576]

[0577] a Characterization was performed using the ninhydrin reagent, and the experiments were carried out in PBS buffer at pH 7.5 and 37 °C. b The conjugated primary and secondary bile acids used as substrates were taurocholic acid (TCA), taurodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA). c Bacteroides thetaiotaomicron did not dissociate TCA. n = 3 biological replicates for each condition. All data are represented as mean ± SEM.

[0578] The BSH from the Gram-positive strain Bifidobacterium longum SBT2928 was also cloned, expressed, and its kinetic parameters were determined (Table 3 and Figure 14). The K 27 values for all recognized substrates were in the low millimolar range, which approximated the concentrations of these bile acids in the intestine. Although the k m values were lower than those reported for the BSH of Lactobacillus salivarius, the K cat values of these enzymes were similar to those of previously characterized BSH cat 。 m 。 27-29 。

[0579] Example 3. α-FMK compound 7 inhibits recombinant BSH as a lead inhibitor

[0580] The ability of the compounds in our library to inhibit the BSH of Bacteroides thetaiotaomicron and Bifidobacterium longum was also evaluated. Riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11), which were previously identified as compounds that inhibit the BSH of Lactobacillus salivarius isolates from chicken intestine in high-throughput screening, were also tested (Figure 14). 30The BSH inhibitory activity was determined by pre-incubating Bacteroides thetaiotaomicron BSH with each inhibitor (100 μM) for 30 min and then adding a mixture of conjugated bile acids (100 μM final concentration). Since BSH shows different reactivity towards different conjugated bile acids, an equimolar combination of two primary and two secondary conjugated bile acids that are predominant in the gallbladder and small intestine of conventional mice was used as our substrate mixture (tauro-β-muricholic acid (TβMC), TCA, TUDCA, and taurodeoxycholic acid (TDCA)). 31 Dissociation of bile acids was monitored over 21 h by ultra-performance liquid chromatography–mass spectrometry (UPLC–MS). Among the synthetic inhibitors, isothiocyanate (1) showed moderate inhibition. Other compounds containing a Michael acceptor warhead (2–6) did not inhibit dissociation. In contrast, incubation with α-FMK-based 7 led to almost complete inhibition of Bacteroides thetaiotaomicron BSH activity for 21 h (>98%, Figures 10a, 15, 16, and Table 4).

[0581] Table 4: % Dissociation of each bile acid determined in experiments using a 4-tauro-conjugated bile acid pool.

[0582]

[0583] Table 4: (Continued)

[0584]

[0585] To verify that the inhibitory activity of 7 was due to the presence of fluorine as a leaving group, a methyl ketone analogue (8) was synthesized 25 This analogue did not show BSH inhibition against the recombinant protein or Bacteroides thetaiotaomicron cultures, indicating that the α-fluoro group is essential for activity (Figures 10a, 17, and Table 4). Riboflavin did not show any inhibitory activity, while CAPE only provided moderate inhibition of Bacteroides thetaiotaomicron BSH.

[0586] The activities of compounds 1, 7, and CAPE against BSH from Bifidobacterium longum were also evaluated. At all time points, compound 7 was again the most active inhibitor, while CAPE was ineffective in inhibiting Bifidobacterium longum BSH (Figures 10b, 15–16, and Table 3). Compound 7 inhibited Bacteroides thetaiotaomicron and Bifidobacterium longum BSH in a dose-dependent manner (IC 50 values were 427 nM and 108 nM, respectively, Figure 18). Overall, these data indicate that compound 7 is an effective inhibitor of purified BSH proteins from Gram-negative and Gram-positive bacterial strains.

[0587] In the presence of a concentration of the substrate equimolar to that of the enzyme and without any pre-incubation of the inhibitor with the enzyme, compound 7 completely inhibited Bacteroides thetaiotaomicron BSH within 15 seconds, two enzymes that are more catalytically effective (Table 2) (Figure 19). In the presence of a large excess (about 80-fold) of the substrate, 7 completely inhibited Bacteroides thetaiotaomicron BSH activity within 15 minutes (the earliest measurable time point for product formation under these conditions). These results indicate that 7 is a kinetically effective inhibitor of BSH activity.

[0588] Example 4. Compound 7 inhibits BSH in intestinal bacterial cultures

[0589] The potency of 7 in growing bacterial cultures was also evaluated. To test the scope of BSH inhibition, three Gram-negative and three Gram-positive strains of human intestinal bacteria containing BSH were used (Gram-negative: Bacteroides thetaiotaomicron, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482; Gram-positive: Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC13124, and Bifidobacterium adolescentis L2-32) 1,16 .

[0590] The bacterial cultures were diluted in the early logarithmic growth phase, and a mixture of the inhibitor (100 μM) and conjugated bile acid (100 μM final concentration) was added simultaneously. Dissociation was monitored by UPLC-MS over 21 hours. Strikingly, while all six bacterial strains dissociated bile acids in the presence of the vehicle control, little dissociation was observed in any of the growing cultures in the presence of 7 (Figure 10c, 20, and Table 3). The isogenic BSH-deficient Bacteroides thetaiotaomicron strain was then 16 incubated with DMSO, 7, or CAPE. Unmetabolized taurine-conjugated bile acid was recovered under all three conditions (Figure 21). These results suggest that the inhibition of BSH by 7 is not due to an effect of this inhibitor on other bile acid utilization processes. Compound 7 did not significantly affect the cell viability of most of the tested strains (Figure 10d), indicating that the observed BSH inhibition is not due to bactericidal activity. The IC 50 values of this inhibitor against Bacteroides thetaiotaomicron and Bifidobacterium adolescentis were determined to be 1070 nM and 237 nM, respectively (Figure 22). These results indicate that 7 is an effective broad-spectrum inhibitor of BSH.

[0591] No BSH inhibition was observed in five of the six bacterial strains grown in the presence of CAPE (Figure 10c). In addition, CAPE inhibited the cell viability of all three tested Gram-negative bacterial strains (Figure 10d). These results suggest that the major effect of CAPE on Gram-negative bacteria is not to inhibit BSH activity but to inhibit growth.

[0592] Finally, to evaluate whether the C12═OH compound is not an effective broad-spectrum inhibitor, a compound cholanic acid (Compound 9, Figure 9d) with an α-FMK warhead appended to the C12═OH bile acid core was synthesized. Compound 9 showed significantly reduced ability to inhibit BSH dissociation in Bacteroides thetaiotaomicron cultures compared to 7 (Figure 17). Thus, the bile acid core structure, particularly the C12 substitution, affects our probe's ability to selectively inhibit BSH. Additionally, these results show that the α-FMK warhead is not broadly reactive but requires proper positioning within the active site.

[0593] Example 5. Compound 7 inhibits BSH activity in mouse feces

[0594] Previous literature has reported significant BSH activity in mouse feces 32 . To further evaluate whether 7 is a pan-inhibitor of BSH, its activity in resuspended feces from conventional mice was tested. This fecal slurry should contain BSH from nearly the entire bacterial community in the distal colon. Compounds 1, 7, and CAPE (20 μM) were added to the fecal suspension in buffer. After 30 minutes, the deuterated substrate glycochenodeoxycholic acid-d4 (GCDCA-d4) was added, and the formation of the dissociation product was quantified using UPLC-MS after 18 hours. Strikingly, incubation with 7 completely inhibited BSH activity in the feces (Figure 10e). CAPE did not provide inhibition of BSH activity in the feces. These results demonstrate that 7 is an effective pan-inhibitor of BSH activity.

[0595] Example 6. Compound 7 covalently modifies the catalytic Cys2 residue.

[0596] Inhibitory mechanism of 7. The Bacteroides thetaiotaomicron BSH containing two cysteine residues, namely Cys2 and Cys67, was also studied. Analysis of the apo crystal structure of this enzyme revealed that both cysteine residues point towards the active site (PDB 3HBC). To confirm that 7 is a covalent inhibitor that modifies Cys2, purified Bacteroides thetaiotaomicron BSH was incubated with an excess of this molecule. Mass spectrometry analysis revealed a mass shift consistent with the addition of a single molecule of 7, confirming the formation of a covalent bond (Figure 23). Subsequent top-down mass spectrometry identified Cys2 as the modified residue (Figure 23).

[0597] The structure of Bacteroides thetaiotaomicron BSH was first determined in its apo form to a resolution, and then with 7 covalently bound to a resolution (Table 5) (PDB 6UFY and 6UH4, respectively).

[0598] Table 5: Data collection and refinement statistics (molecular replacement)

[0599]

[0600] *The highest resolution shell is shown in parentheses. Each data set was collected using a single crystal.

[0601] The structure of the BSH-inhibitor complex contains four copies of the protein in the asymmetric unit. The electron density map was best resolved in two of the four subunits, and in one of these subunits, the electron density of the inhibitor covalently linked to Cys2 was clearly visible (Figures 3a and 3b). Comparison with the apo structure also revealed a repositioned loop (residues 127 - 138) to clamp the inhibitor in the active site in a solvent-exposed channel (Figure 24).

[0602] These data indicate that 7 selectively labels Bacteroides thetaiotaomicron BSH at Cys2. Additionally, the co-crystal structure reveals that the C3-hydroxy group is solvent-accessible, suggesting that this site is amenable to further modification (Figure 11b).

[0603] Example 7. Compound 7 shows minimal off-target effects

[0604] It has been proposed that the non-specific reactivity of covalent inhibitors may lead to problems of acute toxicity 11 Bile acids are ligands for the farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (TGR5). 2 In vitro co-activator recruitment assays showed that 7 is neither an agonist nor an antagonist of FXR at physiologically relevant concentrations (Figure 25). 31 Subsequently, the effect of compound 7 on TGR5 activation was evaluated in a human intestinal cell line (Caco-2). Compound 7 neither agonized nor antagonized TGR5 in the concentration range tested (Figure 25). These results indicate that 7 does not induce off-target effects on these key host receptors.

[0605] It is also known that bile acids are toxic due to their detergent properties. 1,33 The toxicity of the compound against human intestinal cells (Caco-2 and NCI-H716) was also tested. No resulting toxicity was observed when these cells were incubated with up to 50 μM or 100 μM of compound 7, respectively (Figure 25). Since the IC 50 values of 7 are in the range of 237 to 1070 nM, these results suggest that an effective and non-toxic in vivo dose should be achievable. To test the effect of compound 7 on epithelial integrity, Caco-2 cells were differentiated into a polarized monolayer with tight intercellular junctions in transwell inserts. 34Compound 7 was incubated in the apical chamber of transwells, and epithelial integrity was measured by the passive diffusion of 4 kDa FITC-dextran. No significant increase in fluorescence was observed in cells treated with 7 compared to control-treated cells, indicating that 7 did not compromise the integrity of the epithelial monolayer( Figure 26 ).

[0606] It is important to understand the proteome-wide reactivity of 7 35 . To evaluate the target binding and off-target interactions of compound 7, an α-azido moiety 36 was appended to 7 at the solvent-exposed C3 position to generate 7-N 3 to synthesize the "clickable" form of this inhibitor (compound 12, Figure 12A ). Like 7, 7-N 3 effectively inhibited BSH activity in mouse feces( Figure 12B ). These results demonstrate that azid...

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: n is 2; m is 1; X is -C(O)CH 2 F or -NCS; R 1 、R 2 、R 4 、R 6 、R 11 、R 12 、R 15 and R 16 are -H; R 3 is - OR 18 or - OSO 3 R 18 ; R 7 is -H, -OR 18 or -OSO 3 R 18 ; R 17 is C 1 -C 6 alkyl; and Each R 18 is independently -H or an unsubstituted C 1 -C 6 alkyl group.

2. The compound according to claim 1, wherein R 3 is -OR 18 or -OSO 3 R 18 ; and R 7 is -OR 18 .

3. The compound according to claim 1, wherein R 3 and R 7 is -OH.

4. The compound according to claim 1, wherein R 3 is -OSO 3 R 18 ; and R 7 is -OH.

5. The compound according to claim 1, wherein R 18 is -H or C 1 -C 6 alkyl.

6. The compound according to claim 1, wherein R 18 is -H.

7. The compound according to claim 1, wherein the compound is a compound of formula (X): or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 1, wherein the compound is a compound of formula (II): or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 1, wherein the compound is a compound of formula (I-a): or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 1, wherein the compound is a compound having the following formula: or a pharmaceutically acceptable salt thereof.

11. The compound according to claim 1, wherein the compound is a compound of formula (I-e’): compound: or a pharmaceutically acceptable salt thereof, wherein: R 3a and R 7a are independently selected from -OR 18 or -OSO 3 R 18 where each R 18 is independently -H or unsubstituted C 1 -C 6 alkyl.

12. The compound according to claim 11, wherein the compound is a compound having formula (I-e”): or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 1, wherein the compound is a compound having the following formula: or a pharmaceutically acceptable salt thereof.

14. The compound according to claim 1, wherein the compound is a compound of formula (I-f’): or a pharmaceutically acceptable salt thereof, wherein: R 3a Selected from -OR 18 or -OSO 3 R 18 , wherein each R 18 is independently H, or unsubstituted C 1 -C 6 alkyl.

15. The compound according to claim 14, wherein the compound is a compound having formula (I-f”): or a pharmaceutically acceptable salt thereof.

16. The compound according to claim 14, wherein the compound is a compound having formula (I-f”’): or a pharmaceutically acceptable salt thereof.

17. A compound according to any one of claims 1 to 9, 11, 12 or 14 to 16, wherein X is 18. A compound according to any one of claims 1 to 9, 11, 12 or 14 to 16, wherein X is 19. The compound according to claim 1, wherein the compound has the following formula: or a pharmaceutically acceptable salt thereof.

20. A pharmaceutical composition comprising: a compound according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

21. The pharmaceutical composition according to claim 20, wherein the pharmaceutically acceptable excipient limits the delivery of the compound to the gastrointestinal tract.

22. Use of a compound according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20 or 21, in the preparation of a drug for inhibiting bile salt hydrolase (BSH) in a subject.

23. Use of a compound according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20 or 21, in the preparation of a drug for inhibiting bile acid dissociation in a subject.

24. Use of a compound according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20 or 21, in the preparation of a drug for promoting bile acid conjugation in a subject.

25. Use according to any one of claims 22 to 24, wherein the subject is in need of treatment for diabetes or obesity.

26. Use of a compound according to any one of claims 1 to 19, or a pharmaceutical composition according to claim 20 or 21, in the preparation of a drug for modulating bile acids in a subject.

27. Use according to any one of claims 22 to 24 and 26, wherein the subject is a mammal.

28. Use according to any one of claims 22 to 24 and 26, wherein the subject is a human.

29. Use according to any one of claims 22 to 24 and 26, wherein the subject is at risk of having or has diabetes or prediabetes.

30. Use according to claim 29, wherein the diabetes is caused by obesity.

31. Use according to claim 29, wherein the diabetes is type II diabetes.

32. Use according to any one of claims 22 to 24 and 26, wherein the subject is at risk of having or has obesity.

33. Use according to claim 25, wherein the diabetes is caused by obesity.

34. Use according to claim 25, wherein the diabetes is type II diabetes.

35. Use according to any one of claims 22 to 24 and 26, wherein the subject has a body mass index of at least 25 kg / m 2 2.

36. A kit, comprising: a compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 20 or 21; and instructions for using the compound, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

37. An in vitro method of inhibiting bile salt hydrolase (BSH), the method comprising contacting bile salt hydrolase (BSH) with a compound according to any one of claims 1 - 19 or a pharmaceutical composition according to claim 20 or 21.

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