Small molecule modulators of bile acid metabolism of intestinal bacteria

Abstract

Described herein are methods and compositions relating to inhibition of bile salt hydrolase (BSH) and uses thereof. Provided herein are methods 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; a 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; diverticulosis; food intolerance; ulcer; infectious colitis; irritable bowel syndrome; intestinal leakage; the present invention relates to a method for treating a cancer (e.g., digestive cancer, liver cancer), a cancer (e.g., digestive cancer, liver cancer), or an inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcer, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis). The method comprises administering to the subject a compound of Formula (I)-(XVIII).

Description

[0001] This application is a divisional application based on a patent application with application date of May 8, 2020, application number 202080050183.1 (PCT / US2020 / 032016), and invention name: "Small molecule regulators of intestinal bacterial bile acid metabolism".

[0002] Related applications

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. provisional applications USSN 62 / 846,457, filed May 10, 2019, and USSN 62 / 962,048, filed January 16, 2020, each of which is incorporated herein by reference.

[0004] Government support

[0005] This invention was made with government support under Contract Nos. R35 GM128618 and 5P30DK034854-32 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The technology described herein relates to compounds, compositions, and methods for inhibiting bile salt hydrolase (BSH). Background Art

[0007] Bile salt hydrolase (BSH) is widely expressed by human intestinal bacteria and catalyzes a gateway reaction that converts 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 as tools for understanding bile acid physiology in host subjects. Summary of the Invention

[0008] In one aspect, provided herein is a compound of formula (I):

[0009]

[0010] in:

[0011] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0012] m is 1, 2, 3, or 4;

[0013] X is an electrophilic group;

[0014] R1, R2, R3, R4, R6, R7, 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、-CO2H、-CO2R 18 , -NO2, -ONO2, -SO2Cl, -SO3 - 、-OSO3 - 、-NR 18 SO3 - 、-PO3 2- 、-OPO3 2- 、-OSO2R 18 、-SO2N(R 18 )2、-OSO2N(R 18 )2、-NR 18 SO2R 18 、-SO2N(R 18 )2, -NHNH2, -ONH2, or -NHC(O)NHNH2;

[0015] 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;

[0016] or a pharmaceutically acceptable salt thereof.

[0017] In one aspect, the compound of formula (I) is of formula (I'):

[0018]

[0019] in:

[0020] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0021] m is 1, 2, 3 or 4;

[0022] X is an electrophilic group;

[0023] R1, R2, R3, R4, R6, R7, R 11 、R12 、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、-CO2H、-CO2R 18 , -NO2, -ONO2, -SO2Cl, -SO3H, -OSO3H, -NR 18 SO3H, -PO3H2, -OPO3H2, -OSO2R 18 、-SO2N(R 18 )2、-OSO2N(R 18 )2、-NR 18 SO2R 18 、-SO2N(R 18 )2, -NHNH2, -ONH2, 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;

[0024] or a pharmaceutically acceptable salt thereof.

[0025] In another aspect, provided herein is a pharmaceutical composition comprising a compound provided herein and a pharmaceutically acceptable carrier or excipient.

[0026] In another aspect, provided herein is a method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound provided herein.

[0027] In another aspect, provided herein is a method of inhibiting bile acid dissociation in a subject, comprising administering to the subject a therapeutically effective amount of a compound provided herein.

[0028] In another aspect, provided herein is a method of promoting bile acid conjugation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein.

[0029] On the other hand, there is provided herein a method for regulating bile acids in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein. On the other hand, there is provided herein a method 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); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; A method for treating a variety of conditions including colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, kidney cancer or renal cancer, oral cancer, pancreatic cancer, prostate cancer, rectal cancer, gastric cancer, basal cell carcinoma, bile duct cancer, lung cancer, bladder cancer, cervical cancer, endometrial cancer, uterine cancer, and urinary tract cancer), or inflammatory diseases such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcer, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis, comprising administering to a subject in need thereof a compound of Formula (I)-(XVIII), a genetically engineered microorganism that secretes bile acid 7-sulfate, or a population thereof.

[0030] In another aspect, provided are compounds of Formula (I)-(XVIII), or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising compounds of Formula (I)-(XVIII), for use in 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); chylomicrons; and liver cirrhosis). diarrhea; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract 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 intolerances, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0031] In another aspect, a kit is provided, comprising a compound of Formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of Formula (I)-(XVII). In certain embodiments, the kit further comprises instructions for administration (e.g., human administration) and / or use.

[0032] The details of certain embodiments of the present invention are described in the following Detailed Description of Certain Embodiments. Other features, objects and advantages of the present invention will be apparent from the definitions, examples, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] This patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0034] Figure 1A and 1B The chemical and biological effects of enteric bacterial bile salt hydrolase (BSH) are demonstrated. Figure 1AIt was shown that BSH is a gateway enzyme in the conversion of primary (host-produced) to secondary (bacterial-produced) bile acids. Removal or inhibition of BSH should result in a reduction in dissociated primary and secondary bile acids. Figure 1B Specific primary and secondary bile acids have been shown to be ligands for host nuclear hormone receptors (NhRs) and G protein-coupled receptors (GPCRs). By acting as agonists or antagonists of these receptors, these bile acids influence host processes, including metabolic control and immune responses.

[0035] Figures 2A to 2E Demonstrated the rational design of small-molecule broad-spectrum BSH inhibitors. Figure 2A The mechanism of BSH enzymatic amide bond cleavage is shown. Figure 2B Shown is the co-crystal structure of BSH from the Gram-positive enteric bacterium Clostridium perfringens with dissociated taurodeoxycholic acid (TDCA) (PDB 2BJF), which guides inhibitor design. While hydrophobic interactions orient the bile acid core to the active site (magenta residues), the D-ring side chains and amino acids are exposed to the solvent. Figure 2C A representative mechanism of BSH inhibition by rationally designed inhibitors is shown. Attack of the catalytic nucleophilic cysteine ​​residue in the BSH active site can lead to covalent binding of the inhibitor. Figure 2D A library of synthetic inhibitors is shown. Electrophilic warheads, successfully introduced in the design of kinase and protease inhibitors, were appended to the chenodeoxycholic acid bile acid core, resulting in broad-spectrum BSH inhibitors. Figure 2E Shown are the most potent BSH inhibitors identified from high throughput screening, riboflavin and caffeic acid phenethyl ester (CAPE), which were also included in this study.

[0036] Figures 3A to 3B We demonstrate that the screen identified inhibitor 7 as a potent and persistent inhibitor of recombinant BSH. Figure 3A Shown is a screen of an inhibitor library against B. theta BSH, showing % dissociation at 2 and 21 hours. Figure 3B Screening of compounds 1, 7, and CAPE against B. longum BSH is shown, showing % dissociation at 2 and 21 hours. Inhibitors (100 μM) were incubated with 200 nM rBSH for 30 minutes, followed by the addition of taurine-conjugated bile acid substrates (TβMCA, TCA, TUDCA, and TDCA, 25 μM each). Dissociation of the substrates was tracked by UPLC-MS. The assay was performed in biological triplicate. Data are presented as mean ± SEM.

[0037] Figures 4A to 4DCompound 7 was shown to be a potent, non-toxic inhibitor of BSH in growing cultures of Gram-positive and Gram-negative enteric bacteria. Figure 4A Compound 7 was shown to inhibit BSH activity in living Gram-negative (Bacteroides thetaiotaomicron VPI 5482, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482) and Gram-positive (Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32) bacteria. Inhibitors (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 culture medium and the OD values ​​were calculated. 600 0.1 was added to the bacterial culture. The bacterial culture was allowed to grow to the stationary phase, and the percentage of dissociation was determined by UPLC-MS for 24 hours. The assay was performed in biological triplicate. Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.00001. Statistical analysis was not performed for B. vulgatus (common Bacteroides) because the standard error of the replicates was zero. Figure 4B It was shown that compound 7 was not bactericidal. The OD of the bacterial culture was measured at 24 hours. 600 CAPE inhibits the growth of the tested Gram-positive strains. The red downward arrows indicate the percentage reduction compared to the DMSO control. Figure 4C Shown are the dose-response curves and calculated IC for compound 7 incubated with growing cultures of B. thetaiotaomicron (Gram-negative) and B. adolescentis (Gram-positive). 50 The results indicate that compound 7 is a potent and broad-spectrum BSH inhibitor. Figure 4D Representative UPLC-MS traces are shown, demonstrating that inhibitor structure determines BSH inhibitory activity against growing B. 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 culture, and its dissociation into UDCA was followed by UPLC-MS. Inhibitor 9, which has a cholic acid (C12═OH) core and an α-FMK warhead, exhibited significantly reduced activity against B. thetaiotaomicron BSH.

[0038] Figures 5A to 5CCompound 7 was shown to covalently modify B. multiformis BSH at the active site cysteine residue. Figures 5A to 5B Mass spectra showing that compound 7 mono-labeled B. multiformis BSH. Figure 5A Mass spectra (left) and zero charge mass spectra (right, overlay) of BSH treated with DMSO (top, red trace) or 10-fold excess of inhibitor compound 7 (bottom, green trace) for 2 hours. The mass shift of 388 Da is consistent with covalent modification of BSH and loss of HF. Figure 5B Top-down MS of BSH treated with 10-fold excess of inhibitor compound 7. Ions of type c and z are indicated with red and green glyphs, respectively. Ion c3 indicates modification on the N-terminal Cys2 residue. Figure 5C X-ray co-crystal structure of compound 7 bound to B. multiformis BSH, demonstrating that compound 7 is covalently linked to active site Cys2 but not Cys67, and that the C25 fluorine has been eliminated. C3 of the steroid core is exposed to solvent, indicating that this site can be amenable to modification.

[0039] Figures 6A to 6C Compound 7 was shown to exhibit minimal off-target effects. Figure 6A Compound 7 was shown to be neither a farnesoid X receptor (FXR) agonist nor antagonist, as determined by FXR co-activator recruitment assay. Compound 7 was evaluated for FXR agonist activity in the presence of known FXR agonist GW4064 at its EC 50 Compound 7 was evaluated for FXR antagonist activity at FXR in the presence of known FXR agonist GW4064 at its EC value (50 nM). n = 4 biological replicates per concentration. Data represented as mean ± SEM. Figure 6B Compound 7 was shown to be neither a G protein-coupled bile acid receptor (GPBAR1, also known as TGR5) agonist nor antagonist. Endogenous TGR5 agonist activity was measured by incubating Caco-2 cells with different concentrations of compound 7 overnight. Endogenous TGR5 antagonist activity was evaluated in the presence of 10 mM of TGR5 agonist LCA. n > 3 biological replicates per concentration. Data represented as mean ± SEM. Dunnett’s multiple comparison test after one-way ANOVA, ns = not significant. Figure 6C Compound 7 was shown to not exhibit toxicity against Caco-2 cells at concentrations up to 50 mM. n > 3 biological replicates per concentration. Data represented as mean ± SEM. Dunnett’s multiple comparison test after one-way ANOVA, *p < 0.05.

[0040] Figures 7A to 7F Compound 7 was shown to inhibit BSH activity in vitro and in vivo. Figure 7AFecal BSH activity assay design is shown. Freshly collected feces from regular mice (1 mg / mL) were resuspended in PBS and incubated with 20 mM of inhibitor (Compound 1, 7, or CAPE) for 30 minutes. Glycochenodeoxycholic acid-d4 (GCDCA-d4, 100 mM) was added as a substrate and dissociation was measured by UPLC-MS after 18 hours. Figure 7B Compound 7 is shown to effectively inhibit BSH activity in fecal slurry, while CAPE shows minimal inhibitory activity. In line with in vitro results, Compound 1 shows moderate BSH inhibition. Assay was performed in biological triplicate. Data is represented as mean ± SEM. Figures 7C to 7E Treatment of regular mice with a single dose of Compound 7 is shown to result in reversible inhibition of BSH activity and shift towards dissociated bile acids. n = 4 mice per group, Welch’s t-test, *p < 0.05, **p < 0.01, ns = not significant. Figure 7C Design of in vivo BSH inhibition experiment is shown. Male regular C57BL / 6 mice were gavaged with a single dose of Compound 7 (10 mg / kg) or vehicle control. Feces were collected 1 day, 1.5 days, 2 days, and 2.5 days post-gavage. Bile acid analysis was performed 1 day post-gavage. Figure 7D BSH activity in Compound 7 treated groups is shown to be significantly lower compared to control groups 1 day and 1.5 days post-gavage, as measured by BSH activity in feces. BSH recovers 2 days post-gavage. BSH activity was measured by resuspending fresh feces from inhibitor or vehicle treated groups with substrate (GCDCA-d4, 100 mM), incubating for 25 minutes, and quantifying dissociation by UPLC-MS. Figure 7E Fecal bile acid composition 1 day post-gavage is shown. Dissociated bile acids, including the secondary bile acid deoxycholic acid (DCA), are reduced in inhibitor treated groups. Figure 7F Microbial biomass is shown to be not different between inhibitor and vehicle treated groups 1 day or 2.5 days post-gavage. n = 4 mice per group, Mann-Whitney test.

[0041] Figures 8A to 8D Administration of the gut-restricted derivative of Compound 7, 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK), is shown to result in a significant reduction in BSH activity over 1 week when fed in the diet. Figure 8A Structure of 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK) is shown. Figure 8B Design of in vivo BSH inhibition experiment is shown. Male regular C57BL / 6 mice were fed either a normal diet or a diet containing 3S-LCA-FMK (0.03% weight / weight) ad libitum for 7 days. Feces were collected before diet change and on days 3, 4, and 7 after diet change. n = 5 mice per group. Figure 8CIt was shown that BSH activity in feces of mice fed a diet containing 3S-LCA-FMK was significantly reduced. Figure 8D Shown are the concentrations of 3S-LCA-FMK as measured in feces and cecal contents at sacrifice. On day 4, no 3S-LCA-FMK was detected in circulating plasma, indicating that the compound was gut-restricted.

[0042] Figure 9 A shows that the key reaction in the conversion of primary to secondary bile acids is the hydrolysis (cleavage) of the C24 amide bond of the conjugated primary bile acid. Figure 9 B shows that although BSH protein sequences vary significantly among enteric strains, all BSHs have a conserved active site that includes the catalytic cysteine ​​(Cys2). Figure 9 C shows the co-crystal structure of C. perfringens BSH and the substrate taurodeoxycholic acid, which shows that hydrophobic interactions engage the bile acid core and orient the amide towards Cys2, exposing the amino acid to the solvent. Figure 9 D shows a compound of the present disclosure.

[0043] Figure 10 Inhibitors are shown to be effective against Bacteroides thetaiotaomicron BSH ( Figure 10 A) and Bifidobacterium longum BSH ( Figure 10 B) Screening showing the % dissociation of taurobile acid at 2 and 21 hours. Bacterial strains were incubated with 100 μM of conjugated bile acid and plated at 21 hours to assess strain viability ( Figure 10 C). Compound 7 is not bactericidal ( Figure 10 D). CAPE reduces cell viability of the tested Gram-negative strains. Red downward arrows indicate fold reduction compared to the DMSO control. Figure 10 C) and ( Figure 10 D), One-way ANOVA followed by Dunnett's multiple comparison test. ( Figure 10 E) Compound 7 inhibits BSH activity in fecal slurries. All assays were performed in biological triplicates and data are presented as mean ± SEM.

[0044] Figure 11 A shows the X-ray structure of compound 7 bound to Bacteroides thetaiotaomicron BSH. BSH (cyan) is shown in ribbon representation with the indicated side chains (cyan, with heteroatoms in CPK color) presented as sticks. Figure 11 B shows the co-crystal structure of B. thetaiotaomicron BSH and compound 7 shown in ribbon (left, electron density of the compound is shown as blue mesh) and surface (right). Ring A of 7 including C3 hydroxyl group is exposed to solvent. Figures a and b were generated using PYMOL software. Production.

[0045] Figure 12 A shows the structure of the “clickable” 7,7-N3 (12) for hit and off-target studies. Figure 12 B shows that 7-N3 exhibited significant BSH inhibition in normal mouse feces, indicating that the probe retained its function as a BSH inhibitor. Figure 12 C shows that treatment of B. adolescentis L-32 cultures with 7-N3 for 1 hour, followed by cell lysis, click reaction with Fluor 488-alkyne, and in-gel fluorescence visualization revealed labeling of only one protein of approximately 35 kDa (the mass of the annotated B. adolescentis BSH). Figure 12 D shows lysates from cultures of B. adolescentis treated with 7-N3 were reacted with desthiobiotin-alkyne, resolved by SDS-PAGE, and visualized by silver staining. The arrow indicates a band at the predicted molecular weight of BSH (approximately 35 kDa) in the probe-treated sample. Figure 12 E shows that treatment of B. adolescentis cultures with decreasing concentrations of compound 7 followed by 10 μM 7-N3 and click reaction with Fluor 488-alkyne resulted in a dose-dependent increase in fluorescent labeling of annotated B. adolescentis BSH. Figure 12 F shows that treatment of NCI-H716 intestinal cells with 7-N3 for one hour followed by click reaction with Fluor 488-alkyne and visualization by in-gel fluorescence resulted in no significant protein labeling compared to control-treated cells. Figure 12 B, 12C, 12D and 12F), n = 3 biological replicates per condition. Figure 12 B), Data are expressed as mean ± SEM.

[0046] Figure 13 A to 13C show that treatment of conventional mice with a single dose of compound 7 resulted in reversible inhibition of BSH activity and a shift toward conjugated bile acids. n = 4 mice per group, Student's t test. Figure 13 A 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 13 B shows BSH activity was measured in 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 13C shows 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 13 D shows that bacterial OTUs (operational taxonomic units) were not different between inhibitor- and vehicle-treated groups 1 day after gavage. n = 4 mice per group, one-way ANOVA followed by Tukey's multiple comparison test. Figure 13 E shows the structure of intestinal restricted compound 7 (GR-7,13). Figure 13 F 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 a powdered diet alone for 30 hours. Fecal pellets were collected 8 hours after the diet change. n = 10 mice per group. Figure 13 G shows resuspended fresh feces (20 mg / mL) from inhibitor- or control-treated mice incubated with substrate (GCDCA-d4, 100 μM) for 25 minutes, and product formation was quantified by UPLC-MS. Significant inhibition of BSH activity was observed in feces from GR-7-treated mice compared to control-treated mice. Student's t-test. n = 10 mice per group, two-tailed Student's t-test. Figure 13 H shows quantification of GR-7 in tissues and plasma. The inhibitor was detected in feces 8 hours after the dietary change and in cecal contents at sacrifice. GR-7 was not detected in liver or plasma. ND = Not Detected. n = 10 mice per group. All data are expressed as mean ± SEM.

[0047] Figure 14 The purification and kinetic characterization of BSH are shown. ( Figure 14 A) SDS-PAGE of BSH purified from Bacteroides thetaiotaomicron. The experiment was repeated seven times with similar results. Figure 14 B) SDS-PAGE of B. longum BSH purification. Michaelis-Menten analysis of BSH kinetic data. Figure 14 C) and Bifidobacterium longum BSH ( Figure 14 D) Rate vs. substrate concentration curve.

[0048] Figure 15 Identification of compound 7 as a potent broad-spectrum BSH inhibitor is shown. Figure 15 AB shows the inhibitors against Bacteroides thetaiotaomicron BSH ( Figure 15 A) and Bifidobacterium longum BSH ( Figure 15B) Screening, which shows the % dissociation of taurocholic acid at 5 hours. The inhibitor (100 μM) was incubated with 200 nM rBSH for 30 minutes, followed by the addition of taurine-conjugated bile acid substrates (tauro-β-muricholic acid, TβMCA; tauro-cholic acid, TCA; tauro-ursodeoxycholic acid, TUDCA; and taurodeoxycholic acid, TDCA, each 25 μM). The dissociation of the substrate was tracked by UPLC-MS. The assay was performed in biological triplicate, and all data are expressed as mean ± SEM.

[0049] Figure 16 Bile acid quantification for purified BSH protein reported as % dissociation is shown. Figure 16 A) and Bifidobacterium longum BSH ( Figure 16 B) The concentrations of both the product (dissociated bile acid) formed and the unreacted starting material (SM) at each time point. The % dissociation for each sample was then determined using the following formula: % dissociation = product concentration / (product concentration + starting material concentration) * 100.

[0050] Figure 17 Compound structure was shown to influence BSH inhibitory activity against growing Bacteroides thetaiotaomicron cultures. Figure 17 A) Compounds 8 and 9 are less effective inhibitors of B. thetaiotaomicron BSH than compound 7. Inhibitors (10 μM of compound 7, 8, or 9) and 100 μM TUDCA were added to B. thetaiotaomicron cultures at OD 6000.1. Figure 17 B) Structural comparison of compounds 7, 8, and 9. Compound 8 lacks the α-FMK warhead, and compound 9 has a C12=OH hydroxyl group.

[0051] Figure 18 The results show that compound 7 is an effective recombinant BSH inhibitor. The dose response curve and calculated IC value of compound 7. 200 nM recombinant B. thetaiotaomicron BSH ( Figure 18 A, Gram-negative) or Bifidobacterium adolescentis BSH ( Figure 18 B, Gram-positive) were preincubated with different concentrations of compound 7 for 60 min, followed by the addition of conjugated bile acid substrates TUDCA and TDCA, respectively.

[0052] Figure 19A shows the time required for complete inhibition of B. thetaiotaomicron BSH. 100 μM compound 7 and conjugated bile acids (tauro-β-muricholic acid, TβMCA; tauro-cholic acid, TCA; tauro-ursodeoxycholic acid, TUDCA; and taurodeoxycholic acid, TDCA, 25 μM each) were added simultaneously to 200 nM rBSH without a pre-incubation period. The formation of dissociated bile acids was measured using a UPLC-MS-based assay and reported as % conversion. Figure 19 B shows that in the presence of compound 7, no increase in product formation was observed after 15 seconds, indicating that the enzyme activity was inhibited.

[0053] Figure 20 Quantification of bile acids for reporter bacterial cultures as % dissociation is shown. UPLC-MS was used to determine the products (dissociated bile acids) formed in each culture ( Figure 20 A) and unreacted starting material (SM) ( Figure 20 B) The % dissociation of each sample was then determined using the following formula: % dissociation = product concentration / (product concentration + starting material concentration) * 100.

[0054] Figure 21 It is shown that compound 7 did not alter the bile acid pool when incubated with the B. thetaiotaomicron BSH KO strain. ( Figure 21 A) 100 μM of taurine-conjugated bile acid pool (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 culture. ( Figure 21 B) After 24 hours, the test group ( Figure 21 A) Determination of colony forming units (CFU). Compound 7 was not found to be bactericidal against BSH-deficient B. thetaiotaomicron, while CAPE was found to significantly affect the growth of this bacterium.

[0055] Figure 22 The results show that compound 7 is an effective BSH inhibitor in growing bacterial cultures. The dose response curve and calculated IC value of compound 7. Figure 22 A, Gram-negative) and Bifidobacterium adolescentis ( Figure 22 B, Gram-positive) were incubated with conjugated substrate (TUDCA or TDCA) and allowed to grow anaerobically for 48 h and 24 h, respectively.

[0056] Figure 23The mass spectrum is shown, revealing compound 7 singly labeled with B. thetaiotaomicron BSH. ( Figure 23 A) Mass spectra (left) and zero-charge mass spectra (right) of BSH treated for 2 hours with DMSO (top, red trace) or a 10-fold excess of compound 7 (bottom, green trace). The 388 Da mass shift is consistent with covalent modification of BSH and loss of HF. Two independent labeling reactions yielded similar results. ( Figure 23 B) Top-down MS / MS of BSH treated with a 10-fold excess of compound 7. Type c and z ions are represented by red and green glyphs, respectively. Ion c3 indicates modification at the N-terminal Cys2 residue.

[0057] Figure 24 The apo and co-crystal structures of B. thetaiotaomicron BSH are shown. X-ray structure of B. thetaiotaomicron BSH apoprotein ( Figure 24 A) X-ray structure of B. thetaiotaomicron BSH superimposed on the covalently bound compound 7 ( Figure 24 B) (apo magenta, cocrystal structure cyan) is shown as a ribbon representation, with the indicated side chains (magenta or cyan, respectively, with heteroatoms in CPK color) presented as sticks. Compound 7 (green, with heteroatoms in CPK color) is presented in stick form. The box (dashed line) indicates the repositioned loop (residues 127-138) in the cocrystal structure. The figure was created using PYMOL software (Schroedinger).

[0058] Figure 25 It was shown that compound 7 is neither an agonist nor an antagonist of FXR or TGR5 and is non-toxic to human cells. Figure 25 A) Compound 7 is not a farnesoid X receptor (FXR) agonist as determined by the FXR coactivator recruitment assay. n = 4 biological replicates per concentration. ( Figure 25 B) 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 25 C) Compound 7 is not a G protein-coupled bile acid receptor (GPBAR1 / TGR5) agonist. Endogenous TGR5 agonist activity was measured by incubating Caco-2 cells with various concentrations of compound 7 overnight. n = 3 biological replicates per concentration, one-way ANOVA followed by Dunnett's multiple comparison test. ( Figure 25D) Endogenous TGR5 antagonist activity was measured by incubating Caco-2 cells with various concentrations of compound 7 overnight in the presence of 10 μM of the TGR5 agonist LCA. n = 3 biological replicates per concentration, one-way ANOVA followed by Dunnett's multiple comparison test. ( Figure 25 E) 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 biological replicates were determined for each concentration, respectively. One-way ANOVA followed by Dunnett's multiple comparison test was performed. All data are expressed as mean ± SEM.

[0059] Figure 26 Neither compound 7 nor GR-7 significantly affected epithelial barrier integrity. Incubation of compound 7 or GR-7 with differentiated Caco-2 cells for 6 and 12 hours did not impair epithelial monolayer integrity, as measured by passive transport of 4 kDa FITC-dextran. For DMSO controls, n = 2 biological replicates were performed, and for inhibitor-treated conditions, n = 3 biological replicates were performed. All data are presented as mean ± SEM.

[0060] Figure 27 A shows that compound 7-N labels B. adolescentis BSH with minimal off-target reactivity. Figure 27 B shows the quantification of fluorescence intensity of BSH bands. Two-tailed Student's t test was used. Data are expressed as mean ± SEM. Figure 27 C示出 Figure 12 Full SDS-PAGE gel of the experiment described in E. B. adolescentis cultures were treated with decreasing concentrations of compound 7 for 1 hour, followed by treatment with 10 μM compound 7-N for an additional hour. Dose-dependent labeling of BSH was observed with decreasing concentrations of compound 7. The experiment was repeated twice with similar results. Figure 27 D示出用于 Figure 12 Silver-stained gel from the experiment described in D, performed in biological triplicate. Figure 27 E示出通过对 Figure 27 BSH-derived tryptic peptides identified by LC-MS / MS analysis of in-gel digestion performed on the band indicated in D. The amino acids highlighted in the red spectrum are tryptic peptides identified at approximately 1% FDR.

[0061] Figure 28 7-N3 was shown to exhibit minimal off-target labeling in mammalian cells. Figure 12 SDS-PAGE gel of the experiment described in F performed in biological triplicate (ie, NCI-H716 cells treated with 10 μM 7-N3 for 1 hour followed by click reaction with Fluor 488-alkyne).

[0062] Figure 29 Compound 7 was shown to not significantly affect bacterial community composition or microbial biomass in vivo. Figure 29 A) Average relative abundance of the microbiota at the phylum level by taxon-based analysis, n = 4 mice group. Figure 29 B) CFU / g was not different between inhibitor and vehicle treated groups at 0.5, 1, 1.5, 2, or 2.5 days post tube-feeding. n = 4 mice per group, two-tailed Mann-Whitney test. All data presented as mean ± SEM.

[0063] Figure 30 Activity and in vivo effects of GR-7 are shown. Figure 30 A) GR-7 inhibits BSH activity in fecal slurry. Freshly collected feces from regular mice were resuspended in PBS (1 mg / mL) and incubated with 20 or 60 mM of GR-7 for 30 min. Glycocholate-d4 (GCDCA-d4, 100 mM) was added as substrate and product formation was determined by UPLC-MS after 18 h. Assay was performed in biological triplicate. Figure 30 B) 16S rDNA copies / g of cecal content 30 h post dietary change. Microbial biomass was not different between inhibitor and vehicle treated groups. Two-tailed Mann-Whitney test. n = 10 mice per group. All data presented as mean ± SEM.

[0064] Figure 31 3S-LCA-FMK reduced food intake in regular mice compared to mice given vehicle (n = 8 mice per group).

[0065] Definitions

[0066] Chemical Definitions

[0067] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implied by context, the following terms and phrases include the meanings provided below. The definitions are provided to aid 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 discrepancy between the usage of a term in this specification and its definition provided within the specification, the definition provided within the specification shall control.

[0068] Definitions of commonly used 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 (editor), 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, NY, 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 (editor) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (editor), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E.Coligan (Editor), John Wiley and Sons, Inc., 2005; Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety.

[0069] 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, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the 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.

[0070] The compounds described herein can contain one or more asymmetric centers and thus can exist in various stereoisomeric forms. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures as well as mixtures enriched in one or more stereoisomer. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) as well as the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, e.g., 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 p. 268 (E. L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses compounds as pure individual isomers, or as mixtures of various isomers, substantially free of other isomers.

[0071] The chemical structures and formulas set forth herein are constructed according to standard rules of chemical valence known in the chemical arts.

[0072] Where substituent groups are designated by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents, written from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0073] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight-chain (i.e., unbranched) or branched-chain (or carbon) group, or a combination thereof, which can be fully saturated, mono- or poly-unsaturated, and can include 10"alkyl" means a monovalent, saturated straight chain or branched hydrocarbon group having from one to ten carbon atoms. Alkyl groups are un-cyclized chains. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, (cyclohexyl)methyl, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like homologs and isomers of alkyl. Unsaturated alkyl groups have one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-iso- pentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3- propynyl, 3-butynyl, and the like homologs and isomers. Alkoxy is an alkyl group attached to the rest of the molecule by an oxygen linking group (-O-).

[0074] The term "alkylene," by itself or as part of another substituent, means, unless otherwise indicated, a divalent group derived from an alkyl, such as, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups will have from 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms being preferred in the present application. Alkylene is an un-cyclized chain. "Lower alkyl" or "lower alkylene" are shorter chain alkyl or alkylene groups, generally having eight or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise indicated, a divalent group derived from an alkene.

[0075] The term "heteroalkyl," by itself or in combination with another term, means a stable straight chain or branched chain, or combinations thereof, having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms can optionally be oxidized and the nitrogen heteroatom can optionally be quaternized. Heteroalkyl is an un-cyclized chain. One or more heteroatoms O, N, P, S, B, As, and Si can be located at any interior position of the heteroalkyl group or at any position of the heteroalkyl group connecting to the rest of the molecule. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3and -CH2-O-Si(CH3)3.

[0076] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, 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)2R'- represents both -C(O)2R'- and -R'C(O)2-. A heteroalkylene group is an uncyclized chain. As noted above, heteroalkyl groups as used herein include those groups attached to the rest of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR', and / or -S02R'. Where "heteroalkyl" is recited, followed by a recitation of a specific heteroalkyl group, such as -NR'R", etc., it is to be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited for added clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, such as -NR'R", etc.

[0077] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms refer to the cyclic forms of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, a heteroatom may occupy the position where the heterocycle is connected to the rest 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-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" by themselves or as part of another substituent mean a divalent radical derived from cycloalkyl and heterocycloalkyl, respectively.

[0078] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0079] Unless otherwise indicated, the term "acyl" means -C(O)R, where R is 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.

[0080] Unless otherwise indicated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused ring aryl) or covalently linked. A fused ring aryl refers to multiple rings fused together, wherein at least one fused ring is an aryl ring. The term "heteroaryl" refers to an aryl group (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 heteroaryls (i.e., multiple rings fused together, wherein at least one fused ring is a heteroaryl ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, 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 heteroaryl refers to two rings fused together, one of which has 6 members and the other has 5 members, and at least one of which is a heteroaryl ring. The heteroaryl group can be connected to the rest 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- The substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the group consisting of the following acceptable substituents. "Arylene" and "heteroarylene," by themselves or as part of another substituent, mean a divalent radical derived from an aryl group and a heteroaryl group, respectively. A heteroaryl substituent may be -O- bonded to a ring heteroatom nitrogen.

[0081] "Fused ring aryl-heterocycloalkyl" is an aryl group fused to a heterocycloalkyl group. "Fused ring heteroaryl-heterocycloalkyl" is a heteroaryl group fused to a heterocycloalkyl group. "Fused ring heterocycloalkyl-cycloalkyl" is a heterocycloalkyl group fused to a cycloalkyl group. "Fused ring heterocycloalkyl-heterocycloalkyl" is a heterocycloalkyl group fused to a cycloalkyl group. "Fused ring heterocycloalkyl-heterocycloalkyl" is a heterocycloalkyl group fused to another heterocycloalkyl group. A fused ring aryl-heterocycloalkyl, fused ring heteroaryl-heterocycloalkyl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl group may each independently be unsubstituted or substituted with one or more substituents described herein. A fused ring aryl-heterocycloalkyl, fused ring heteroaryl-heterocycloalkyl, fused ring heterocycloalkyl-cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl group 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 group. A spirocycle is two or more rings in which adjacent rings are connected by a single atom. In spirocycle, each ring can be the same or different. Each ring in spirocycle can be substituted or unsubstituted, and can have a substituent different from other single rings in one group of spirocycle. When not being a part of spirocycle, the possible substituent of each ring in spirocycle is the possible substituent of the same ring (for example, a substituent of a cycloalkyl or heterocycloalkyl ring). Spirocycle can be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene or substituted or unsubstituted heterocycloalkylene, and each ring in spirocyclic group can be any one of the preceding lists, including all rings with a type (for example, all rings are substituted heterocycloalkylene, wherein each ring can be the same or different substituted heterocycloalkylene). When referring to spirocycle ring system, heterocycle spirocycle means that at least one ring is a heterocycle and wherein each ring can be a spirocycle of different rings. When referring to spirocycle ring system, the spirocycle substituted means that at least one ring is substituted and each substituent can be optionally different.

[0082] As used herein, the term "oxo" means an oxygen double-bonded to a carbon atom.

[0083] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical.

[0084] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), boron (B), arsenic (As), and silicon (Si).

[0085] As used herein, "substituent" means a group selected from the following moieties:

[0086] (A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC═(O)NHNH2, -NHC═(O)NH2, -NHSO2H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0087] (B) an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group substituted with at least one substituent selected from the group consisting of:

[0088] (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC═(O)NHNH2, -NHC═(O)NH2, -NHSO2H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0089] (ii) an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, and a heteroaryl group substituted with at least one substituent selected from the group consisting of:

[0090] (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2,

[0091] -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2,

[0092] -NHC═(O)NHNH2, -NHC═(O)NH2, -NHSO2H, -NHC═(O)H,

[0093] -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0094] (b) an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group substituted with at least one substituent selected from the group consisting of oxo, halogen, -CF3, -CN, -OH,

[0095] -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H,

[0096] -SO2NH2, -NHNH2, -ONH2, -NHC═(O)NHNH2, -NHC═(O)NH2,

[0097] -NHSO2H, -NHC═(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0098] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ in the structural arrangement or configuration of the atoms.

[0099] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another.

[0100] It will be apparent to one skilled in the art that a particular compound of the present invention may exist in tautomeric forms and all such tautomeric forms of the compounds are within the scope of the present invention.

[0101] 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.

[0102] The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present application contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present application contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids, and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzylic, salicylic, sulfanilic, toluenesulfonic, citric, tartaric, methanesulfonic, formic, and the like. Also included are salts of amino acids like arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al, "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present application contain both basic and acidic functionalities, allowing the compounds to be converted into either base or acid addition salts.

[0103] The term "salt" as used herein refers to an acid or base salt of a compound used in the methods of the present application. Illustrative examples of salts include mineral acid (hydrochloric, hydrobromic, phosphoric, etc.) salts, organic acid (acetic, propionic, glutamic, citric, etc.) salts, quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. The term salt also refers to the formation of a salt between two compounds.

[0104] The term "metabolic disorder" refers to any disorder involving an alteration of the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof. Metabolic disorders are associated with a deficiency or excess of metabolic pathways, resulting in an imbalance of nucleic acid, protein, lipid, and / or carbohydrate metabolism. Factors affecting metabolism include, but are not limited to, endocrine (hormonal) control systems (e.g., the insulin pathway, gut endocrine hormones including GLP-1, PYY, etc.), neural control systems (e.g., GLP-1 in the brain), and the like. 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.

[0105] The term "obesity" refers to an excess of body fat. Obesity can be determined by any measure accepted and utilized by those skilled in the art. At present, the accepted measure for obesity is body mass index (BMI), i.e., the 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 about 18.5 to 24.9 is considered normal, a BMI of about 25.0 to 29.9 is considered overweight, a BMI of about 30.0 or more is considered fat, and a BMI of about 40 or more is considered morbidly obese (see, for example, Gallagher et al. (2000) Am J Clin Nutr 72:694-701.). These BMI ranges are based on the impact of body weight on the risk of disease that is increased. Some common conditions relevant to high BMI and obesity include cardiovascular disease, high blood pressure (i.e., hypertension), osteoarthritis, cancer, and diabetes. Although BMI is related to body fat, the relationship between BMI and actual body fat varies with age and sex. For example, for the same BMI, women are more likely to have a higher body fat percentage than men. In addition, the BMI thresholds that distinguish normal, overweight, and obese may vary, for example, with factors such as age, sex, race, health, and body type. In some embodiments, a subject with obesity may be a subject with a body weight of at least about 25 kg / m2 prior to administration of a treatment as described herein. 2 In some embodiments, a subject with obesity may 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. 2 The body mass index of the subjects.

[0106] As used herein, the term "inflammation" or "inflamed" or "inflammatory" refers to the activation or recruitment of the immune system or immune cells (e.g., T cells, B cells, macrophages). Tissue with inflammation may become red, white, swollen, hot, painful, show loss of function, or have a membrane 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 a bacterium selected from the group consisting of Staphylococcus; Helicobacter pylori; Escherichia coli; Salmonella; Campylobacter; Yersinia enterocolitica; Shigella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus.

[0107] As used herein, the term "inflammatory disease" refers to any disease that affects the immune system. An inflammatory disease may cause at least one symptom of the disease. Symptoms may include, but are not limited to, diarrhea, vomiting, nausea, stomach upset, pain, joint swelling, malaise, fever, weight loss, weight gain, bleeding, any change in bowel movements or stool consistency or frequency, or any other symptom associated with an inflammatory disease in a subject. In some embodiments, the inflammatory disease is an autoimmune disease.

[0108] In some embodiments of any of the aspects, the inflammatory disease is selected from: an infection; Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcer, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis, or any other inflammatory disease known in the art.

[0109] As used herein, the term "gastrointestinal disease" refers to any disease affecting the gastrointestinal tract or intestinal tract. Gastrointestinal disease 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 the consistency or frequency of bowel movements or any other symptom associated with the subject's gastrointestinal disease. The non-limiting examples of gastrointestinal disease include gastrointestinal infection, 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, ulcer, infectious colitis, irritable bowel syndrome, leaky gut, pancreatitis, diabetes, hepatitis, liver disease and cancer.

[0110] As used herein, the term "liver disease" refers to any disease that affects the liver.

[0111] Liver disease can cause at least one symptom of the disease. These symptoms include, but are not limited to, bile acid imbalance, fatigue, weight loss, pain, yellowing of the skin and / or eyes, or dark urine. Examples of liver disease 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.

[0112] As used herein, the term "cancer" refers to a cell hyperproliferation that exhibits a loss of normal cellular 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 an abnormal growth of cells or tissues, e.g., of a malignant or benign type. Non-limiting examples of cancer include digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatocellular carcinoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary system cancer.

[0113] As used herein, "subject" means a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, livestock, or a game animal. Primates include, for example, chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include, for example, cattle, horses, pigs, deer, bison, buffaloes, cat species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, a subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0114] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, in which the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease. The term "treatment" includes reducing or alleviating at least one symptom or effect associated with diabetes. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, relief (whether partial or total) and / or reduction of death rates whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from a disease's symptoms or side effects (including palliative treatment).

[0115] As used herein, the term "small molecule" refers to an organic or inorganic molecule, natural (i.e., occurring in nature) or non-natural (i.e., not occurring in nature), which can include, but is not limited to, a peptide, a peptide mimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound having a molecular weight of less than about 10,000 grams / mole (e.g., including heteroorganic and organometallic compounds), an organic or inorganic compound having a molecular weight of less than about 5,000 grams / mole, an organic or inorganic compound having a molecular weight of less than about 1,000 grams / mole, an organic or inorganic compound having a molecular weight of less than about 500 grams / mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Examples of "small molecules" that occur naturally include, but are not limited to, paclitaxel, dynemicin, and rapamycin. In certain other preferred embodiments, natural product-like small molecules are utilized.

[0116] As used herein, a "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 chemical, such as a drug, can reduce dissociation of primary and secondary bile acids as provided herein.

[0117] As used herein, the term "derivative" refers to any chemical, conservative substitution, or structural modification of an agent. A derivative can improve the characteristics of an agent or small molecule, such as pharmacodynamics, pharmacokinetics, absorption, distribution, delivery, targeting to a specific receptor, or efficacy. For example, for a small molecule, a derivative can essentially consist of at least one chemical modification to about ten modifications. A derivative can also be a corresponding salt of an agent. A derivative can be a prodrug of a small molecule as provided herein.

[0118] As used herein, the term "bile acid" refers to a steroid acid that acts as an emulsifier for fats to aid digestion and may also play a role in various systemic endocrine hormone-like functions. Bile acids in mammals are synthesized as primary bile acids from cholesterol in the liver and metabolized into secondary bile acids by specific mammalian intestinal microorganisms. Bile acids are stored in the gallbladder and released into the duodenum when food is ingested, 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 of the intestinal bacteria reside. Intestinal bacteria then enzymatically modify the primary bile acids to produce a class of molecules called secondary bile acids.

[0119] Bile acids in mammals regulate metabolic pathways by activating farnesoid X receptors and G protein-coupled receptors (GPCRs) such as TGR5. By activating these various signal transduction pathways, bile acids can regulate their own enterohepatic circulation and 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 acid known in the art. The term "bile acid" or "bile salt" may further refer to the salt forms of bile acids, sulfated bile acids and other metabolites.

[0120] As used herein, "bile salt hydrolase" or "BSH" refers to an enzyme ubiquitously expressed by mammalian intestinal bacteria that converts host-produced primary bile acids into bacterially modified secondary bile acids. Figure 1AExamples of primary and secondary bile acids being dissociated by BSH and converted to secondary bile acids by bacterial bile acid modification enzymes are provided. 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 may 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 enterobacterial BSH ( Figure 1A ).

[0121] As used herein, "suitable control" refers to untreated otherwise identical cells or colonies (e.g., subjects who have not been administered an agent provided herein, or who have only been administered a subset of an agent provided herein, compared to non-control cells). As used herein, the term "pharmaceutical composition" may include any material or substance that allows the component to retain biological activity and not react with the subject's immune system when combined with an active ingredient (e.g., Compound 7 or its derivatives). 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 compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment.

[0122] As used herein, an "agent" is a chemical molecule of synthetic or biological origin. In the context of the present invention, an agent is typically a molecule that can be used in a pharmaceutical composition.

[0123] 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, which is involved in carrying or transporting the subject agent from one organ or part of the body to another organ or part of the body. The term "pharmaceutically acceptable carrier" does not include tissue culture medium. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, for example, the carrier does not reduce the effect of the agent on the treatment. In other words, the carrier is pharmaceutically inert. The terms "physiologically tolerable carrier" and "biocompatible delivery vehicle" are used interchangeably. Non-limiting examples of drug carriers include particle- or polymer-based vehicles, such as nanoparticles, microparticles, polymer microspheres, or polymer-drug conjugates.

[0124] As used herein, the term "limiting delivery of a composition to the gastrointestinal tract" refers to a formulation that allows or facilitates delivery of an agent or pharmaceutical composition described herein to the colon, large intestine, or small intestine in a live form. Enteric coatings or micro- or nanoparticle formulations may facilitate such delivery, as may, for example, buffers or other protective formulations.

[0125] The term "effective amount" can be used interchangeably with the term "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 derivatives thereof) in a pharmaceutical composition, at a dosage and for a period of time necessary to achieve the desired therapeutic outcome (e.g., "relieve," reduce, or stop at least one symptom of diabetes, obesity, or an inflammatory disease). For example, an effective amount for use in the methods disclosed herein would be considered 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, slow the progression of a symptom of a disease), or reverse a symptom of a disease in a subject suffering from diabetes, prediabetes, hyperglycemia, obesity, or an inflammatory disease. Thus, as used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a compound of Formulas (I)-(XVIII) or derivatives thereof) of 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 an amount of an agonist that exerts a beneficial effect on the symptoms of, for example, a disease (e.g., inflammatory disease, 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 shape), the extent of the symptoms, concurrent treatments, the frequency of treatment, and the desired effect. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the therapeutic agent. The effective amount in each individual case can be determined empirically by a skilled artisan according to methods established in the art without undue experimentation. In general, the phrases "therapeutically effective" and "effective for treating, preventing, or inhibiting" are intended to qualify agonists 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.

[0126] As used herein, the terms "co-administered," "co-administered," and the like are intended to encompass administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

[0127] As used herein, the term "unit dosage form" refers to a dosage suitable for a single administration. By way of example, a unit dosage form can be an amount of therapeutic agent disposed in a delivery device, such as a syringe or an intravenous drip bag. In one embodiment of any aspect, the unit dosage form is administered as a single administration. In another embodiment, more than one unit dosage form can be administered simultaneously.

[0128] The terms "administering" and "subjecting" are used interchangeably in the context of treating a disease or disorder.

[0129] In jurisdictions that prohibit patenting of methods practiced on humans, the meaning of "administering" a composition to a human subject should be limited to providing that the human subject will self-administer a controlled substance by any technique (e.g., oral, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the law or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit patenting of methods practiced on humans, "administering" a composition includes both the method practiced on humans and the aforementioned activities.

[0130] As used herein, the term "administration" refers to placing a composition into 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 can 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.

[0131] As used herein, the phrases "parenteral administration" and "parenteral administration" refer to, but are not limited to, modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection, infusion, and other injection or infusion techniques. Without limitation, oral administration can be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, mouthwashes, powders, and the like.

[0132] As used herein, the term "modulate" is intended to include effects that increase or decrease a given parameter, as those terms are defined herein.

[0133] As used herein, the term "contacting" when used with reference to a cell or organ encompasses both the introduction or administration of an agent, surface, hormone, etc., to a cell, tissue, or organ in a manner that allows the cell to physically contact the agent, surface, hormone, etc., and the introduction of an element (such as a genetic construct or vector) that allows the expression of an agent (such as a miRNA, polypeptide, or other expression product) in a cell. It should be understood that a cell genetically modified to express an agent is "contacted" with the agent, such as the progeny of a cell that expresses the agent.

[0134] The term "statistically significant" or "significant" refers to statistical significance, and generally means a difference of two standard deviations (2SD) or greater.

[0135] As used herein, the terms "comprising" or "comprises" are used to refer to compositions, methods, and their respective components that are essential to the method or composition, but are open to including unspecified elements, whether essential or not.

[0136] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention. DETAILED DESCRIPTION

[0137] Human-related bacteria play a vital role in health and disease. Microbial imbalance is relevant to a wide range of disease states. Studies in germ-free mice colonized with single strains, multiple strains, or limited bacterial communities have revealed the ability of intestinal bacteria to affect host processes (including metabolism, immune function, and neural responses). Compounds that selectively change specific bacterial metabolites and protein levels can be used to evaluate how bacterial products affect host physiology in fully developed animals with complex microbial communities, and are used as therapeutic agents for the treatment of diseases such as metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases, cancers (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).

[0138] 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.

[0139] The compounds provided herein are selective, potently 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 the host subject.

[0140] Compound

[0141] In one aspect, provided herein is a compound of formula (I):

[0142]

[0143] in:

[0144] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0145] m is 1, 2, 3 or 4;

[0146] X is an electrophilic group;

[0147] R1, R2, R3, R4, R6, R7, 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、-CO2H、-CO2R 18 , -NO2, -ONO2, -SO2Cl, -SO3 - 、-OSO3 - 、-NR 18 SO3 - 、-PO3 2- 、-OPO3 2- 、-OSO2R 18 、-SO2N(R 18 )2、-OSO2N(R 18 )2、-NR 18 SO2R 18 、-SO2N(R 18 )2, -NHNH2, -ONH2, 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;

[0148] or a pharmaceutically acceptable salt thereof.

[0149] In certain embodiments, the compound of formula (I) is of formula (I'):

[0150]

[0151] in:

[0152] n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0153] m is 1, 2, 3 or 4;

[0154] X is an electrophilic group;

[0155] R1, R2, R3, R4, R6, R7, 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、-CO2H、-CO2R 18 , -NO2, -ONO2, -SO2Cl, -SO3H, -OSO3H, -NR 18 SO3H, -PO3H2, -OPO3H2, -OSO2R 18 、-SO2N(R 18 )2、-OSO2N(R 18 )2、-NR 18 SO2R 18 、-SO2N(R 18 )2, -NHNH2, -ONH2, 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;

[0156] or a pharmaceutically acceptable salt thereof.

[0157] In certain embodiments, the compound of formula (I) is of formula (Ia):

[0158]

[0159] in:

[0160] Each R 18and R 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.

[0161] In certain embodiments, the compound of formula (Ia) is of formula (I-a'):

[0162]

[0163] in:

[0164] Each R 18 and R 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.

[0165] In certain embodiments, the compound of formula (I) is of formula (Ib):

[0166]

[0167] in:

[0168] Each R 18 and R 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.

[0169] In certain embodiments, the compound of formula (Ib) is of formula (I-b'):

[0170]

[0171] in:

[0172] Each R 18 and R 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.

[0173] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ic):

[0174]

[0175] or a pharmaceutically acceptable salt thereof.

[0176] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Id):

[0177]

[0178] or a pharmaceutically acceptable salt thereof.

[0179] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ie):

[0180]

[0181] or a pharmaceutically acceptable salt thereof.

[0182] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (If):

[0183]

[0184] or a pharmaceutically acceptable salt thereof.

[0185] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ig):

[0186] or a pharmaceutically acceptable salt thereof.

[0187] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ih):

[0188] or a pharmaceutically acceptable salt thereof.

[0189] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ii):

[0190] or a pharmaceutically acceptable salt thereof.

[0191] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-c'):

[0192] or a pharmaceutically acceptable salt thereof,

[0193] in:

[0194] R 3a 、R 7a and R 12a Independently selected from -OR 18 、-SO3R 18 、-OSO3R18 , -PO3(R 18 )2, -OPO3(R 18 )2, -OSO2R 18 , and -SO2N(R 18 )2, wherein 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 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, wherein 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 , -SO3H, and -OSO3H, wherein R 18 is H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a , R 7a , and R 12a are independently selected from -OH and -OSO3H.

[0195] In certain embodiments, the compound of Formula (I) or Formula (I’) is of Formula (I-d’):

[0196]

[0197] or a pharmaceutically acceptable salt thereof,

[0198] wherein:

[0199] R 3a and R 12a are independently selected from -OR 18 , -SO3R 18 , -OSO3R 18 , -PO3(R 18 )2, -OPO3(R 18 )2, -OSO2R 18 , and -SO2N(R 18 )2, wherein each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 12a are independently selected from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, wherein R 18is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 12a In certain embodiments, the compound of formula (I) or formula (I') is of formula (Id"):

[0200]

[0201] or a pharmaceutically acceptable salt thereof.

[0202] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-e'):

[0203]

[0204] or a pharmaceutically acceptable salt thereof,

[0205] in:

[0206] R 3a and R 7a Independently selected from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(R 18 )2, where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 7a Independently selected from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 7a In certain embodiments, R 3a for -OSO3H, and R 7a Select from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a is -OH, and R 7a Select from -OR 18, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 are independently H, or substituted or unsubstituted alkyl.

[0207] In certain embodiments, the compound of Formula (Ie) is Formula (Ie"):

[0208]

[0209] or a pharmaceutically acceptable salt thereof.

[0210] In certain embodiments, the compound of formula (Ie") is the following:

[0211]

[0212] or a pharmaceutically acceptable salt thereof.

[0213] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-f'):

[0214]

[0215] or a pharmaceutically acceptable salt thereof,

[0216] in:

[0217] R 3a Select from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(R 18 )2, where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a Select from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a In certain embodiments, R 3a It is -OH.

[0218] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (If"):

[0219]

[0220] or a pharmaceutically acceptable salt thereof.

[0221] In certain embodiments, the compound of formula (I-f') is of formula (If'"):

[0222]

[0223] or a pharmaceutically acceptable salt thereof.

[0224] In certain embodiments, the compound of formula (If'") is the following:

[0225]

[0226] or a pharmaceutically acceptable salt thereof.

[0227] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-g'):

[0228]

[0229] or a pharmaceutically acceptable salt thereof,

[0230] in:

[0231] R 3a 、R 6a and R 7a Independently selected from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(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 Independently selected from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a 、R 6a and R 7a Independently selected from -OH and -OSO3H.

[0232] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ig"):

[0233]

[0234] or a pharmaceutically acceptable salt thereof.

[0235] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-h'):

[0236]

[0237] or a pharmaceutically acceptable salt thereof,

[0238] in:

[0239] R 7a and R 12a Independently selected from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(R 18 )2, where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 7a and R 12a Independently selected from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 7a and R 12a Independently selected from -OH and -OSO3H.

[0240] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ih"):

[0241]

[0242] or a pharmaceutically acceptable salt thereof.

[0243] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (I-i'):

[0244]

[0245] or a pharmaceutically acceptable salt thereof,

[0246] in:

[0247] R3a and R 6a Independently selected from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(R 18 )2, where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 6a Independently selected from -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H and -SO2NH2, where R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a and R 6a Independently selected from -OH and -OSO3H.

[0248] In certain embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ii"):

[0249]

[0250] or a pharmaceutically acceptable salt thereof.

[0251] In certain embodiments, the compound of any one of Formula (I-c'), (I-d'), (I-e'), (I-f'), (I-g'), (I-h'), or (I-i') may contain a substituent R 3a 、R 6a 、R 7a , or R 12a In certain embodiments, R 3a 、R 6a 、R 7a and R 12a Independently selected from -OR 18 、-SO3R 18 、-OSO3R 18 、-PO3(R 18 )2、-OPO3(R 18 )2, -OSO2R 18 and -SO2N(R 18 )2, where each R 18 is independently H, or substituted or unsubstituted alkyl. In certain embodiments, R 3a 、R 6a 、R 7a, or R 12a At least one example of is independently -OR 18 In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -SO3R 18 In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -OSO3R 18 In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -PO3(R 18 )2. In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -OPO3(R 18 )2. In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -OSO2R 18 In certain embodiments, R 3a 、R 6a 、R 7a , or R 12a At least one example is independently -SO2N(R 18 )2. In certain embodiments, R 3a is -OH, and R 6a 、R 7a and R 12a In certain embodiments, R 3a for -OSO3H, and R 6a 、R 7a and R 12a Independently selected from -OH and -OSO3H.

[0252] In some embodiments of the various aspects disclosed herein, the compound of formula (I) may be a compound of any one of formulas (II)-(XV):

[0253]

[0254]

[0255]

[0256] In the compounds of formula (I)-(XV), X is an electrophilic group. The terms "electrophile" and "electrophilic" refer to functional groups that are susceptible to nucleophilic attack, i.e., readily react with introduced nucleophilic groups (e.g., thiols, amines). Generally speaking, an electrophilic group is a group of atoms, one or more of which are electron-deficient. Typically, an electrophilic group includes an electron-withdrawing group. Examples of electron-withdrawing groups include, but are not limited to, halo, nitro, cyano, ester, aldehyde, ketone, sulfone, or amide groups. One or more electron-deficient atoms are referred to as electrophilic centers, representative examples of which include carbonyl, thiocarbonyl, phosphinyl, and thiophosphinyl. Exemplary electrophilic groups include, but are not limited to, acyl halides, isothiocyanates, isocyanates, epoxy, and anhydride groups.

[0257] 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 susceptible to nucleophilic attack by the lone electron pair on the sulfur atom of the thiol group or the thiolate anion. Examples of thiol-reactive electrophilic groups include groups with good leaving groups. For example, α-halocarbonyl, isothiocyanate, isocyanate, alkyl groups with halides or alkoxy groups attached, and electron-deficient vinyl groups. In some embodiments, X is an α-halocarbonyl or isothiocyanate group.

[0258] In some embodiments of the various aspects disclosed herein, X is -C(O)R 19 、-NCS、-NHC(O)R 19 、-CH=C(CN)CO2R 20 , or -CN, where R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, and R 20 For alkyl.

[0259] In certain embodiments, X is -C(O)R 19 , where R 19 In certain embodiments, X is -C(O)R 19 , where R 19 In certain embodiments, X is -NCS. In certain embodiments, X is -NHC(O)R 19 , where R 19 is alkyl (eg, Me, Et, Pr), haloalkyl (eg, CH2F). In certain embodiments, X is -CH=C(CN)CO2R 20 , where R 20 In certain embodiments, X is -CN.

[0260] In certain embodiments, X is selected from electrophilic groups.

[0261] 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

[0262] In some embodiments, X is -C(O)R 19 or -NCS, where R 19 For example, X is -C(O)CH2F or -NCS.

[0263] In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently H. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted alkyl. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted heteroalkyl. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted cycloalkyl. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted heterocycloalkyl. In certain embodiments, R1, R2, R3, R4, R6, R7, R11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted aryl. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently substituted or unsubstituted heteroaryl. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently OR 18 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently N(R 18 )2. In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently for SR 18 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently -CO2R 18 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently -SO3 - In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently -OSO3 - In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 are independently -NR 18 SO3 - In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 Independently -PO3 2-In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -OPO3 2- In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -OSO2R 18 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -SO2N(R 18 )2 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -OSO2N(R 18 )2 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -NR 18 SO2R 18 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -SO2N(R 18 )2 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17 are independently -NHNH2 In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 , and R 17In certain embodiments, R1, R2, R3, R4, R6, R7, R 11 、R 12 、R 15 、R 16 and R 17 is independently -NHC(O)NHNH2.

[0264] In certain embodiments, R 18 is H. In certain embodiments, R 18 is a substituted or unsubstituted alkyl group. In certain embodiments, R 18 In certain embodiments, R 18 is a substituted or unsubstituted cycloalkyl group. 18 In certain embodiments, R 18 is a substituted or unsubstituted aryl group. In certain embodiments, R 18 is a substituted or unsubstituted heteroaryl group.

[0265] In the compound of formula (I), R1, R2, R4, R6, R 11 、R 15 and R 16 At least one of R1, R2, R4, R6, R 11 、R 15 and R 16 One, two, three, four, five, six, or all seven of R1, R2, R4, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R4, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15 11 、R 15 and R 16 All are H.

[0266] In some compounds of formula (I), R3, R7 and R 12 At least one of them may be -OR 18 For example, R3, R7 and R 12 One, two or all three can be -OR 18 Thus, in some embodiments of the various aspects disclosed herein, R3 is -OR 18 In some embodiments of the various aspects disclosed herein, R7 is -OR 18 In some embodiments of the various aspects disclosed herein, R 12 For-OR 18 In some embodiments, R3 and R7 are -OR 18 In some embodiments, R3 and R 12 For-OR18 In some embodiments, R7 and R 12 For-OR 18 In some embodiments, R3, R7 and R 12 All can be -OR 18 .

[0267] In some embodiments of the various aspects disclosed herein, at least one of R3 and R7 is -OR 18 , and R 12 H or OR 18 For example, at least one of R3 and R7 is -OH, and R 12 It is H or -OH.

[0268] In some additional embodiments of the various aspects disclosed herein, R3 and R7 are -OR 18 , and R 12 H or -OR 18 For example, R3 and R7 are -OH, and R 12 It is H or -OH.

[0269] In some compounds of formula (I), R3, R6, R7 and R 12 At least one of them may be -OSO3 - 、-NR 18 SO3 - , or -OPO3 2- In other embodiments of these, R3, R6, R7 and R 12 At least one of -OSO3 - In some embodiments, R3 is -OSO3 - .

[0270] Exemplary R 18 Groups include, but are not limited to, H and C1-C6 alkyl. In some embodiments of the various aspects disclosed herein, R 18 For H.

[0271] In the compound of formula (I), R 17 It can be a C1-C6 alkyl group. For example, R 17 Can be methyl, ethyl, propyl, isopropyl, butyl, pentyl. In some embodiments of the various aspects disclosed herein, R 17 It is a methyl group.

[0272] 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.

[0273] In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4.

[0274] In compounds of Formula (I), n can be 1 or 2. In some exemplary compounds of Formulas (I)-(XVIII), n is 2.

[0275] In compounds of Formula (I), m can be 1, 2, or 3. In some exemplary compounds of Formulas (I)-(XVIII), m is 1.

[0276] In some embodiments of the various aspects described herein, the compound of Formula (I) is of Formula (XVI):

[0277]

[0278] wherein R 19 is haloalkyl; R1, R2, R3, R4, R6, 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; R1, R2, R4, R6, R 11 , R 15 , R 16 is H; R3 and R7 are OH; R 12 is H or -OH; R 17 is methyl; and R 19 is haloalkyl, e.g., -CH2F. In some other exemplary compounds of Formula (XVI), m is 1 or 2; n is 1 or 2; R1, R2, R4, R6, R 11 , R 15 , R 16 is H; R3 is -OSO3 - ; R7 is OH; R 12 is H or -OH; R 17 is methyl; and R 19 is haloalkyl, e.g., -CH2F.

[0279] In some embodiments of the various aspects described herein, the compound of Formula (I) is of Formula (XVII):

[0280]

[0281] wherein R 19 is haloalkyl; R1, R2, R3, R4, R6, 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; R1, R2, R4, R6, R 11 , R 15 , R 16 is H; R3 and R7 are OH; R 12 is H or OH; R 17 is methyl; and R 19 is haloalkyl. In some other exemplary compounds of Formula (XVII), m is 1 or 2; n is 1 or 2; R1, R2, R4, R6, R 11 , R 15 , R 16 is H; R3 is -OSO3 - ; R7 is OH; R 12 is H or -OH; R 17 is methyl; and R 19 is haloalkyl, e.g., -CH2F.

[0282] In some embodiments of the various aspects described herein, the compound of Formula (I) is of Formula (XVIII):

[0283]

[0284] wherein R 19 is haloalkyl; R1, R2, R3, R4, R6, R7, 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 (XVIII), m is 1 or 2; n is 1 or 2; R1, R2, R4, R6, R 11 , R 15 , R 16 is H; R3 and R7 are OH; R 12 is H or OH; R 17 is methyl; and R 19is a halogenated alkyl group, such as -CH2F. In some other exemplary compounds of formula (XVIII), m is 1 or 2; n is 1 or 2; R1, R2, R4, R6, R 11 、R 15 、R 16 is H; R3 is -OSO3 - ; R7 is OH; R 12 is H or OH; R 17 is methyl; and R 19 is a halogenated alkyl group, such as -CH2F.

[0285] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-a):

[0286]

[0287] in:

[0288] 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

[0289] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. 18 H and R 19 is a haloalkyl group (eg -CH2F).

[0290] In certain embodiments, the compound of formula (XVIII-a) is of formula (XVIII-a'):

[0291]

[0292] in:

[0293] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. 19 is a haloalkyl group (eg -CH2F).

[0294] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-b):

[0295]

[0296] in:

[0297] R 18is 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

[0298] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. 18 H and R 19 is a haloalkyl group (eg -CH2F).

[0299] In certain embodiments, the compound of formula (XVIII-b) is of formula (XVIII-b'):

[0300]

[0301] in:

[0302] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. 19 is a haloalkyl group (eg -CH2F).

[0303] In certain embodiments, the compound of formula (XVIII) is of formula (XVIII-c):

[0304]

[0305] in:

[0306] 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

[0307] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. 18 H and R 19 is a haloalkyl group (eg -CH2F).

[0308] In certain embodiments, the compound of formula (XVIII-c) is of formula (XVIII-c'):

[0309]

[0310] in:

[0311] R 19alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH2F).

[0312] In certain embodiments, the compound of Formula (XVIII) is of Formula (XVIII-d):

[0313]

[0314] wherein:

[0315] 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

[0316] 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., -CH2F).

[0317] In certain embodiments, the compound of Formula (XVIII-d) is of Formula (XVIII-d’):

[0318]

[0319] wherein:

[0320] R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, or a pharmaceutically acceptable salt thereof. In certain embodiments, R 19 is haloalkyl (e.g., -CH2F).

[0321] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F, -NCS, -C(O)CH=CH2, -C(O)C=CH, -NHC(O)CH=CH2, -CN, -CH=C(CN)CO2Et, or -C(O)CH3; R1, R2, R4, R 16 , R 11 , R 15 , and R 16 are H; R3 is -OH or -OSO3 - ; R7 is -OH; and R 12 is H or -OH.

[0322] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F, -NCS, -C(O)CH=CH2, -C(O)C=CH, -NHC(O)CH=CH2, -CN, -CH=C(CN)CO2Et, or -C(O)CH3; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH or -OSO3H; R7 is -OH; and R 12 It is H or -OH.

[0323] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F or -NCS; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH or -OSO3 - ; R7 is -OH; and R 12 It is H or -OH.

[0324] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F or -NCS; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH or -OSO3H; R7 is -OH; and R 12 It is H or -OH.

[0325] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH or -OSO3 - ; R7 is -OH; and R 12 It is H or -OH.

[0326] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH or -OSO3H; R7 is -OH; and R 12 It is H or -OH.

[0327] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is -OH and R7 is OH; and R 12 It is H or -OH.

[0328] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is –OSO3 - ; and R7 is -OH; and R 12 For H.

[0329] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is -C(O)CH2F; R1, R2, R4, R 16 、R 11 、R 15 and R 16 is H; R3 is –OSO3H; and R7 is –OH; and R 12 For H.

[0330] In embodiments of the various aspects disclosed herein, the compound of formula (I) does not modulate the activity of TGR5. In other words, the compound of formula (I) is neither an agonist nor an antagonist of TGR5.

[0331] Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, —halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —C02R′, —CONR′ R″, –OC(O)NR′R″, –NR″C(O)R′, –NR′–C(O)NR″R′″, –NR″C(O)2R′, –NR–C(NR′R″R′″)=NR″″, –NR–C( NR′R″)=NR′″, –S(O)R′, –S(O)2R′, –S(O)2NR′R″, –NRSO2R′, –NR′NR″R′″, –ONR′R″, –NR′C=(O)NR″N R'"R"", -CN, -NO2, -NR'S02R", -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 a group. R', R', R", R'" and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aromatic hydrocarbons. When the compounds of the present invention include more than one R group, for example, when more than one of these groups is present, each R group is independently selected as each R', R", R'", and R"" group. When R' and R" are attached to the same nitrogen atom, they can be combined 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. Based on 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 groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0332] Similar to the substituents described for alkyl groups, substituents for aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR', -halogen, -SiR'R"R'", -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR"R")-NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR"R ′″, –ONR′R″, –NR′C═(O)NR″NR′″R″″, –CN, –NO2, –R′, –N3, –CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, –NR′S02R″, –NR′C═(O)R″, –NR′C(O)–OR″, –NR′OR″, in a number ranging 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 a compound of the invention includes more than one R group, for example, when more than one of these groups is present, each R group is independently selected as each R′, R″, R′″ and R″″ group.

[0333] Substituents for rings (such as cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylidene or heteroarylene) can be described as substituents on the ring rather than on the specific atom of the ring (commonly referred to as floating substituents). In this case, the substituent can be connected to any ring atom (following chemical valence rules), and in the case of condensed rings or spirocycles, the substituent (floating substituent on a single ring) associated with a member of the condensed rings or spirocycles can be a substituent (floating substituent on multiple rings) on any condensed rings or spirocycles. When the substituent is connected to the ring but non-specific atom (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 condensed rings, different spirocycles, and each substituent can be optionally different. In the case where the point of attachment of the ring to the rest of the molecule is not limited to a single atom (floating substituent), the point of attachment can be any atom of the ring, and in the case of condensed rings or spirocycles, it is any atom of any condensed ring or spirocycle, while following chemical valence rules. When a ring, condensed ring, or spirocycle contains one or more ring heteroatoms and the ring, condensed ring, or spirocycle is shown as having one or more floating substituents (including but not limited to points of attachment to the rest of the molecule), the floating substituent may 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 hydrogen) in a structure or formula with a floating substituent, when the heteroatom is bonded to a floating substituent, the substituent will be understood to replace the hydrogen while following the rules of chemical valence.

[0334] In some embodiments, the cyclic substituents are connected to the adjacent members of the basic structure. For example, two cyclic substituents connected to the adjacent members of the cyclic basic structure form a condensed ring structure. In another embodiment, the cyclic substituents are connected to the single member of the basic structure. For example, two cyclic substituents connected to the single member of the cyclic basic structure form a spirocyclic structure. In another embodiment, the cyclic substituents are connected to the non-adjacent members of the basic structure.

[0335] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a group of the formula -TC(O)-(CRR') q -U-, wherein 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 the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) rwherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally 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)2NR'-. 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.

[0336] In certain embodiments, the compound of formula (I) is as follows:

[0337]

[0338]

[0339] or pharmaceutically acceptable salts thereof.

[0340] In certain embodiments, the compound of formula (I) is as follows:

[0341]

[0342] or pharmaceutically acceptable salts thereof.

[0343] In certain embodiments, the compound of formula (I) is as follows: In certain embodiments, the compound of formula (I) is as follows: or a pharmaceutically acceptable salt thereof.

[0344] In certain embodiments, the compound of formula (I) is not:

[0345]

[0346] In certain embodiments, the compound of formula (I) is not:

[0347]

[0348] In some embodiments, each substituted group described in the compounds herein is substituted by at least one substituent. More specifically, in some embodiments, each substituted alkyl, substituted assorted alkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted sub-assorted alkyl, substituted cycloalkylene, substituted sub-heterocycloalkylene, substituted arylene and / or substituted sub-heteroaryl described in the compounds herein is substituted by at least one substituent. In other embodiments, at least one or all of these groups are substituted by at least one size-restricted substituent. In other embodiments, at least one or all of these groups are substituted by at least one low substituent.

[0349] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl group may be a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, and / or each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, and / or each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene.

[0350] In some embodiments, each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2- to 8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3-C7 cycloalkyl group, and / or each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group. In some embodiments, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C8 alkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 2- to 8-membered heteroalkylene group, each substituted or unsubstituted cycloalkylene group is a substituted or unsubstituted C3-C7 cycloalkylene group, and / or each substituted or unsubstituted heterocycloalkylene group is a substituted or unsubstituted 3- to 7-membered heterocycloalkylene group.

[0351] Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms (which can be defined in terms of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- for amino acids), and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those known in the art to be too unstable to synthesize and / or separate. 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 olefinic bonds or other centers of geometric asymmetry, unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers.

[0352] Thus, the compounds of the present invention may exist as salts, such as salts formed with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts formed with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art.

[0353] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0354] In addition to salt forms, the present invention also provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when placed in a transdermal patch reservoir together with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into the compounds of the present invention.

[0355] The specific compounds of the present invention can exist in unsolvated forms and solvated forms (including hydrated forms). Generally speaking, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. The specific compounds of the present invention can exist in multiple crystalline or amorphous forms. Generally speaking, 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.

[0356] Unless otherwise indicated, structures shown herein are also intended to include all stereochemical forms of the structure; that is, the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0357] Unless otherwise indicated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, a structure having the present invention but with the replacement of hydrogen by deuterium or tritium, or with the replacement of carbon by 13 C- or 14 C-enriched carbon-substituted compounds are within the scope of the present invention.

[0358] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with, for example, radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0359] Pharmaceutical compositions, kits, and administration

[0360] In yet another aspect, provided herein is a pharmaceutical composition comprising a compound of Formula (I)-(XVIII) and a pharmaceutically acceptable carrier or excipient.

[0361] In some embodiments of any aspect, the agents or compounds provided herein are formulated in a pharmaceutical composition. In another embodiment of any aspect, the pharmaceutical composition is formulated for the treatment of a disease (e.g., a metabolic disorder (e.g., diabetes, obesity), a gastrointestinal disease (e.g., a gastrointestinal infection; inflammatory bowel disease (IBD); appendicitis; Crohn’s disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease (e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcer; infectious colitis; irritable bowel syndrome; leaky gut syndrome; and cancer), a cancer (e.g., a digestive system cancer; a liver malignancy; a liver cancer; a colon cancer; an esophageal cancer; a stomach cancer; a hepatocellular carcinoma; a kidney cancer or renal cancer; an oral cancer; a pancreatic cancer; a prostate cancer; a rectal cancer; a stomach cancer; a basal cell carcinoma, a bile duct cancer; a lung cancer; a bladder cancer; a cervical cancer; an endometrial cancer; a uterine cancer; and a urinary system cancer), or 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, ulcer, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis), for example.

[0362] In another aspect of any embodiment, provided herein is a composition comprising an agent that inhibits a bile salt hydrolase (BSH) in a subject.

[0363] In another embodiment of any aspect, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0364] The present disclosure provides pharmaceutical compositions comprising a compound of Formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical compositions described herein comprise a compound of Formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0365] In some embodiments, the pharmaceutical composition is a liquid dosage form or a 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 compound of any one of formula (I)-(XVIII), the liquid dosage form may also include inert diluents such as those commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (particularly 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 inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and aromatics.

[0366] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, a compound of any one of Formulas (I)-(XVIII) is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glyceryl monostearate, h) absorbents, 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 comprise buffering agents.

[0367] Solid compositions of similar types also can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycol. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared into coatings and shells, such as other coatings known in the field of enteric coatings and pharmaceutical preparations. They can optionally contain an emulsifier and can also be compositions that optionally release one or more active ingredients only or preferentially in a particular part of intestinal tract in a delayed manner. The example of spendable embedded composition comprises polymeric substances and wax. Solid compositions of similar types also can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycol.

[0368] The compound of any one of formula (I)-(XVIII) can also be in the form of microencapsulation with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared to have coatings and shells, such as enteric coatings, controlled release coatings and other coatings known in the field of pharmaceutical preparations. In such solid dosage forms, the compound of any one of formula (I)-(XVIII) can be mixed with at least one inert diluent such as sucrose, lactose and starch. As in normal practice, such dosage forms can also include additional substances except 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 can also include a buffer. They can optionally contain an emulsifier and can also be a composition that optionally releases one or more active ingredients in a delayed manner only or preferentially in a specific part of the intestinal tract. The example of an embedding composition that can be used includes polymeric substances and waxes.

[0369] In some embodiments, the carrier or excipient restricts delivery of the composition to the gastrointestinal tract.In some embodiments, the compositions provided herein are restricted to the gastrointestinal tract by adding sulfate groups or polar groups to the compound.

[0370] 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 administered orally but does not degrade or activate before the device enters the intestine. Such methods can utilize coatings or capsules that degrade in a pH-dependent manner, for example, thereby allowing the delivery device and the agent to be administered or implanted to be protected throughout the gastrointestinal tract until the device reaches the alkaline pH of the intestine (e.g., cecum or colon).

[0371] An enteric coating can control where the agent is released in the digestive system. Thus, an enteric coating can be used so that the pharmaceutical composition does not dissolve and release the agent in the stomach, but rather travels to the intestine, where it dissolves and releases the agent in an environment most conducive to inhibiting BSH (e.g., targeting bacteria located in the cecum, ileum, large intestine, or colon). Enteric coatings can be stable at low pH (such as in the stomach) and can dissolve at higher pH (such as in the intestine). Materials that can be used for enteric coatings 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 entireties. The enteric coating can be an aqueous enteric coating. Examples of polymers that can be used for enteric coatings include, for example, shellac (commercially available under the trade names EmCoat 120N, Marcoat 125); cellulose acetate phthalate (commercially available under the trade names AQUACOAT TM , AQUACOAT ECD TM , SEPIFILM TM , KLUCEL TM , and METOLOSE TM ); polyvinyl acetate phthalate (commercially available under the trade name SURETERIC TM ); and methacrylic acid (commercially available under the trade names EUDRAGIT TM , EUDRAGIT L 100-55 TM , from Evonik Industries, Germany).

[0372] Another example of a method known in the art that allows a pharmaceutical composition to be restricted to the intestine includes enteric magnesium micro-motors (EMgM). EMgM are described in the art, for example, in Li et al., ACS NANO, (2016).

[0373] The pharmaceutical composition includes formulations suitable for oral administration, which can be provided as discrete units, such as tablets, capsules, cachets, syrups, elixirs, prepared food, microemulsions, solutions, suspensions, lozenges, or gelatin-coated ampoules, each containing a predetermined amount of the active compound; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or oil-in-water or water-in-oil emulsions.

[0374] Thus, formulations suitable for rectal administration may be used, including gels, creams, lotions, aqueous or oily suspensions, dispersible powders or granules, emulsions, soluble solid materials, lavages, and the like. The formulation is preferably provided as a unit dose suppository comprising the active ingredient in one or more solid carriers (e.g., cocoa butter) forming a suppository base. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycol, alcohol, and combinations thereof. Alternatively, a colon wash having the rapid recolonization deployment agent of the present disclosure may be formulated for colonic or rectal administration.

[0375] 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 (e.g., an aqueous solution) for intravenous injection. In certain embodiments, the pharmaceutical composition is a liquid (e.g., an aqueous solution) for subcutaneous injection.

[0376] After being formulated with appropriate pharmaceutically acceptable excipients at the desired dose, the pharmaceutical compositions of the present disclosure can be administered to humans and other animals orally, parenterally, intracisternal, intraperitoneally, topically, buccally, etc., depending on the disease or condition to be treated.

[0377] In certain embodiments, the pharmaceutical composition comprising the compound of formula (I)-(XVIII) is administered orally or parenterally at a dosage level sufficient to deliver each pharmaceutical composition of about 0.001mg / kg to about 200mg / kg, with one or more doses administered for one or several days (depending on the mode of administration). In certain embodiments, the effective amount of each dose is from about 0.001mg / kg to about 200mg / kg, about 0.001mg / kg to about 100mg / kg, about 0.01mg / kg to about 100mg / kg, about 0.01mg / kg to about 50mg / kg, preferably about 0.1mg / kg to about 40mg / kg, preferably about 0.5mg / kg to about 30mg / kg, about 0.01mg / kg to about 10mg / kg, about 0.1mg / kg to about 10mg / kg subject weight changes, once a day or multiple times, to obtain desired treatment and / or preventive effect. In certain embodiments, the compounds described herein can be sufficient to deliver a dosage level of about 0.001 mg / kg to about 200 mg / kg, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 40 mg / kg, preferably about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, and more preferably about 1 mg / kg to about 25 mg / kg of the subject's body weight per day, once or more per day to achieve the desired therapeutic and / or prophylactic effect. The desired dose can be delivered three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can 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 by the compositions described herein is lower than the dose at which the compound or agent causes nonspecific effects.

[0378] In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.001 mg to about 1000 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.01 mg to about 200 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.01 mg to about 100 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.01 mg to about 50 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.01 mg to about 10 mg per unit dose. In certain embodiments, the pharmaceutical composition is administered at a dosage of about 0.1 mg to about 10 mg per unit dose.

[0379] 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 or one or more other auxiliary ingredients, and then, if necessary and / or desired, forming and / or packaging the product into a desired single-dose or multi-dose unit.

[0380] The pharmaceutical composition can be prepared, packaged, and / or sold in bulk 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 containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would 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.

[0381] 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 on the route by which the composition is administered. By way of example, the composition may contain 0.1% to 100% (w / w) active ingredient.

[0382] Pharmaceutically acceptable excipients for use in the manufacture of the provided pharmaceutical compositions include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavorings, and fragrances may also be present in the compositions.

[0383] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0384] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (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.

[0385] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween 1996)), and cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose). 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80)), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethyl stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor TM ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (Brij 30)), poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, Poloxamer-188, cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0386] Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol shell mucilage, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl pyrrolidone), magnesium aluminum silicate (Veegum), and pine wood polysaccharide), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohols, and / or mixtures thereof.

[0387] 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.

[0388] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0389] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., 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, bromopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hydroxybenzoate, imidazole, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0390] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0391] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethanol.

[0392] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0393] Other preservatives include tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.

[0394] Exemplary buffers include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, 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 hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0395] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0396] Exemplary natural oils include almond, apricot, avocado, carnauba, bergamot, black currant seed, borage, juniper, chamomile, canola, coriander, carnauba wax, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grapeseed, hazelnut, hyssop, isopropyl myristate, jojoba, macadamia, lavender, saffron, cumin, thyme ... Examples of the present invention include lavender, lemon, wild pepper, macadamia nut, mallow, mango seed, white pondweed seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, Japanese tsubaki, 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.

[0397] 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 form may also contain, for example, inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly 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 inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and fragrances. In a specific embodiment for parenteral administration, the agent of the present invention is mixed with a solubilizer, such as CREMOPHOR®. (polyethoxylated castor oil), alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

[0398] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be employed are water, Ringer's solution USP and isotonic sodium chloride solution. In addition, sterile fixed oils are typically used as solvents or suspending media. For this reason, any mild fixed oil can be employed, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injections.

[0399] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0400] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium 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) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glyceryl monostearate, h) absorbents, 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 comprise buffering agents.

[0401] Solid compositions of similar types also can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycol. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared into coatings and shells, such as other coatings known in the field of enteric coatings and pharmaceutical preparations. They can optionally contain an emulsifier and can also be compositions that optionally release one or more active ingredients only or preferentially in a particular part of intestinal tract in a delayed manner. The example of spendable embedded composition comprises polymeric substances and wax. Solid compositions of similar types also can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or sugar paste and high molecular weight polyethylene glycol.

[0402] Active agent can also be the microencapsulated form with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared into coatings and shells, such as other coatings known in the field of enteric coatings, controlled release coatings and pharmaceutical preparations. In such solid dosage forms, active agent can be mixed with at least one inert diluent such as sucrose, lactose or starch. As in normal practice, such dosage forms can also include the additional substances except 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, dosage forms can also include buffers. They can optionally contain an emulsifier and can also be compositions that optionally release one or more active ingredients only in or preferentially in a specific part of the intestinal tract in a delayed manner. The example of spendable embedding composition includes polymeric substances and wax.

[0403] Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, gels, spreads, 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 with a dermatologically acceptable carrier such as a lotion, cream, ointment, or soap. Useful carriers are capable of forming a film or layer on the skin to localize application and hinder removal. For topical administration to internal tissue surfaces, the agent can be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to tissue surfaces. For example, the use of hydroxypropylcellulose or fibrinogen / thrombin solutions can be beneficial. Alternatively, tissue coating solutions such as pectin-containing formulations can be used. Ophthalmic preparations, ear drops, and eye drops are also contemplated within the scope of the present disclosure. In addition, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the agent to the body. Such dosage forms can be prepared by dissolving or dispensing the agent in an appropriate medium. Absorption enhancers can also be used to increase the flux of the agent across the skin.The rate can be controlled by either providing a rate controlling membrane or by dispersing the agent in a polymer matrix or gel.

[0404] In addition, carriers for topical formulations may be in the form of hydroalcoholic systems (e.g., liquids and gels), anhydrous oil or silicone-based systems, or emulsion systems, including but not limited to oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions. Emulsions can cover a wide range of consistencies, including thin lotions (which may also be suitable for spray or aerosol delivery), creamy lotions, light creams, heavy creams, and the like. Emulsions may also include microemulsion systems. Other suitable topical carriers include anhydrous solids and semisolids (e.g., gels and sticks); and water-based mousse systems.

[0405] The present disclosure also encompasses kits (e.g., pharmaceutical packaging). The provided kits may include or contain a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampoule, bottle, syringe, and / or dispenser packaging, or other suitable container). In some embodiments, the provided kits may also optionally include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound provided herein in the first and second containers are combined to form a unit dosage form.

[0406] Thus, in one aspect, a kit is provided, comprising a first container comprising a compound or pharmaceutical composition of Formula (I)-(XVIII) as described herein. In certain embodiments, the kit can be used to treat a disease (e.g., 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); celiac disease; diverticulitis; food intolerance; ulcers; infectious diseases) in a subject in need thereof. colitis; irritable bowel syndrome; leaky gut; and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancers), inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis). In certain embodiments, the kits are useful for preventing a disease (e.g., 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); celiac disease; diverticulitis; food intolerances; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancers), inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).In certain embodiments, the kits can be used to reduce the risk of developing a disease (e.g., metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases, cancer, inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis)) in a subject in need thereof.

[0407] In certain embodiments, kit as herein described also includes instructions for use of the kit. Kit as herein described may also include information as required by regulatory agencies such as the U.S. Food and Drug Administration (US Food and Drug Administration) (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions provide for treating diseases (such as metabolic disorders (such as diabetes, obesity), 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 kits and instructions provide for preventing a disease (e.g., 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); celiac disease; diverticulitis; food intolerances; ulcers; infectious colitis; and cancers of the digestive system (e.g., liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract 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 intolerances, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis). In certain embodiments, the kits and instructions provide for reducing the risk of developing a disease (e.g., a metabolic disorder (e.g., diabetes, obesity), a gastrointestinal disease, a cancer (e.g., liver cancer), or 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) in a subject in need thereof. The kits described herein may include one or more additional pharmaceutical compounds described herein in separate compositions.

[0408] Treatment

[0409] In one aspect, provided herein is a method of modulating bile acids in a subject. In another aspect, provided herein is a method of inhibiting dissociation of bile acids in a subject. In yet another aspect, provided herein is a method of promoting conjugation of bile acids in a subject.

[0410] In one aspect of any embodiment, provided herein is 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 Formulae I-XVIII, a derivative thereof, or a pharmaceutical composition provided herein.

[0411] 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.

[0412] In another embodiment of any aspect, the agent or inhibitor is a compound of Formulae (I)-(XVIII) or a derivative thereof; Compound 1-9 or a derivative thereof; riboflavin; or caffeic acid phenethyl ester (CAPE). Compound 1-9 is also shown in Figure 2D .

[0413] In another embodiment of any aspect, the inhibitor is selected from the group consisting of a small molecule, an antibody, a peptide, a genome editing system, an antisense oligonucleotide, shRNA, and siRNA.

[0414] 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 way of selective post-transcriptional gene silencing by molecules that bind to mRNA and inhibit mRNA processing, for example, inhibit translation of mRNA or cause degradation of mRNA. 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, independent of the mechanism of downstream processing of the RNA.

[0415] In some embodiments of any aspect, the agent 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 generally 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. Antisense oligonucleotides as described herein are complementary nucleic acid sequences designed to hybridize to a gene under cellular conditions. Therefore, oligonucleotides are selected that are sufficiently complementary to the target, i.e., hybridize well enough under the conditions of the cellular environment and have sufficient specificity to give the desired effect. For example, an antisense oligonucleotide that directly or indirectly inhibits BSH levels or activity 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 for bacterial BSH. In addition, antisense oligonucleotides can target transcription factors that regulate bacterial BSH expression.

[0416] In some embodiments, the agent 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. The antibody reagent may comprise an antibody or a polypeptide containing the antigen binding domain of an antibody. In some embodiments of any aspect, the antibody reagent may comprise a monoclonal antibody or a polypeptide containing the antigen binding domain of a monoclonal antibody. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, the 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, CDRs, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; the entirety of which is incorporated herein by reference)) as well as intact antibodies. Antibodies may have the structural features of IgA, IgG, IgE, IgD, or IgM (and subtypes and combinations thereof). Antibodies may be from any source, including mouse, rabbit, pig, rat, 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.

[0417] In other embodiments, the agent that inhibits BSH is a polypeptide. As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides", and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to, "peptide", "dipeptide", "tripeptide", "protein", "enzyme", "amino acid chain" and "contiguous amino acid sequence" are all included within the definition of "polypeptide", and the term "polypeptide" can be used to replace any of these terms or to be used interchangeably with them. 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. Conventional nomenclature for polynucleotide and polypeptide structures exists in the art. 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 disclosed herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue as long as the desired properties of the polypeptide are retained.

[0418] In another embodiment of any aspect, BSH is inhibited in the genome of a bacterial cell using any genome editing system, including but not limited to zinc finger nucleases, TALENS, meganucleases, and CRISPR / Cas systems. In some embodiments of any aspect, the genome editing system used to introduce nucleic acids 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 low amounts of Cas enzymes / proteins. It is also contemplated herein that the Cas enzymes or sgRNAs are each expressed under the control of different inducible promoters, thereby allowing for the temporary expression of each to prevent such interference. The gene editing system can directly or indirectly regulate the level or activity or expression of BSH.

[0419] In one aspect of any of the embodiments, provided herein is a method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound provided herein.

[0420] In some embodiments of any aspect, the agent is an inhibitor of bile salt hydrolase (BSH). In another embodiment of 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 of 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 of any aspect, the agent is a bile acid or a derivative thereof. In another embodiment of any aspect, the agent is chenodeoxycholic acid (CDCA) or a derivative thereof.

[0421] In some embodiments, inhibition of BSH results in a reduction 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.

[0422] In some embodiments of 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.

[0423] Imbalance 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.

[0424] In some embodiments of any of the aspects, the subject is at risk of 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).

[0425] In some embodiments of any aspect, the disease is a gastrointestinal disease. In certain embodiments, the gastrointestinal disease is a gastrointestinal infection. A gastrointestinal infection is an infection caused by bacteria selected from the group consisting of Staphylococcus, Helicobacter pylori, Escherichia coli, Salmonella, Campylobacter, Yersinia enterocolitica, Shigella, Clostridium, Bacteroides, Lactobacillus, Parabacterium, 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 a 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 a food intolerance. In certain embodiments, the gastrointestinal disease is an 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. In certain embodiments, the gastrointestinal disease is cancer.

[0426] In another embodiment of any aspect, the gastrointestinal disease is a 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.

[0427] In another embodiment of any of the aspects, the subject is at risk for or has obesity.As used herein, the term "obesity" refers to excess body fat.

[0428] In some embodiments of any aspect, the subject with obesity may be a subject having a body mass index of at least about 25 kg / m2 prior to administration of a treatment as described herein. 2In some embodiments, a subject with obesity may 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. 2 The body mass index of the subjects.

[0429] In another embodiment of any aspect, the subject is at risk of 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, ulcer, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0430] In one aspect, provided herein is a method of treating diabetes in a subject.

[0431] In some embodiments, the diabetes is type I diabetes, type II diabetes, neonatal diabetes, maturity-onset diabetes of the young, or gestational diabetes.

[0432] In some embodiments, the diabetes is caused by obesity.In one aspect, provided herein is a method of treating obesity in a subject.

[0433] 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 stomach cancer. In certain embodiments, the cancer is hepatoma. 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 stomach cancer. In certain embodiments, the cancer is basal cell carcinoma. In certain embodiments, the cancer is bile duct cancer. 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 a urinary tract cancer.

[0434] In some embodiments, the inflammatory disease is selected from the group consisting of 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 or 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.

[0435] 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.

[0436] In another embodiment of any aspect, the subject is at risk for or has cancer. The conversion of primary to secondary bile acids can lead to a reduction in tumor suppressors in the liver. It is expected that this mechanism can be extended to other types of cancer. See, for example, Ma et al. Science (2018), which is incorporated herein by reference in its entirety.

[0437] The methods and compositions provided herein can also be used to treat or prevent prediabetes in a subject. The subject may also be suffering from or at risk of developing diabetes or prediabetes. The cause of diabetes may be due to genetic mutations, inherited diabetes, obesity, lifestyle, or idiopathic conditions.

[0438] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds preferably lies within the range that includes the ED 50 The dosage may vary within this range depending upon the dosage form employed and the route of use or administration utilized.

[0439] Effective doses can be estimated initially from cell culture assays. Doses can be formulated in animals. Generally, the compositions are administered such that the compounds disclosed herein are administered at 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 5 In some embodiments, the present invention relates to an agent that is used or administered at a dose of 0 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 will be understood that the ranges given herein include all intermediate ranges, for example, 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. Doses 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 ranges intermediate to those given above are within the scope of the present disclosure, for example, within the range of 1 mg / kg to 10 mg / kg, for example, uses 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.

[0440] The compounds described herein can be administered once, or can be divided into multiple smaller doses to be administered at intervals. It should be understood that the precise dosage and duration of treatment will be a function of the position of the parenteral administration composition, the carrier, and other variables that can be determined empirically using known test protocols or by extrapolating from in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​can also vary with the age of the individual being treated. It should also be understood that for any particular subject, a specific dosing regimen may need to be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the formulation. Therefore, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulation.

[0441] In one embodiment of any aspect, the agent, compound, or composition is administered continuously (e.g., at a constant level over a period of time). Continuous administration of an agent or compound can be achieved, for example, by an epidermal patch, a continuous release formulation, or an in vivo syringe.

[0442] The compound can be administered as a single bolus or multiple boluses, continuous infusion, or a combination thereof. For example, the compound can be initially administered as a single bolus and then administered as a continuous infusion after the bolus. The infusion rate can be any desired rate. Some envisioned infusion rates include 1 μg / kg / minute to 100 mg / kg / minute or 1 μg / kg / hour to 1000 mg / kg / hour. The infusion rate can include 0.2 to 1.5 mg / kg / minute, or more specifically 0.25 to 1 mg / kg / minute, or even more specifically 0.25 to 0.5 mg / kg / minute. It should be understood that the infusion rate can be determined based on the dosage required to maintain an effective plasma concentration and the elimination rate of the compound, so that the compound is administered via infusion at a rate sufficient to safely maintain an effective plasma concentration of the compound in the bloodstream.

[0443] The dosage of an agent or compound as described herein can be determined by a physician and adjusted as needed to accommodate the observed therapeutic effect. Regarding the duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when treatment provides a therapeutic effect and to determine whether to administer additional agents, interrupt 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 patient's age, condition, and sex, and can be determined by one skilled in the art. In the event of any complications, the dosage can also be adjusted by an individual physician.

[0444] In one embodiment of any aspect, reagent as herein described, compound or composition are used as monotherapy.In another embodiment of any aspect, reagent as herein described or compound can be used in combination with other known reagents and therapies for diabetes.As used herein, " combination " administration means delivering two (or more) different treatments to the subject during the process of the subject suffering from the obstacle, for example, after the subject has been diagnosed with obstacle (such as diabetes) and before the obstacle has been cured or eliminated or treatment stops for other reasons, two or more treatments are delivered.In some embodiments, the delivery of a treatment still occurs when the delivery of the second treatment begins, so that there is overlap in terms of administration.This is sometimes referred to as "simultaneously" or "parallel delivery" herein.

[0445] In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments of either scenario, the treatment is more effective due to combined administration. For example, the second treatment is more effective, for example, 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 with the first treatment. In some embodiments, delivery results in a greater reduction in symptoms or other parameters related to the disorder than observed when one treatment is delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered. The compounds and agents described herein and at least one additional therapy may be administered simultaneously in the same or separate compositions or sequentially. For sequential administration, the agents described herein may be administered first, and the additional agents may be administered second, or the order of administration may be reversed. The agents and / or other therapeutic agents, procedures, or modalities may be administered during the active disorder period or during remission or during a less active disease period. The agents may be administered before, concurrently with, after, or during remission of the disorder.

[0446] Current therapeutic agents used to treat or prevent gastrointestinal disorders, inflammatory disorders, liver diseases, and metabolic disorders (e.g., obesity) include, but are not limited to, insulin therapy, sulfonylureas (e.g., glyburide), meglitinides (e.g., nateglinide), SGLT2 inhibitors (e.g., canaglifozin), bile acid sequestrants (e.g., cholestyramine), dopamine-2-agonists (e.g., bromocriptine), biguanides (e.g., metformin), DPP-4 inhibitors (e.g., alogliptin, linagliptin, etc.), alpha-glucosidase inhibitors (e.g., acarbose and miglitol), thiazolidinediones (e.g., rosiglitazone), antibiotics (e.g., aminosalicylic acid, norfloxacin, penicillin, cephalosporin), antivirals (e.g., zanamivir, oseltamivir), vaccines, corticosteroids (e.g., hydrocortisone, prednisone, prednisolone, budesonide), analgesics (e.g., paracetamol, ibuprofen), nonsteroidal anti-inflammatory drugs (e.g., mesalazine), anti-inflammatory drugs (e.g., sulfasalazine), immunosuppressants (e.g., infliximab, azathioprine, adalimumab, mercaptopurine), dietary supplements (e.g., iron), surgical procedures (e.g., colostomy, ileostomy, colectomy, ileocolonic resection, gastric bypass), ursodeoxycholic acid (UDCA, also known as ursodiol, INN, NAN, AAN, or USAN), cholestyramine, stanozolol, naltrexone, rifampin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinol esters, vitamin A, liver dialysis, or liver transplantation, intravenous infusions, enemas, other therapeutic agents known in the art.

[0447] 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. One skilled in the art can readily identify the chemotherapeutic agent for use (e.g., see 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 DS, Knobf MF, Durivage HJ (eds.): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

[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. One skilled in the art can readily identify the chemotherapeutic agents for use (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 DS, Knobf MF, Durivage HJ (eds.): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

[0449] When administered in combination, reagent or composition and additional reagent (such as the second or third reagent) or all with higher, lower or identical amount or dosage than the amount of each reagent used alone (for example, as a monotherapy) or dosage or dosage or administration.In certain embodiments, reagent, additional reagent (such as the second or third reagent) or all dosage or dosage or dosage than the amount of each reagent used alone or dosage low (for example at least 20%, at least 30%, at least 40% or at least 50%).In other embodiments, cause the reagent of desired effect (for example, treatment diabetes), additional reagent (such as the second or third reagent) or all amount or dosage than the amount of each reagent needed for realization same therapeutic effect or dosage low (for example, low at least 20%, at least 30%, at least 40% or at least 50%).

[0450] Drug administration

[0451] In some embodiments of any aspect, the agent is administered by direct injection, subcutaneous injection, intramuscular injection, oral administration, or nasal administration. In some embodiments, administration of an agent or pharmaceutical composition provided herein reduces glucose levels in the serum of a subject.

[0452] Exemplary modes of administration include but are not limited to injection, infusion, instillation, suction or intake." injection " includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, through trachea, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intravertebral, intracerebrospinal and intrasternal injection and infusion. In specific preferred embodiments, compositions are administered orally. In some embodiments, reagent or compositions provided herein are directly injected into the portal vein. For example, the systemic side effects of reagent or pharmaceutical composition can be limited in the portal vein. In some embodiments, compositions provided herein are implanted in the portal vein for sustained release. In some embodiments, compositions are administered via injection ports.

[0453] Because administration of parenteral dosage forms typically bypasses a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a 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.

[0454] Suitable vehicles that can be used to provide the parenteral dosage forms of the present disclosure are well known to those skilled in the art. Examples include, but are not limited to, sterile water; water for injection, USP; saline solution; dextrose solution; 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.

[0455] In some embodiments of any aspect, described herein are agents or pharmaceutical compositions that are administered to a subject in a controlled or sustained-release manner. Ideally, the use of optimally designed controlled-release preparations in medical treatment is characterized by using the least amount of drug substance to cure or control the condition in the shortest amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosing frequency; 3) increased patient compliance; 4) less total drug usage; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved therapeutic efficacy; 9) reduced loss of toxicity or drug activity; and 10) improved control speed of the 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 the compound of formula (I). In particular, controlled or extended release dosage forms or formulations can be used to ensure that maximum effectiveness of an agent is achieved while minimizing potential side effects and safety issues that may occur from underdosing the drug (i.e., below the minimum therapeutic level) as well as exceeding toxic levels of the drug.

[0456] A variety of known controlled or extended release dosage forms, formulations, and devices may be suitable for use with any of the agents 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 may be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose in varying proportions, other polymer matrices, gels, permeable membranes, osmotic systems (e.g., (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or combinations thereof to provide the desired release characteristics. In addition, ion exchange materials can be used to prepare fixed adsorbed salt forms of the disclosed compounds, thereby 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).

[0457] effect

[0458] The efficacy of reagents as described herein, for example, for treating a disease can be determined by a skilled practitioner. However, if one or more of the signs or symptoms of diabetes, obesity, gastrointestinal disease, cancer, or inflammatory disease are changed in a beneficial manner, other clinically acceptable symptoms are improved or even improved, or after treatment according to the methods described herein, an expected reaction of at least 10% is induced, then treatment is considered to be "effective treatment" as used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or morbidity of the illness treated according to the methods described herein or any other suitable measurable parameter (such as glucose levels or glucose tolerance). Efficacy can also be measured by the absence of an individual worsening, as assessed by the need for hospitalization or medical intervention (i.e., the progression of symptoms). Methods for measuring these indices are known to those skilled in the art, and / or are described herein.

[0459] Efficacy can be assessed in animal models of the conditions described herein, e.g., a mouse model or an appropriate animal model of a disease provided herein, as appropriate. When an experimental animal model is used, efficacy of the treatment is demonstrated when statistically significant changes in markers are observed, e.g., reduced blood glucose levels in a diabetes model.

[0460] It should be understood that the present disclosure is not limited to the specific methodology, protocols, reagents, etc. provided herein and may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0461] In certain embodiments, provided herein are methods 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); celiac disease; diverticulitis; food intolerances; ulcers; infectious colitis; intestinal and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancer), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).

[0462] In certain embodiments, provided herein are methods for preventing 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); celiac disease; diverticulitis; food intolerances; ulcers; infectious colitis; intestinal and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancer), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).

[0463] The present disclosure also provides compounds of Formula (I)-(XVIII) or pharmaceutically acceptable salts thereof for use in 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); celiac disease; diverticulitis; food intolerance; ulcers; Infectious colitis; irritable bowel syndrome; leaky gut; and cancers), cancers (e.g., digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancer), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).

[0464] The present disclosure also provides a compound of Formula (I)-(XVIII) or a pharmaceutically acceptable salt thereof for use in the manufacture of a pharmaceutical composition 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); celiac disease; diverticulitis; food intolerance; ulcers; In some embodiments, the present invention relates to a drug that is used to treat a variety of conditions, including but not limited to: infectious colitis; irritable bowel syndrome; leaky gut; and cancers; cancers such as digestive system cancers; liver malignancies; liver cancer; colon cancer; esophageal cancer; stomach cancer; hepatoma; kidney cancer or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, bile duct cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and urinary tract cancers; or inflammatory diseases such as Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerances, enteritis, ulcers, and gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis.

[0465] 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.

[0466] 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 or hepatitis. In certain embodiments, the inflammatory disease is appendicitis. In certain embodiments, the inflammatory disease is gastritis or 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, ulcers, or 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.

[0467] In certain embodiments, the methods of the present disclosure comprise 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.

[0468] The ad hoc methods described herein may include administering one or more additional agents in combination with a compound as described herein. 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 one or more any additional agents may be in the same administration schedule or different administration schedules. All or some doses of the compound of formula (I)-(XVIII) may be administered before all or some doses of the additional agent, after all or some doses of the additional agent, within the administration schedule of the additional agent, or a combination thereof. For different additional agents, the administration schedule of the compound of formula (I)-(XVIII) and the additional agent may be different.

[0469] Example

[0470] Assay protocol

[0471] Bacterial culture. All bacterial strains were cultured in Cullen-Haiser Gut (CHG) medium (which was derived from brain heart infusion medium (Bacto TMBHI, BD) supplemented with 1% BBL vitamin K1-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 uL / ​​L vitamin K1) 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 and 20% carbon dioxide and nitrogen. Escherichia coli was grown aerobically in LB medium supplemented with ampicillin at 37 ° C to select for the pET21b plasmid.

[0472] UPLC-MS analysis. Bile acid analysis was performed by UPLC-MS using published methods. 16 Correction factors for extraction efficiency were used and determined by extracting known concentrations of the relevant bile acid from buffer or bacterial culture medium and comparing them to a standard curve. Detection limits for individual bile acids were determined using commercial standards / synthetic compounds dissolved in 1:1 MeOH / water and were as follows: βMCA, 0.03 pmol / μL; TβMCA, 0.01 pmol / μL; CA, 0.04 pmol / μL; TCA, 0.01 pmol / μL; UDCA, 0.04 pmol / μL; TUDCA, 0.01 pmol / μL; DCA, 0.04 pmol / μL; TDCA, 0.05 pmol / μL; GCDCA-d4, 0.1 pmol / μL; CDCA-d4, 0.1 pmol / μL; 7-oxo-CA, 0.5 pmol / μL; 7, 1.0 pmol / μL; GR-7, 0.05 pmol / μL.

[0473] Protein expression and purification.

[0474] The gene encoding BT_2086 of B. thetaiotaomicron rBSH. (without a leader sequence) of E. coli was codon optimized and cloned into a pET-21b (+) vector containing a C-terminal His6 tag (primers are shown in Table 2). The expression plasmid was then transformed into BL21 (DE3) pLysS E. coli (New England Biolabs) cells under ampicillin selection. Overnight cultures grown in LB medium containing ampicillin (50 μg / mL) were diluted 1:1000 with fresh LB medium containing ampicillin and grown at 37°C. The cells were grown at an OD of 0.6-0.7 by adding 1 mM isopropyl-1-thio-D-pyranogalactoside (IPTG). 600 Induce expression and further incubate overnight at 18°C. By centrifugation at 7,000g for 20 minutes at 4°C, the cells are precipitated. The precipitated cells are then resuspended in a PBS buffer (with 5% glycerol) containing 20mM imidazole, 1mM phenylmethylsulfonyl fluoride (PMSF) and 0.25mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The resuspended cells are ultrasonically treated and precipitated by centrifugation at 16,000g for 20 minutes at 4°C. The supernatant is then mixed with preformed Ni-NTA at 4°C for 45 minutes. The nickel-bound protein is eluted with a PBS buffer (with 0.25mMTCEP and 5% glycerol) containing imidazole, the concentration of which is gradually increased. The purity of the collected fractions is tested by SDS-PAGE. The pure fractions are combined and concentrated, and then dialyzed using a storage buffer (PBS with a pH of 7.5 of 0.25mM TCEP and 5% glycerol).

[0475] For crystallization purposes, the protein was further purified on a BioRad FPLC using an S200 size exclusion column (from GE) in 50 mM Tris buffer, pH 7.5, containing 300 mM NaCl, 0.25 mM TCEP and 5% glycerol.

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

[0477] Enzyme kinetics. Characterization of enzymes using a modified BSH activity assay 26. To 144.8 μL PBS buffer (containing 10 mM TCEP and 5% glycerol) was added 35.2 μL of rBSH to obtain final concentrations of 6.2 μM and 7.0 μM for B. thetaiotaomicron BSH and B. 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 a 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. for 18 minutes in a BioRad thermal cycler. The resulting solution was cooled at 4° C. for 20 minutes, and the absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices).

[0478] Inhibitor screening was performed using rBSH. 200 nM rBSH was incubated with 100 μM inhibitor in 3 mL PBS buffer (pH 7.5) containing 0.25 mM TCEP and 5% glycerol at 37°C for 30 minutes. Bile acid pool (100 μM) was added to the above solution and incubated at 37°C. At time intervals, 1 mL of the above buffer solution was acidified to pH = 1 using 6 M HCl and extracted twice with 1 mL ethyl acetate. The combined organic layers were then 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 bile acid concentrations obtained were used to determine the % dissociation.

[0479] Formula for calculating % dissociation.

[0480] % dissociation = concentration of detected dissociated bile acid / (concentration of detected dissociated bile acid + concentration of detected conjugated bile acid)*100.

[0481] Compound 7 kinetic studies. The assay was run in PBS buffer (containing 0.25 mM TCEP and 5% glycerol), and all reactions were incubated at 37°C prior to the reaction start time. Bacteroides thetaiotaomicron BSH (200 nM) was added to a 100 μM bile acid pool and a 100 μM pool of 7. At designated time points, 500 μL aliquots were removed and flash-frozen in liquid nitrogen. After thawing, the solution was acidified to pH = 1 using 6 M HCl and then treated as described in "Inhibitor Screening Using rBSH." The procedure was repeated with 8.2 mM TUDCA.

[0482] Determination of the IC of compound 7 against recombinant protein 50 200 nM rBSH was incubated with increasing concentrations of 7 in 1 mL of PBS buffer (pH 7.5) containing 0.25 mM TCEP and 5% glycerol at 37°C for 1 hour. 100 μM bile acid (TUDCA for B. thetaiotaomicron BSH and TDCA for B. longum BSH) was added to the above solution and incubated at 37°C for 2 hours. The solution was acidified to pH = 1 using 6 M HCl and then processed as described in "Inhibitor Screening Using rBSH".

[0483] Inhibitor screening in bacteria. Bacterial cultures were incubated with 4 mL BHI containing 100 μM taurine-conjugated bile acid pool, 100 μM inhibitor + Dilute to an OD of 0.1 600 These cultures were then grown anaerobically at 37°C. After 21 hours, serial dilutions were plated in BHI + The cells were plated on agar to determine cell viability (CFU / mL). 1 mL of the entire bacterial culture was acidified to pH = 1 using 6 M HCl, and then 2 mL of ethyl acetate was added and vortexed. The culture was centrifuged at 2,500 g for 5 minutes to obtain better separation. The organic layer was then 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 concentration obtained was used to determine % dissociation.

[0484] IC of compound 7 in bacterial culture 50 Determination of the OD value. Note that due to the slow growth of Bifidobacterium longum, Bifidobacterium adolescentis was used for the bacterial growth study. Overnight cultures of Bacteroides thetaiotaomicron and B. adolescentis were diluted to an OD value of 0.1 with 2 mL of fresh CHG medium (see "Bacterial Culture"). 600, the fresh CHG medium contained 100 μM TUDCA or TDCA, respectively, and increasing concentrations of inhibitor 7. In the inhibitor screening in bacterial assays, Bacteroides thetaiotaomicron and Bifidobacterium adolescentis dissociated TUDCA and TDCA, respectively, to the greatest extent of any conjugated substrate, and therefore these substrates were used to determine IC 50 The cultures were then grown anaerobically at 37°C for 24 hours (B. adolescentis) or 48 hours (B. thetaiotaomicron). B. thetaiotaomicron required a longer incubation time because significant BSH activity was only observed during the stationary phase for this bacterium. The cultures were extracted and analyzed as described in "Inhibitor Screening in Bacteria".

[0485] Screening for inhibitors in conventional mouse feces. Quantification of BSH activity in fecal pellets using a modified version of a published method. 45 . Fecal pellets (approximately 10-20 mg) were broken into fine particles in buffer (10% PBS, 90% sodium acetate, pH 5.2) to obtain a concentration of 1 mg / mL. The indicated concentrations of inhibitors were added to the fecal slurry, and the mixture was incubated at 37°C for 30 minutes. 100 μM glycochenodeoxycholic acid-d4 (GCDCA-d4) was added to the mixture and incubated at 37°C for 18 hours. The tubes were then frozen in dry ice for 5 minutes and diluted with an equal volume of methanol after thawing. The slurry was centrifuged at 12,500g for 10 minutes. 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 are reported directly.

[0486] Crystallization, data collection, and structure determination. Crystals of BSH and BSH complexed with 7 were grown in 24-well hanging drop cultures 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, crystals of the BSH-7 complex (5.0 mg / mL) were grown in 21% PEG 3350 and 100 mM X citrate trisodium salt dihydrate pH 5.0. The crystals were cryoprotected by supplementing the mother solution with 10% 2-methyl-2,4-pentanediol (v / v). Crystals were obtained at 100 K using an ion exchanger at the Advanced Photon Source NE-CAT beamline 24ID-C. Data collection was performed at a wavelength of 100 nm. The diffraction images were processed and scaled using XDS. To obtain the phase of the apo BSH structure, 3HBC was used as the search model and the Phaser software 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 of apo BSH as a search model. Iterative modeling and refinement of the atomic B-factors of the BSH-7 group were performed using the Kh–L applied twinning rule. The model quality of both structures was evaluated using composite omission density maps. In the final cycle of model building, NCS restraints were removed. The final model quality was assessed using MolProbity. 48 For 6UFY, 97% of the residues are in the favored region of the Ramachandran plot, 3% are in the allowed region, and none are in the outlier region; for 6UH4, 89.3% of the residues are in the favored region, 10.3% are in the allowed region, and 0.4% are outliers. All crystallographic data processing, refinement, and analysis software are compiled and supported by the SBGrid Consortium. 49 Using Pymol Prepare the diagram.

[0487] Mass spectrometry analysis for identification of labeled residues on BSH. BSH protein was incubated with DMSO or a 10-fold molar excess of inhibitor 7 for 2 hours at room temperature. The reactions were then analyzed by LC-MS using a Shimadzu LC interfaced with an LTQ ion trap mass spectrometer (ThermoFisher Scientific, San Jose, CA) and autosampler system (Shimadzu, Marlborough, MA).

[0488] To determine the modification site, the protein modified by compound 7 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 a continuous cycle, including a 1MS scan (m / z 300-2000, distribution mode, electron multiplier detection), followed by an ETD MS / MS scan of the +41 charge state precursor of the protein modified by compound 7 (ETD reagent target = 200ms, image current detection at 60K resolution, target value = 2E6, ETD reaction time = 100 or 200ms). Ion assignment was performed using mzStudio software 50 .

[0489] Effects of 7 on FXR. The effects of 7 on FXR were 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 (agonism assay) or at its EC50 (50.3 nM, measured in this assay) was added (antagonism assay). After incubation for 1 hour at room temperature, the 520 / 495 TR-FRET ratio was measured using a PerkinElmer Envision fluorescence plate reader using the following filter settings: excitation 340 nm, emission 495 nm, and emission 520 nm. A 100 μsec delay followed by a 200 μsec integration time was used to collect time-resolved signals.

[0490] Cell culture. Caco-2 cells and NCI-H716 cells were obtained from the American Type Culture Collection (Manassas, VA). Caco-2 cells were maintained in Minimum 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 culture media were supplemented with 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin (GenClone). Cells were grown in "complete" medium supplemented with FBS and antibiotics at 37°C in an atmosphere of 5% CO2.

[0491] Plasmids and transient transfection. For luciferase reporter gene assays, vectors expressing human reporter gene constructs were used. pGL4.29[luc2P / CRE / Hygro] plasmids (Promega Corporation) were transiently transfected into Caco-2 cells at a concentration of 2 μg / ml of culture medium for each study of TGR5 activation. pGL4.74[hRluc / CMV] plasmids (Promega Corporation) were used as transfection efficiency controls at a concentration of 0.05 μg / ml of culture 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 transfections were performed in antibiotic-free MEM medium containing 10% FBS. After overnight incubation, 7 and / or bile acids were added to complete culture medium. 7 and / or bile acids were diluted with DMSO and the concentration of DMSO was kept constant. To investigate TGR5 antagonism, 10 μM LCA was added along with 7 and incubated overnight. The next day, cells were harvested for luciferase assay.

[0492] Luciferase reporter gene assay. Dual-Luciferase Reporter assay system (Promega Corporation) is used to measure luminescence according to the manufacturer's instructions. Cells are gently washed with PBS and cracked in the PLB deriving from the test kit. At the ICCB-Longwood Screening Facility of HMS, SpectraMax M5 plate reader (Molecular Devices, San Jose, CA) is used to measure luminescence. Luminescence is normalized to Renilla luciferase activity, and relative luminescence percentage is calculated compared with DMSO control.

[0493] Cell viability assay. Caco-2 and NCI-H716 cells were treated with the indicated compounds diluted in DMSO in complete MEM and RPMI medium, 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% CO2 atmosphere. The next day, cells were treated with 0.25% trypsin in HBSS (GenClone) at 37°C for 10 minutes. Cell viability was measured in a Countess II automated cell counter (Invitrogen). Relative viability percentages were calculated compared to the DMSO control.

[0494] Epithelial permeability assay. Undifferentiated Caco-2 cells were seeded in 24-well transwells (0.4 μM pore size, Costar) at 200,000 cells / transwell. The culture medium was 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 FITC-dextran permeability assay. Briefly, 7 and GR-7 at specified concentrations in PBS were added to the top chamber of transwells containing differentiated Caco-2 cells and incubated for 6 or 12 hours. The top chamber of the transwells contained a 100ul volume of PBS and compound or DMSO control, while the basolateral chamber contained 500uL of PBS. Caco-2 epithelial integrity was determined by measuring the passive diffusion of 4kDa FITC-dextran (SigmaAldrich) added to the top chamber at a concentration of 5uM. At the ICCB-Longwood Screening Facility at HMS, diffusion from the top side to the basolateral side was measured by fluorescence readings in PBS on the basolateral side of the transwell system using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA). Fluorescence readings were normalized to DMSO controls.

[0495] Target Validation and Off-Target Analysis of 7-N3 in Bifidobacterium adolescentis. Pilot studies of 7-N3 were performed using B. adolescentis (Gram-positive) and B. thetaiotaomicron (Gram-negative). We chose to use B. adolescentis due to the strong total fluorescence signal detected by in-gel fluorescence. B. adolescentis cultures were diluted to an OD of 0.1 with 6 mL of fresh CHG medium containing 100 μM taurine-conjugated bile acid pool. 600 . The culture was grown anaerobically at 37°C for 21 hours. 10 μM 7-N3 (10 mM stock solution in DMSO) or 6 μL DMSO (to the control tube) was then added to the culture and incubated anaerobically at 37°C for 1 hour. The culture was centrifuged at 2,500 g for 15 minutes at 4°C. The culture 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 minutes 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 90s*2, 6500 speed 60s) and centrifuged at 15,000 for 20 minutes at 4°C. The supernatant was removed and the protein concentration in the lysate was quantified by Bradford assay. The lysate was then 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 mass spectrophotometer-based quantification and identification.

[0496] Dose-dependent labeling of BSH in B. adolescentis by competition with 7 and 7-N3. B. adolescentis cultures were diluted to an OD of 0.1 with 6 mL of fresh CHG containing 100 μM taurine-conjugated bile acid pool. 600 The cultures were allowed to grow anaerobically at 37°C for 21 hours. Decreasing concentrations of 7 were added to different tubes, and the cultures were incubated anaerobically at 37°C for 1 hour. 10 μM 7-N3 was then added to the cultures and incubated anaerobically at 37°C for an additional hour. The cultures were further processed according to the methods reported in "Target Validation and Off-Target Analysis in Bifidobacterium adolescentis Using 7-N3" and "Click Chemistry for In-Gel Fluorescence Imaging."

[0497] Off-target analysis using 7-N3 in mammalian cells. The human epithelial cell line NCI-H716 was used to study the interaction with mammalian proteins. 10 μM 7-N3 (10 mM stock in DMSO) or 1 μL DMSO (for control) was added to approximately 8 × 10 6Cells were collected in 15 ml Falcon tubes and washed twice in 15 ml DBPS by centrifugation at 500 g for 5 minutes. TM The third wash was performed by centrifugation in DPBS with a protease inhibitor cocktail (Roche, Switzerland). The cells were resuspended in TM The cells were washed with 250 μl of DPBS containing 1% protease inhibitor cocktail and sonicated at 50% amplitude for 2 seconds, followed by 30 seconds on ice for 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. The lysate was then subjected to a click reaction according to the "Click Chemistry for In-Gel Fluorescence Imaging" for in-gel fluorescence and the "Click Chemistry for MS / MS of Mammalian Lysates" for quantification and identification based on mass spectrophotometry.

[0498] Click chemistry for in-gel fluorescence imaging. Click reactions were performed on a 25 μL scale. Lysates pretreated with 10 μM compound 7-N3 (normalized to 1.5 mg / mL for both bacteria and mammalian cells) were incubated with 100 μM fluor 488-alkyne (10 mM stock in DMSO), 100 μM CuBr (5 mM stock in DMSO), and 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (5 mM stock in 4:1 t-BuOH:DMSO) at 37°C in the dark for 1 hour. 10 μL of 2x Laemmli buffer (containing 5% β-mercaptoethanol) was added to the reaction, and the tubes were heated at 95°C for 10 minutes. 15 μL of protein samples were then resolved by 10% SDS-PAGE. The ladder was diluted 100-fold and 10 μL was loaded. The gel was destained with 40% methanol, 50% acetic acid, 10% water for 30 minutes and visualized using a Bio-Rad ChemiDoc MP imaging system. The gel was stained in Coomassie blue for 20 minutes and destained for 2 hours before imaging.

[0499] Click chemistry for MS / MS of bacterial lysates. Click reactions were performed at a 100 μL scale. Lysates pretreated with 10 μM 7-N3 (normalized to 1.3 mg / mL) were incubated with 100 μM desthiobiotin-PEG4-alkyne (10 mM stock in DMSO), 1 mM CuBr (50 mM stock in DMSO), and 1 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (50 mM stock in 4:1 t-BuOH:DMSO) for 1 hour at 37°C. Samples were then processed for further analysis as described in "Proteomic Analysis of Click-Tagged Proteins."

[0500] Click chemistry for MS / MS of mammalian lysates. Click reactions were performed at a 100 μL scale. Lysates pretreated with 10 μM 7-N3 (1.5 mg / mL for mammalian cells) were incubated with 100 μM desthiobiotin-PEG4-alkyne (10 mM stock in DMSO), 100 μM CuBr (5 mM stock in DMSO), and 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (5 mM stock in 4:1 t-BuOH:DMSO) for 1 hour at 37°C. Samples were then processed for further analysis as described in "Proteomic Analysis of Click-Tagged Proteins."

[0501] Proteomic analysis of click-tagged proteins. Pull-down precipitation and bead digestion of desthiobiotinylated proteins were performed similarly to the previously described protocol. 52 After resuspension of tryptic peptides in 5% acetonitrile containing 0.1% formic acid, peptides were analyzed by nanoflow LC-MS / MS as described. 53 Using multiplierz 54 The raw data were converted to .mgf and searched against forward reversed databases of human or B. 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. Normalized spectral abundance factors were obtained as described. 55 Data for proteins with more than 5 spectral counts (average of biological triplicates) were filtered for 7-N3 treated samples. In a separate experiment, hit bacterial lysate proteins were subjected to avidin enrichment and washed as described above. Proteins were then eluted with LDS loading buffer and 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 .

[0502] Animal studies. C57BL / 6 mice obtained from Jackson Laboratory were maintained under a strict 12 h / 12 ​​h light / dark cycle with constant temperature (21 ± 1°C) and humidity (55–65%). All experiments were performed on 8-9 week old male mice.

[0503] Single 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 uM: (0.00005 M) × (approximately 10 mL volume per 1 mouse gastrointestinal tract) × (1 mmol compound 7 / 408 mg) = 0.2 mg / mouse × (1 mouse / approximately 0.02 kg) = 10 mg / kg.

[0504] Mice were maintained on a standard diet (LabDiet, catalog number 5053) for the duration of the experiment. Mice were divided into two groups of four mice each and gavaged with either 200 μL of corn oil containing 5% DMSO (vehicle group) or 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.

[0505] Feeding a GR-7-containing diet for one day. Mice were fed a powdered standard chow (LabDiet, catalog number 5053) for the duration of the experiment. The mice were divided into two groups of ten mice each and maintained on a powdered diet (control group) or fed a powdered diet containing 0.09% (w / w) GR-7 (experimental group). Feces of these mice were collected at 8 hours as described above. 30 hours after receiving the powdered diet containing or not containing GR-7, the mice were euthanized using carbon dioxide. Blood samples were collected by cardiac puncture and placed in EDTA-coated tubes on ice. The liver and cecal contents were then collected from each mouse, snap-frozen in liquid nitrogen, and stored at -80°C until further analysis. The blood samples were then centrifuged at 2500g for 15 minutes at 4°C. The resulting supernatant (plasma) was collected and stored at -80°C until analysis.

[0506] BSH activity in feces. BSH activity in fecal pellets was quantified using a modified version of a published method. 45 Fecal pellets (approximately 10-20 mg) were suspended in a buffer solution (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. The samples were processed and analyzed as described in "Screening of inhibitors in conventional mouse feces". The product concentrations detected from these assays were reported directly.

[0507] Quantification of bile acids in tissues and plasma. Bile acids were extracted from tissues and plasma collected from mouse experiments using previously published methods. 16 .

[0508] Microbial biomass was determined 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.

[0509] Isolation and 16S rRNA gene sequencing analysis of fecal bacterial microbiota. TM ) Isolate mouse fecal microbial DNA. Amplify variable region 4 of the 16S rRNA gene using primers: forward 5'-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3'

[0510] Reverse 5'-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3'. According to the manufacturer's instructions, Quant-IT dsDNA high sensitivity assay (Invitrogen) quantitative PCR products were used. The success of PCR amplification was checked using gel electrophoresis. The concentration of PCR products was measured by Quan-IT dsDNA high sensitivity assay. Approximately 120ng of DNA from each PCR product was pooled together to generate an aggregation library for downstream processing. According to the manufacturer's instructions, a 300-500bp PCR DNA amplicon was selected from the aggregation library on a target size selection platform (pippin prep 1.5% agarose cassette, from Sage Sciences). The size of the DNA amplicon was characterized on an Agilent Technologies 2100 bioanalyzer trace. The DNA concentration of the aggregation library was measured by Quant-IT dsDNA high sensitivity assay. The DNA in the library was denatured by NaOH and diluted to 7.5pM with the HT buffer provided in the Illumina test kit. 600ul of denatured and diluted library containing 20% ​​spiked phiX (120ul, 7.5pM phiX) was loaded onto a MiSeq V2 reagent column (Illumina) and sequenced with paired-end 250bp reads using the custom primers described above. After running the MiSeq, demultiplexed fastq files were generated using the default parameters of the Illumina MiSeq control software and quality controlled by the Massachusetts Host-Microbiome Center pipeline. The resulting FASTQ sequences were then quality filtered and analyzed according to QIIME_mothur_DADA2. 56-59 Operational taxonomic units (OTUs) with 97% sequence similarity were selected. The phylogenetic relationships of each OTU were compared with the Greengenes reference database and 99% ID.

[0511] Quantification of bacterial 16S rDNA copy number. Bacterial DNA was isolated from mouse cecal contents using the AllPrep Bact. DNA / RNA / Protein kit (QIAGEN). 16S rDNA was then amplified using 10 μM of the following primer pair: forward 5'-AGAGTTTGATCCTGGCTCAG-3', reverse 5'-CTGCTGCCTYCCGTA-3'. Amplification was performed using the LightCycler 480 SYBR Green I Master on a QuantStudio 7Flex Real-Time PCR System according to the provided qPCR protocol. The cycle threshold value of each sample was compared to a standard curve obtained from serial dilutions of Bacteroides thetaiotaomicron genomic DNA. 60 .

[0512] Example 1. Development of broad-spectrum covalent inhibitors of enteric bacterial bile salt hydrolases

[0513] Development of broad-spectrum covalent inhibitors of enteric bacterial bile salt hydrolases

[0514] This article describes the development of broad-spectrum covalent inhibitors of enteric 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 enteric bacterial cultures, a lead inhibitor with an α-fluoromethylketone warhead was identified. Another BSH inhibitor, caffeic acid phenethyl ester (CAPE), was determined to inhibit the growth of Gram-negative enteric bacteria, but lacked the same broad-spectrum activity as the other BSH inhibitors described herein. Mass spectrometry and X-ray crystallography confirmed that the inhibitor covalently monolabeled the protein at the catalytic cysteine ​​residue. Remarkably, the lead inhibitor completely abolished BSH activity in the feces of conventional mice. Conventional mice gavaged with a single dose of the lead inhibitor showed a loss of BSH activity in the feces and a reduction in dissociated bile acids. In summary, these studies demonstrate the potential of covalent BSH inhibitors to serve as chemical tools for regulating bile acid composition in vivo.

[0515] introduction

[0516] Human-associated bacteria play crucial roles in health and disease. Microbial imbalances are associated with a wide range of disease states, including inflammatory bowel disease. 1 ,cancer 2 , autism 3 and obesity 4 However, the ways in which bacterial guests influence their human hosts at the molecular level are poorly understood. Studies in germ-free mice colonized with a single strain, multiple strains, or a defined bacterial community have revealed that gut bacteria influence host processes including metabolic 5 , immune function 6,7and neural responses 8 While germ-free mice are a useful tool, they display physiological differences compared to conventional animals, including altered food processing for energy. 9 Defects in immune cell balance, especially in the intestine 10,11 , and altered stress response behaviors 12 These differences can complicate determining whether effects observed in germ-free animals can be extrapolated to conventional animals and humans. Chemical tools that selectively alter the levels of specific bacterial metabolites and proteins may allow researchers to study how these bacterial products affect host physiology in fully developed animals with complex microbial communities. The use of small molecules as chemical tools may also provide therapeutic opportunities. Indeed, small molecule inhibitors of enteric 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 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.

[0517] Bacteria in the gastrointestinal tract are bathed in 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 in the liver from cholesterol 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 when food is ingested, 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 of the intestinal bacteria reside. Intestinal 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 have been detected in human feces. Due to the higher concentrations of bile acids released into the small intestine, the resulting concentrations of these molecules in the lower intestine are still in the low millimolar range. 17 As a result, even less abundant secondary bile acids are present in physiologically relevant concentrations.

[0518] Although bile acids were initially studied for their cleansing properties, it was later recognized that these compounds can act as signaling molecules by binding to host receptors, including nuclear hormone receptors (NhRs) and G protein-coupled receptors (GPCRs). Figure 1B Primary and secondary bile acids influence host processes by acting as agonists or antagonists at these receptors, including 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. 18-22 Specifically, by binding to host receptors, bile acids regulate host metabolism, including energy expenditure and glucose and lipid homeostasis. 18,23 , and host immune responses, including innate and adaptive immunity 24,25 Furthermore, 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.

[0519] Importantly, individual primary and secondary bile acids have different binding affinities for host receptors, suggesting that the specific composition of the bile acid pool in vivo dictates 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 the conjugated primary bile acid ( Figure 1A This reaction is carried out by intestinal bacterial bile salt hydrolase (BSH). 16 BSH (EC 3.5.1.24) is widely distributed in 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 a population indigenous to Tanzania. These results indicate that BSH activity is a conserved function of the human gut metagenome. Therefore, broad-spectrum, non-toxic, small-molecule inhibitors of gut 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 shift in the bile acid pool toward conjugated bile acids and away from dissociated bile acids and secondary bile acids ( Figure 1A ). These compounds as described herein can be used to study how secondary bile acids produced by bacteria affect physiology in fully colonized hosts.

[0520] This article describes the development of broad-spectrum covalent inhibitors of bacterial BSH, identified using a rational design approach. Importantly, the compounds described herein significantly inhibit BSH activity in normal mouse feces, demonstrating their activity as broad-spectrum inhibitors of BSH.

[0521] Experimental results

[0522] Rational design and synthesis of covalent small molecule inhibitors of bile salt hydrolase

[0523] With the goal of generating potent and durable BSH inhibitors, we developed and characterized covalent inhibitors of these enteric bacterial enzymes. Covalent inhibitors have gained widespread interest in drug discovery because they can inactivate their protein targets with high potency and selectivity, even in the presence of high concentrations of their natural substrates. 30 The substrates of BSH, namely conjugated bile acids, are present in high concentrations (1-10 mM) in the colon. 17 , suggesting that covalent inhibition could be an effective strategy for targeting these enzymes. Furthermore, recently developed bacterial cutC inhibitors are irreversible and block trimethylamine production in vivo with minimal off-target effects. 14 This work demonstrates that covalent inhibitors of bacterial enzymes can be effective in the intestine, thus further validating the methods of the present invention.

[0524] Although BSH protein sequences vary significantly among enteric strains, all BSHs share 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 targeting this highly conserved Cys residue, a broad-spectrum BSH inhibitor was developed. Structural data and biochemical information from the Gram-positive species Clostridium perfringens aided the design plan. The co-crystal structure of C. perfringens BSH and the substrate taurodeoxycholic acid (TDCA) shows that while hydrophobic interactions hold the bile acid core in place and orient the amide bond to the conserved cysteine, the amino acid is solvent-exposed ( Figure 2B ) 31 Furthermore, purified C. perfringens BSH tolerates a large degree of variability in amino acid side chains, including long-chain conjugates. 32 These results suggest that the bile acid D-ring side chain is a possible site for introducing electrophilic groups into inhibitors.

[0525] Next, a small library of potential inhibitors was designed containing a bile acid core motif that selectively targets BSH and a side-group electrophilic warhead that irreversibly binds the enzyme Figure 2C ). While previous literature suggests that BSH hydrolyzes all conjugated bile acids with amide bond cleavage regardless of the steroidal core 16,26 , it was recently determined that a species from the abundant Gram-negative phylum Bacteroidetes cleaves the C12=H rather than the C12=OH primary bile acid Figure 1A 33 Since the goal was to develop a BSH inhibitor that targets both Gram-negative and Gram-positive strains, the steroidal portion of the human primary bile acid chenodeoxycholic acid (CDCA, C12=H) was used as a scaffold for the inhibitors described herein Figure 2C

[0526] For the electrophilic trapping group, warheads that have been successfully employed in the development of selective and potent protease and kinase inhibitors were chosen 34,35 , including isothiocyanates (1) 36-38 , cyanoacrylates (2) 39,40 , a, b-unsaturated systems (3 and 4) 41 , acrylamides (5) 42 , and nitriles (6) 43,44 . Inhibitors with a-fluoromethylketone warheads (FMK) (7) were chosen for the library. Covalent inhibitors with this warhead have shown high potency and selectivity 45-47 . In contrast to the more electrophilic a-iodo, a-bromo-, and a-chloromethylketone 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 show minimal off-target effects 45,49 .

[0527] All compounds in the library were obtained from commercially available bile acid chenodeoxycholic acid (CDCA, 12) in 3-9 steps (Scheme 1). Utilizing a modified one-pot Curtius rearrangement, isothiocyanates (1) and acrylamides (5) were synthesized from CDCA in 3 steps to install the C23-substituted primary amine (Scheme S1) 50 . Synthesis of cyanoacrylates (2), a, b-unsaturated systems (3 and 4), and nitriles (6) compounds proceeded rapidly from the bis-methoxymethyl ether (MOM)-protected C24-aldehyde CDCA via Grigard addition or condensation reactions in 2-3 steps (Scheme S1). To obtain compound 7, the bis-MOM protected CDCA (13) was coupled with benzyl fluoromalonate to provide the b-keto-a-fluorobenzyl ester product 14 in 66% yield 51 . Hydrogenation followed by deprotection gave the target compound 7.​​

[0528]

[0529] Scheme 1: Synthesis of compound 7, which contains an α-fluoromethylketone warhead on a chenodeoxycholic acid core. Abbreviations: SOCl2, thionyl chloride; DIPEA, N,N-diisopropylethylamine; MOMCl, methyl chloromethyl ether; THF, tetrahydrofuran; CDI, 1,1'-carbonyldiimidazole; Pd / C, palladium on carbon; R = methoxymethyl ether.

[0530] Biochemical characterization of BSH

[0531] With regard to inhibitors 1-9 in progress, the next goal is to evaluate the biochemical activity of these compounds against both Gram-negative and Gram-positive BSH. Specifically, these compounds were tested against selective Bacteroides BSH, as the more limited substrate range of this enzyme may make it more difficult to target. To date, biochemical characterization has been primarily limited to inhibitors from Gram-positive bacteria. 16,26,52 , including Lactobacillus spp. 53 , Bifidobacterium 54 , Clostridium 31 and Enterococcus spp. 55 Among Gram-negative bacteria, only BSH from Bacteroides vulgaris and Bacteroides fragilis have been biochemically characterized, and no corresponding genes have been identified. 56,57 In addition, these strains do not have selective BSH selectivity 33 BT2086 was recently identified as the gene responsible for selective BSH activity in the intestinal bacterium Bacteroides thetaiotaomicron VPI-5482 (B. theta) 33 To test compounds against this selective BSH, the enzyme encoded by BT2086 was heterologously expressed, purified, and molecularly cloned. Because the enzyme had not been previously characterized, 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 The taurine-conjugated substrate was chosen because taurine conjugates are present in both mice and humans, whereas sugar-bile acids are essentially absent in mice. 28 Consistent with previous results with B. thetaiotaomicron cultures, purified B. thetaiotaomicron BSH showed a preference for TDCA dissociation over TCA dissociation (Table 1). 33 These results suggest that the enzyme selectivity observed in B. thetaiotaomicron whole-cell cultures is due to intrinsic biochemical properties of BSH and not to differences in transport or substrate accessibility to the enzyme.

[0532] To test the potency of inhibitors against the Gram-positive BSH, the known B. longum SBT2928 BSH was cloned and expressed 54 , and the same set of taurine-conjugated bile acids substrates was used to determine the kinetic parameters of the enzyme (Table 1). It is worth noting that the K m values of all the recognized substrates were in the low millimolar range, which approximates the concentration of these bile acids in the intestine. The K m values established here for B. longum are higher than those previously reported 54 . This difference can be a result of the conditions under which the assays were performed, i.e. the physiological pH (7.5) in the present work versus the pH (6) optimized for activity in previous studies. In summary, both enzymes showed kinetic parameters comparable to those of previously characterized BSHs 53,54,56 .

[0533] Table 1: Kinetic characterization of the Gram-negative B. theta BSH and the Gram-positive B. longum BSH.

[0534]

[0535] a Characterization was performed using the ninhydrin reagent, and the experiments were performed 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), tauro-ursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA).

[0536] Biochemical evaluation identified a-FMK compound 7 as a lead inhibitor

[0537] Next, the ability of the compounds in the library to inhibit BSHs from B. theta and B. longum was evaluated. Two additional compounds were included in the assay, i.e. riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11) Figure 2E . These molecules have been previously identified as BSH inhibitors by high-throughput screening against BSH from a chicken intestinal isolate of Lactobacillus salivarius 59 . To determine the BSH inhibitory activity of these compounds, B. theta BSH was incubated with each inhibitor (100 μΜ) for 30 min, followed by the addition of equimolar amounts of the four conjugated bile acids (TβMCA, TCA, TUDCA, and TDCA, total 100 μΜ).

[0538] The conversion of conjugated to dissociated bile acid was monitored by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) over a total of 21 hours ( FIG3 ). Among the synthesized inhibitors, isothiocyanate ( 1 ) showed moderate inhibition during the experiment. Other compounds containing the Michael receptor warhead ( inhibitors 2–6 ) did not inhibit dissociation ( Figure 3A In contrast, incubation with the α-fluoromethylketone-based inhibitor 7 resulted in almost complete inhibition of B. thetaiotaomicron BSH activity by 21 h (>98%, Figure 3A To verify that the inhibitory activity of compound 7 is due to the presence of fluorine as a leaving group, a methyl ketone analogue lacking a fluorine atom (8) was synthesized. 49 This analog did not show BSH inhibition, indicating that the α-fluorine group is required for activity. Riboflavin, a previously identified BSH inhibitor, did not show any inhibitory activity, while CAPE provided only moderate inhibition of B. thetaiotaomicron BSH.

[0539] Next, the activity of the two most potent inhibitors against B. thetaiotaomicron BSH was evaluated. Compounds 1 and 7, as well as CAPE ( Figure 3B ). These compounds showed the same specific effectiveness against B. longum BSH as observed for B. 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 at inhibiting the dissociation of B. 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, because the activity of CAPE and riboflavin against genera other than Lactobacillus was not determined, the activity of CAPE and riboflavin against genera other than Lactobacillus was not determined. 59 , these results suggest that these molecules may not be effective broad-spectrum inhibitors.

[0540] Compound 7 inhibits BSH activity in growth cultures of enteric bacteria

[0541] Given that compound 7 showed activity against purified BSH from Bacteroides thetaiotaomicron and Bifidobacterium longum, the potency of this inhibitor in growing bacterial cultures was evaluated. To test the scope 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 vulgaris ATCC 8482; Gram-positive: Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32). 16,33 .

[0542] Bacterial cultures were diluted in the early logarithmic growth phase and a mixture of inhibitors (100 μM) and conjugated bile acids (100 μM final concentration; TCA, TβMCA, TDCA, and TUDCA) were added simultaneously. Dissociation was monitored over 24 hours using UPLC-MS. Remarkably, while all six bacterial strains dissociated bile acids in the presence of vehicle controls, almost no detectable dissociation was observed in any growing culture in the presence of compound 7. These results indicate that compound 7 exhibits potent BSH inhibition against both Gram-negative and Gram-positive bacteria ( Figure 4A Compound 7 did not significantly affect the growth of any tested strains ( Figure 4B ), indicating that the observed BSH inhibition was not due to antibacterial activity. To quantify the potency of compound 7, the IC values ​​of this inhibitor against the Gram-negative strain Bacteroides thetaiotaomicron and the Gram-positive strain Bifidobacterium adolescentis were calculated. 50 The values ​​were determined to be 913 nM and 227 nM ( Figure 4C ). Taken together, these results indicate that compound 7 is a potent broad-spectrum inhibitor of BSH.

[0543] In contrast, no inhibition of dissociation was observed over the course of 21 h for five of the six bacterial strains grown in the presence of CAPE (100 μM). Figure 4A CAPE was found to inhibit dissociation in L. plantarum, a result consistent with the hypothesis that this compound inhibits lactobacilli BSH but is not a broad-spectrum BSH inhibitor. Furthermore, in contrast to inhibitor 7, CAPE inhibited the growth of all three tested Gram-negative strains ( Figure 4B These results indicate that the main effect of CAPE on Gram-negative bacteria is not inhibition of BSH activity but inhibition of growth.

[0544] To evaluate the hypothesis that the C12=OH compounds were not potent, broad-spectrum inhibitors because they did not inhibit B. thetaiotaomicron BSH activity, inhibitory bile acids were synthesized in which the α-fluoromethylketone warhead from the most potent inhibitor, compound 7, was appended to a C12=OH bile acid core (compounds 9, Figure 2D Next, growing cultures of B. thetaiotaomicron were incubated with compound 9 (1 μM or 10 μM) and a conjugated bile acid substrate (GUDCA, 100 μM), and dissociation was monitored using UPLC-MS. While incubation with 10 μM of compound 7 resulted in almost complete inhibition of dissociation, significant dissociation was observed in the presence of the same concentration of compound 9 ( Figure 4DThese results support the hypothesis that the bile acid core structure, particularly the C12 substitution, influences the probe's ability to act as a broad-spectrum inhibitor. Furthermore, these results suggest that the α-fluoromethylketone warhead is not broadly reactive but rather requires proper positioning within the active site, which can be further tested using mass spectrometry and crystallography studies.

[0545] Compound 7 covalently binds to the catalytic cysteine ​​residue of BSH

[0546] With the efficacy of compound 7 established, the mechanism of its inhibition was investigated. In order to confirm that compound 7 is a covalent inhibitor and that it modifies Cys2 (the catalytic cysteine ​​residue), mass spectrometry experiments were performed. B. thetaiotaomicron BSH contains two cysteine ​​residues, Cys2 and Cys67. Analysis of the apo crystal structure of the enzyme revealed that both cysteine ​​residues point toward the active site, indicating that either residue could be a potential binding site for compound 7 (PDB 3HBC). It was found that further incubation of B. thetaiotaomicron BSH with compound 7 resulted in a full mass shift of the protein by 388 mass units. This mass shift is consistent with the addition of a single equivalent of inhibitor to the protein ( Figure 5A Although no labeled peptides were identified by trypsin or Lys-C digestion, a top-down approach revealed Cys2 as a modified residue, as indicated by the c3 ion ( Figure 5B ).

[0547] To understand the spatial arrangement of the inhibitor in the binding pocket and guide further inhibitor design, the cocrystal structure of B. thetaiotaomicron BSH covalently bound to compound 7 was constructed using The cocrystal structure was determined at 1.5 Å resolution. Consistent with the mass spectrometry data, the cocrystal structure revealed that Cys2 is bound to the C25-methylene group of the bile acid structure, with the fluorine atom eliminated. Together, these data indicate that compound 7 selectively labels B. thetaiotaomicron BSH at the nucleophilic cysteine ​​residue in the protein's active site. Furthermore, the cocrystal structure reveals that the C3-hydroxyl group is solvent-exposed, suggesting that this site is amenable to further modification.

[0548] Compound 7 showed minimal off-target effects

[0549] Although covalent inhibitors have been shown to be highly effective, concerns have been raised about the nonspecific reactivity of these compounds, which may lead to 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 to be ligands for host nuclear hormone receptors (NhRs) and G protein-coupled receptors (GPCRs). 18 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 could act as a ligand for FXR, an in vitro coactivator recruitment assay was performed ( Figure 6A ) 28 The assay measures the ability of compounds to enhance the binding of recombinant FXR ligand binding domain (LBD) to a coactivator peptide (SRC2-2) as measured by an increase in the time-resolved fluorescence resonance energy transfer (TR-FRET) signal. While the known FXR agonist GW4064 showed a clear dose-dependent increase in SRC2-2 binding to FXR (EC 50 =50 nM), but the binding of SRC2-2 to FXR was not increased in the presence of compound 7, indicating that this inhibitor does not activate FXR. 50 Compound 7 did not show a dose-dependent curve in the presence of 10 μM concentrations, 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 did not agonize TGR5 within the concentration range tested. In addition, in the presence of the known TGR5 agonist LCA (10 μM), compound 7 did not antagonize TGR5 ( Figure 6B These results suggest that inhibitor 7 does not induce off-target effects via binding to any of these key host receptors.

[0550] In addition to their effects on host receptors, bile acids are known to be toxic to cells due to their cleaning properties. 16,60 Because the expected in vivo region of inhibitor 7 is the lower intestine, the toxicity of this compound was tested against human intestinal cells (Caco-2). No resulting toxicity was observed when these cells were incubated with up to 50 μM of compound 7 ( Figure 6C ). Because the IC of compound 7 against bacterial BSH 50 With values ​​ranging from 227 nM to 913 nM, these results suggest that effective in vivo dosing should be achievable at concentrations that do not cause toxicity to intestinal cells. Overall, these results demonstrate that inhibitor 7 is non-toxic and selective for bacterial BSH relative to potential host targets.

[0551] Compound 7 inhibits BSH activity in normal mouse feces

[0552] While the results demonstrate the effectiveness of inhibitor 7 against growing cultures of six different strains of gut bacteria, there are hundreds of bacterial species in the human gut. 61 Previous literature reported significant BSH activity in mouse feces. 62To further strengthen the finding that compound 7 is a broad-spectrum BSH inhibitor, the activity of compound 7 was tested in resuspended feces from conventional (i.e., fully colonized) mice. Compounds 1, 7, and CAPE (20 μM) were added to the fecal suspension 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 resulted in reduced dissociation, incubation with compound 7 completely inhibited BSH activity in feces ( Figure 7B Consistent with the in vitro results, CAPE did not inhibit BSH in normal mouse feces. These results further demonstrate that the lead inhibitor compound 7 is a potent broad-spectrum inhibitor of enteric bacterial BSH activity.

[0553] A single dose of compound 7 inhibited BSH activity in conventional mice

[0554] Having established the in vitro efficacy of compound 7, the activity of this inhibitor was evaluated in conventional mice. C57B1 / 6 mice were gavaged with a dose of compound 7 (10 mg / kg) or vehicle control, and BSH activity was monitored in half-day increments until 2.5 days after gavage ( Figure 7C While not wishing to be bound by a particular theory, it is expected that if compound 7 is active in vivo, an initial decrease in BSH activity will be observed, followed by a recovery of BSH activity. This expected effect was observed.

[0555] A significant decrease in fecal BSH activity was noted at 1 and 1.5 days after gavage, whereas recovery of activity was observed at subsequent time points (2 and 2.5 days after gavage). Figure 7D ). Based on the initial assumption ( Figure 1A ), and without being bound by a particular theory, it is expected that changes in the bile acid pool should be observed following BSH inhibition. A significant decrease in conjugated bile acids and an increase in dissociated bile acids were observed 1 day after gavage. Notably, a decrease in the dissociated secondary bile acid deoxycholic acid (DCA) was observed at this time point ( Figure 7E ).

[0556] Bacterial culture results indicated that compound 7 did not significantly inhibit bacterial growth. Consistent with this result, no significant reduction in bacterial biomass was observed at any time point after the initial gavage ( Figure 7F ). Taken together, these results indicate that compound 7 inhibits gut bacterial BSH activity in vivo in the mouse gastrointestinal tract without significantly inhibiting the overall growth of the gut bacterial community.

[0557] A derivative of compound 7, 3-sulfated-lithocholic acid-fluoromethyl ketone (3S-LCA-FMK), was generated to restrict the delivery of BSH inhibitors to the intestine ( Figure 8A Male conventional C57Bl / 6 mice were fed a normal diet or a diet containing 3S-LCA-FMK (0.03% w / w) ad libitum for 7 days. Feces were collected before and on days 3, 4, and 7 after the dietary change. n = 5 mice per group ( Figure 8B ). It was found that BSH activity in the feces of mice fed a diet containing 3S-LCA-FMK was significantly reduced, and on day 4, no 3S-LCA-FMK was detected in the circulating plasma ( Figures 8C-8D ). In summary, these results demonstrate that the 3S-LCA-FMK compound is gut-restricted and maintains inhibition of bile acid dissociation in an animal model. 3S-LCA-FMK also showed that it reduced food intake in conventional mice compared to mice given vehicle (n=8 mice per group). Mice given 3S-LCA-FMK showed suppressed BSH activity and a significant reduction in food consumption ( Figure 31 ).

[0558] Summarize

[0559] This article describes the development of a chemical tool: a potent, selective, broad-spectrum inhibitor of enteric bacterial BSH. We identified a lead inhibitor, Compound 7, that potently inhibited the dissociation of purified BSH protein, growing cultures of BSH-containing Gram-negative and Gram-positive human enteric strains, and resuspended conventional mouse feces. We also demonstrated that a single dose of Compound 7 administered to conventional mice reduced BSH activity and predictably shifted the bile acid pool in vivo. Importantly, Compound 7 did not significantly affect the growth of these bacteria.

[0560] 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.

[0561] A 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 The introduction of exogenous bacterial strains that overexpress proteins from different bacterial sources into the gut is a significant perturbation to the natural ecosystem, however, complicating the interpretation of how BSH functions in natural systems. Another study found that treatment of conventional mice with the antioxidant compound TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) resulted in reduced BSH activity and weight gain in Lactobacillus species.62 However, TEMPOL has not been shown to act directly as a BSH inhibitor, and it may exert its metabolic effects via BSH-independent mechanisms.

[0562] Furthermore, in recent work, it was shown that deletion of the BSH-encoding gene from the Gram-negative gut commensal strain Bacteroides thetaiotaomicron resulted in reduced weight gain, lower liver and blood lipid levels, and a decreased respiratory exchange ratio in mice colonized with this bacterium compared to wild-type strains of Bacteroides thetaiotaomicron. 33 However, these experiments were performed in monocolonized germ-free mice and did not reveal how the restricted activity of all BSHs affects metabolism in conventional animals. Without being bound by a particular theory, it is hypothesized that the reduced weight gain phenotype in B. thetaiotaomicron BSH knockout (KO)-colonized mice is due to reduced food intake. Administration of chemical inhibitors such as compound 7 to mice in metabolic cages can determine the origin of the metabolic effects of inhibiting individual BSHs in monocolonized mice as well as all BSHs in conventional mice.

[0563] In addition to facilitating the study of bile acid effects on host metabolism, selective BSH inhibitors will also enable the investigation of how primary and secondary bile acids influence host immune responses, particularly in the context of liver cancer. Recent studies have proposed a causal relationship between bacterial bile acid metabolism (particularly the conversion of primary to secondary bile acids) and a reduction in the tumor suppressive environment in the liver. 64 By feeding mice bile acids, treating them with antibiotics, and colonizing them with bile acid-metabolizing bacteria, the researchers provide support for a model in which secondary bile acids reverse the beneficial NKT cell accumulation and liver tumor growth inhibition promoted by primary bile acids. The use of BSH inhibitors in a mouse model of liver cancer could further test this hypothesis by converting the endogenous body bile acid pool to primary bile acids without significantly perturbing the enterohepatic system and microbial community. If such conversion of the bile acid pool limits liver tumor growth, bacterial BSH inhibitors could be developed as novel cancer therapeutics.

[0564] Finally, in the development of BSH inhibitors, two molecules previously identified by high-throughput screening as inhibitors of BSH from a chicken gut isolate of Lactobacillus salivarius, riboflavin and CAPE, were also evaluated. 59. In contrast to compound 7, riboflavin and CAPE showed no significant inhibitory activity against any Gram-negative strain and only one of the three Gram-positive enteric bacterial strains (which were also from the genus Lactobacillus). In addition, while compound 7 (20 μM) almost completely inhibited BSH activity in resuspended mouse feces, CAPE at concentrations of 20 μM or 100 μM did not significantly reduce dissociation in this assay. CAPE significantly inhibited the growth of the tested Gram-negative enteric bacterial strains. The use of CAPE to inhibit BSH in mice has been reported, and this has led to the investigation of how the shift to a more FXR-antagonistic bile acid pool affects host metabolism, particularly hepatic gluconeogenesis. 65 In light of these results, and especially the finding that CAPE has antibiotic properties, the conclusions from previous in vivo results obtained with CAPE should be reexamined 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 effects on host physiology.

[0565] 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 their natural substrates. 11 The substrates of BSH, namely conjugated bile acids, are present in high concentrations in the colon (1-10 mM). 4 Furthermore, recent work has demonstrated that irreversible inhibitors of bacterial enzymes can be effective in the gut. 12 .

[0566] Although BSH protein sequences vary significantly among enteric strains, all BSHs share a conserved active site consisting of the catalytic cysteine ​​(Cys2) ( Figure 9 b) 1,10 Therefore, compounds targeting this conserved residue may be effective pan-BSH inhibitors. The co-crystal structure of C. perfringens BSH and the substrate taurodeoxycholic acid shows that hydrophobic interactions engage the bile acid core and orient the amide toward Cys2, exposing the amino acid to the solvent ( Figure 9 c) 13 Furthermore, C. perfringens BSH tolerates a large degree of variability in amino acid side chains, including long-chain conjugates. 14 .

[0567] A small library of potential inhibitors containing both a bile acid core motif and a side-group electrophilic warhead was developed ( Figure 9 d) Without wishing to be bound by any particular theory, previous literature indicates that amino acid identity of the conjugate may largely drive BSH specificity. 1 , and the core configuration of sterols also affects BSH reactivity 15In addition, some Bacteroidetes species cleave C12=H but not C12=OH primary bile acids ( Figure 9 a) 16 .

[0568] Selection of several electrophilic capture groups 17 , including isothiocyanates (1) 18 、Cyanoacrylate(2) 19 、α,β-unsaturated system (3 and 4) 20 、Acrylamide(5) 21 and nitrile (6) 22 Inhibitors with an α-fluoromethylketone warhead (FMK) were also synthesized (7). The weak leaving group ability of fluorine makes the FMK warhead less reactive and therefore more selective, as opposed to the more electrophilic α-iodo-, α-bromo-, and α-chloromethylketone warheads. 23,24 FMK-based inhibitors shown to cause minimal off-target effects 23,25 .

[0569] Example 2. Biochemical Characterization of BSH

[0570] Then, the activity of inhibitors 1-9 against Gram-negative and Gram-positive BSH was evaluated using a selective Bacteroides BSH for inhibitor optimization. Therefore, a selective BSH (BT_2086) was heterologously expressed and purified (Table 2 and Figure 14 ) 16 .

[0571] Table 2: Primers used for BSH gene amplification.

[0572]

[0573] Determination of kinetic parameters using a ninhydrin-based assay 26 Purified B. thetaiotaomicron BSH showed a preference for tauro-ursodeoxycholic acid (TUDCA) dissociation over tauro-cholic acid (TCA) dissociation (Table 3 and Figure 14 ) 16 .

[0574] Table 3: Kinetic parameters of BSH of B. theta and Bifidobacterium longum.

[0575]

[0576] a Characterization was performed using ninhydrin reagent, and experiments were performed in PBS buffer at pH 7.5 and 37°C. bThe conjugated primary and secondary bile acids used as substrates were taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA). c Bacteroides thetaiotaomicron does not break down TCA. n = 3 replicates per condition. All data are presented as mean ± SEM.

[0577] We also cloned and expressed the BSH from the Gram-positive strain Bifidobacterium longum SBT2928 27 The kinetic parameters of BSH were determined (Table 3 and Figure 14 ). K of all recognized substrates m The values ​​are in the low millimolar range, which approximate the concentrations of these bile acids in the intestine. cat The value is lower than the k reported for Lactobacillus salivarius BSH cat , but the K m Values ​​are similar to those of previously characterized BSH 27-29 .

[0578] Example 3. α-FMK compound 7 as a lead inhibitor inhibits recombinant BSH

[0579] The compounds in our library were also evaluated for their ability to inhibit BSH of Bacteroides thetaiotaomicron and Bifidobacterium longum. Riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11), compounds previously identified in high-throughput screening to inhibit BSH of Lactobacillus salivarius chicken intestine isolates ( Figure 14 ) 30 BSH inhibitory activity was determined by preincubating B. thetaiotaomicron BSH with each inhibitor (100 μM) for 30 minutes before adding a mixture of conjugated bile acids (100 μM final concentration). Because BSH shows different reactivity to different conjugated bile acids, an equimolar combination of two primary and two secondary conjugated bile acids that predominate 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 . The dissociation of bile acids was monitored by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) over 21 h. Among the synthesized inhibitors, isothiocyanate (1) showed moderate inhibition. Other compounds containing Michael receptor warheads (2-6) did not inhibit dissociation. In contrast, incubation with α-FMK-based 7 resulted in almost complete inhibition of B. thetaiotaomicron BSH activity by 21 h (>98%, Figure 10 a, 15, 16 and Table 4).

[0580] Table 4: % dissociation of each bile acid determined in experiments using a pool of four tauro-conjugated bile acids.

[0581]

[0582] Table 4: (continued)

[0583]

[0584] 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 analog showed no BSH inhibition against recombinant protein or B. thetaiotaomicron culture, indicating that the α-fluoro group is required for activity ( Figure 10 a. Figure 17 and Table 4). Riboflavin did not show any inhibitory activity, while CAPE provided only moderate inhibition against B. thetaiotaomicron BSH.

[0585] Compounds 1, 7, and CAPE were also evaluated for their activity against BSH from B. longum. Compound 7 was again the most active inhibitor at all time points, while CAPE was ineffective in inhibiting B. longum BSH ( Figure 10 b, 15-16 and Table 3). Compound 7 inhibited BSH of Bacteroides thetaiotaomicron and Bifidobacterium longum in a dose-dependent manner (IC 50 The values ​​were 427nM and 108nM, Figure 18 ). Taken together, these data indicate that compound 7 is a potent inhibitor of purified BSH proteins from both Gram-negative and Gram-positive bacterial strains.

[0586] At equimolar concentrations to the substrate and without any preincubation of the inhibitor with the enzyme, compound 7 completely inhibited the B. thetaiotaomicron BSH, the more catalytically efficient of the two enzymes (Table 2) within 15 seconds ( Figure 19 ). In the presence of a large excess (approximately 80-fold) of substrate, 7 completely inhibited B. thetaiotaomicron BSH activity within 15 minutes (the earliest measurable time point of product formation under these conditions). These results indicate that 7 is a kinetically effective inhibitor of BSH activity.

[0587] Example 4. Compound 7 inhibits BSH in intestinal bacterial cultures

[0588] The efficacy of 7 in growing bacterial cultures was also evaluated. To test the extent 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 vulgaris ATCC 8482; Gram-positive: Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32). 1,16 .

[0589] Bacterial cultures were diluted in the early logarithmic growth phase and a mixture of inhibitor (100 μM) and conjugated bile acids (100 μM final concentration) were added simultaneously. Dissociation was monitored over 21 hours using UPLC-MS. Remarkably, while all six bacterial strains dissociated bile acids in the presence of vehicle controls, almost no dissociation was observed in any of the cultures growing in the presence of 7 ( Figure 10 c, 20 and Table 3). Then the isogenic BSH-deficient Bacteroides thetaiotaomicron strain 16 Incubated with DMSO, 7 or CAPE. Under all three conditions, unmetabolized taurine-conjugated bile acid ( Figure 21 These results suggest that the inhibition of BSH by 7 is not due to the effect of this inhibitor on other bile acid utilization processes. Compound 7 did not significantly affect the cell viability of most tested strains ( Figure 10 d), indicating that the observed BSH inhibition was not due to bactericidal activity. The IC values ​​of this inhibitor against Bacteroides thetaiotaomicron and B. adolescentis were 50 The values ​​were determined to be 1070 nM and 237 nM ( Figure 22 ). These results indicate that 7 is a potent broad-spectrum inhibitor of BSH.

[0590] No BSH inhibition was observed in five of the six bacterial strains grown in the presence of CAPE ( Figure 10 c). In addition, CAPE inhibited the cell viability of all three Gram-negative bacterial strains tested ( Figure 10 d). These results indicate that the main effect of CAPE on Gram-negative bacteria is not inhibition of BSH activity but inhibition of growth.

[0591] Finally, to evaluate whether the C12=OH compound was not an effective broad-spectrum inhibitor, a compound containing a C12=OH bile acid core with an α-FMK warhead attached to it was synthesized (compound 9, Figure 9 d). Compound 9 showed a significantly reduced ability to inhibit BSH dissociation in B. thetaiotaomicron culture compared to 7 ( Figure 17 ). Thus, the bile acid core structure, particularly the C12 substitution, affects the ability of our probe to selectively inhibit BSH. Furthermore, these results show that the α-FMK warhead is not broadly reactive but requires proper positioning within the active site.

[0592] Example 5. Compound 7 inhibits BSH activity in mouse feces

[0593] Previous literature reported significant BSH activity in mouse feces 32To further evaluate whether 7 is a pan-inhibitor of BSH, its activity was tested in resuspended feces from conventional mice. This fecal slurry should contain BSH from almost the entire bacterial community of 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 dissociation products was quantified after 18 hours using UPLC-MS. Remarkably, incubation with 7 completely inhibited BSH activity in feces ( Figure 10 e). CAPE did not inhibit BSH activity in feces. These results demonstrate that 7 is a potent pan-inhibitor of BSH activity.

[0594] Example 6. Compound 7 covalently modifies the catalytic Cys2 residue.

[0595] The inhibitory mechanism of 7 was also investigated. Bacteroides thetaiotaomicron BSH, which contains two cysteine ​​residues, Cys2 and Cys67, was also investigated. Analysis of the apo crystal structure of the enzyme revealed that both cysteine ​​residues point toward the active site (PDB 3HBC). To confirm that 7 is a covalent inhibitor of Cys2 modification, purified Bacteroides thetaiotaomicron BSH was incubated with an excess of this molecule. Mass spectrometry 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 analysis identified Cys2 as the modified residue ( Figure 23 ).

[0596] The structure of B. thetaiotaomicron BSH was first investigated in its apo form. resolution, and then covalently bound to 7 resolution (Table 5) (PDB 6UFY and 6UH4, respectively).

[0597] Table 5: Data collection and refinement statistics (molecular replacement)

[0598]

[0599] *The highest resolution shell is shown in parentheses. Each data set was collected using a single crystal.

[0600] The structure of the BSH-inhibitor complex contains four copies of the protein in the asymmetric unit. The electron density map is best resolved in two of the four subunits, and in one of these subunits, the electron density for the inhibitor covalently linked to Cys2 is clearly visible (Figures 3a and 3b). Comparison with the apo structure also suggests the presence of a repositioned loop (residues 127-138) that clamps the inhibitor in the active site in a solvent-exposed channel (Figure 3b). Figure 24 ).

[0601] These data indicate that 7 selectively labels B. thetaiotaomicron BSH at Cys2. Furthermore, the cocrystal structure reveals that the C3-hydroxyl group is solvent accessible, suggesting that this site may be amenable to further modification ( Figure 11 b).

[0602] Example 7. Compound 7 shows minimal off-target effects

[0603] The nonspecific reactivity of covalent inhibitors has been raised as a potential cause 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 coactivator recruitment assays showed that 7 was neither an agonist nor an antagonist of FXR at physiologically relevant concentrations ( Figure 25 ) 31 Next, 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 within the concentration range tested. Figure 25 These results suggest that 7 does not induce off-target effects on these key host receptors.

[0604] Bile acids are also known to be toxic due to their cleansing properties 1,33 The toxicity of the compound was also tested against human intestinal cells (Caco-2 and NCI-H716). No toxicity was observed when these cells were incubated with compound 7 at concentrations up to 50 μM or 100 μM, respectively. Figure 25 ). Because the IC of 7 50 The values ​​ranged from 237 to 1070 nM, and these results suggest that effective non-toxic in vivo doses should be achievable. To test the effect of compound 7 on epithelial integrity, Caco-2 cells were differentiated into polarized monolayers with tight intercellular junctions in transwell inserts. 34 Compound 7 was incubated in the top chamber of transwells, and epithelial integrity was measured by passive diffusion of 4 kDa FITC-dextran. No significant increase in fluorescence was observed in 7-treated cells compared to control-treated cells, indicating that 7 did not impair the integrity of the epithelial monolayer ( Figure 26 ).

[0605] Understanding the proteome-wide reactivity of 7 is important 35 To evaluate the target binding and off-target interactions of compound 7, the α-azido moiety was incorporated into the solvent-exposed C3 position. 36 The "clickable" form of this inhibitor (compound 12, Figure 26 a). Like 7, 7-N3 effectively inhibited BSH activity in mouse feces ( Figure 26b). These results demonstrate that azide substitution does not significantly interfere with the BSH inhibitory activity of this molecule. To investigate on-target and off-target effects in bacterial cells, cultures of B. adolescentis L2-32 were treated with 10 μM 7-N3 (i.e., the concentration at which 7 inhibits BSH in bacterial cultures) for 1 hour. Figure 17 The lysed bacterial supernatant was then reacted with Fluor 488-alkyne under copper-catalyzed azide-alkyne cycloaddition conditions, and the proteins were visualized by in-gel fluorescence. Only one fluorescent band at a mass of approximately 35 kDa (the predicted mass of B. adolescentis BSH) was visible ( Figure 12 c and 27). To identify the protein, the clarified lysate was hit with desthiobiotin-alkyne and a streptavidin pull-down was performed. Bound proteins from control and treated samples were resolved by SDS-PAGE and visualized by silver staining ( Figure 12 d and 27). A single silver-stained band at the predicted molecular weight of BSH (approximately 35 kDa) was observed. This band, along with the corresponding region of the control lane, was excised, digested with trypsin, and subjected to LC-MS / MS. BSH was identified with high confidence in the gel band, and semi-quantitative analysis of these data indicated a 4.5-fold enrichment of 7-N3- relative to vehicle-treated bacterial cultures.

[0606] To assess off-target binding on the bacterial proteome, streptavidin-bead-bound proteins isolated from treated and control bacterial cultures were digested. Label-free LC-MS / MS analysis identified BSH as 3.6-fold enriched in probe-treated cultures. No other proteins exceeded the 2-fold enrichment threshold in biological triplicate experiments. Competition of 7 with 7-N3 showed dose-dependent labeling of B. adolescentis BSH ( Figure 12 e and 27), further confirming the on-target activity of 7.

[0607] The off-target effects of compound 7 were analyzed in mammalian intestinal cells (NCI-H716). These cells were also treated with 7-N3 and processed in the same manner as bacterial cells. Click reaction with Fluor 488-alkyne did not show enrichment of any band of fluorescence in the gel ( Figure 12 f and 28). Based on label-free LC-MS / MS analysis, there was no protein enrichment (≥2-fold) in the probe-treated lysates. In summary, our data demonstrate on-target BSH binding of 7 and limited off-target activity against other bacterial or mammalian proteins in intestinal cells.

[0608] Example 8. A single dose of 7 inhibits BSH activity in vivo

[0609] C57BL / 6 mice were gavaged with a single dose of 7 (10 mg / kg, see online methods for dose calculations) or vehicle control, and fecal BSH activity was monitored over time in half-day increments ( Figure 13 a). A significant decrease in fecal BSH activity was observed 1 day and 1.5 days after gavage, whereas BSH activity recovered at subsequent time points ( Figure 13 b), and a significant increase in fecal conjugated bile acids and a decrease in dissociated bile acids 1 day after gavage ( Figure 13 c). 16S rDNA sequencing and plating of fecal samples from these mice indicated that compound 7 did not significantly affect intestinal bacterial OTUs, biomass, or community composition ( Figure 13 d and 28). Taken together, our results indicate that a dose of 7 can inhibit gut bacterial BSH activity and modulate bile acid pools in vivo without significantly affecting the gut bacterial community.

[0610] Proof of Concept for Intestinal Restriction of Example 9.7

[0611] To further minimize the possibility of 7 inducing off-target effects, it is ideal to restrict the molecule to the gastrointestinal tract. A 3-sulfated variant (intestinal restricted 7 or GR-7, compound 13) was synthesized. Figure 11 b, 13e).

[0612] Evaluation of GR-7 in mouse feces revealed that GR-7 remains a potent pan-BSH inhibitor ( Figure 29 C57BL / 6 mice were fed a powdered diet containing 0.09% GR-7 (w / w) for 1 day or a powdered diet alone ( Figure 13 f). Significant inhibition of BSH activity was observed in the feces of inhibitor-treated mice 8 hours after the diet change ( Figure 13 g) GR-7 was detected in feces collected at 8 hours, indicating that the inhibitor was excreted at a rate consistent with its colonic transit time in mice. 38 The compound was detected at 20 pmol / mg wet weight (approximately 20 μM) in the cecal contents (mean, Figure 13 h). This concentration was effective for BSH inhibition in the mouse fecal assay and was below the 100 μM toxicity threshold of 7 ( Figure 24 and 29 Furthermore, GR-7 (60 μM) did not affect the epithelial barrier integrity of Caco-2 cells, indicating that this compound is relatively nontoxic ( Figure 25 GR-7 also did not affect microbial biomass ( Figure 29GR-7 was not detected in the serum and liver of inhibitor-treated mice (Figure 5h). Taken together, these results provide proof of concept that 7 can be chemically modified to minimize absorption and that gut-restricted 7 derivatives can inhibit BSH activity when fed with food.

[0613] Example 10. Compound Synthesis [061...

Claims

1. A compound of formula (I): in: n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 1, 2, 3 or 4; X is an electrophilic group; R1, R2, R3, R4, R6, R7, 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、-CO2H、-CO2R 18 , -NO2, -ONO2, -SO2Cl, -SO3 - 、-OSO3 - 、-NR 18 SO3 - 、-PO3 2- 、-OPO3 2- 、-OSO2R 18 、-SO2N(R 18 )2、-OSO2N(R 18 )2、-NR 18 SO2R 18 、-SO2N(R 18 )2, -NHNH2, -ONH2, or -NHC(O)NHNH2; 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.

2. The compound according to claim 1, which has the formula: or pharmaceutically acceptable salts thereof.

3. The compound according to any one of claims 1 to 2, wherein the compound is of formula (I-e'): or a pharmaceutically acceptable salt thereof, in: R 3a and R 7a Independently selected from –OR 18 、–SO3R 18 、–OSO3R 18 , –PO3H2, –OPO3H2, –OSO2R 18 and –SO2N(R 18 )2, where each R 18 are independently H, or substituted or unsubstituted alkyl.

4. The compound according to any one of claims 1 to 3, wherein the compound is of formula (I-f'): or a pharmaceutically acceptable salt thereof, in: R 3a Selected from –OR 18 、–SO3R 18 、–OSO3R 18 , –PO3H2, –OPO3H2, –OSO2R 18 and –SO2N(R 18 )2, where each R 18 are independently H, or substituted or unsubstituted alkyl.

5. A pharmaceutical composition comprising: a compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or excipient.

6. A method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with the compound according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 5.

7. A method of inhibiting bile acid dissociation in a subject, the method comprising: A therapeutically effective amount of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 is administered to a subject.

8. A method of promoting bile acid conjugation in a subject, the method comprising: A therapeutically effective amount of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 is administered to a subject.

9. A method of regulating bile acids in a subject, the method comprising: A therapeutically effective amount of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 is administered to a subject in need thereof.

10. A kit comprising: The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 5; and Instructions for using the compound, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

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