Small molecule modulators of enterobacterial bile acid metabolism

Abstract

Described herein are methods and compositions relating to the inhibition of bile salt hydrolase (BSH) and uses thereof.SOLUTION: Provided herein are methods for treating metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases (such as gastrointestinal infections; inflammatory bowel diseases (IBD); appendicitis; Crohn's disease (CD); ulcerative cholitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetics; hepatits; hepatic diseases (such as non-alcoholic fatty liver disease (NAFLD); non-alcoholic fat hepatits (NASH); hepatits A; hepatits B; hepatits C; autoimmune hepatits; and cirrhosis of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndromes; intestinal wall leakage; and cancers); Methods for treating cancer (e.g., cancer of the digestive system, liver cancer) or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof comprising administering to the subject a compound represented by Formulae (I) - (XVIII).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. (USSN) 62 / 846,457, filed May 10, 2019, and U.S. Provisional Application No. (USSN) 62 / 962,048, filed January 16, 2020, each of which is incorporated herein by reference.

[0002] Government support 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.

[0003] FIELD OF THE INVENTION FIELD OF THE INVENTION The technology described herein relates to compounds, compositions, and methods for inhibiting bile salt hydrolase (BSH). [Background technology]

[0004] Background of the Invention Bile salt hydrolase (BSH) enzymes are widely expressed by human intestinal bacteria and catalyze the gateway reaction leading to the conversion of primary bile acids produced by the host to 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. Currently, there is an unmet need for potent and selective agents that inhibit BSH for the treatment of diseases such as cancer, inflammation, obesity, diabetes, and gastrointestinal disorders, and to be used as tools to understand bile acid physiology in host subjects. Summary of the Invention

[0005] SUMMARY OF THE INVENTION In one aspect, provided herein is a compound of formula (I): [ka] During the ceremony: 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.

[0006] In one aspect, the compound of formula (I) has the formula (I'): [ka] During the ceremony: 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, -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, where 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.

[0007] In another aspect, provided herein is a pharmaceutical composition comprising a compound provided herein and a pharmaceutically acceptable carrier or excipient.

[0008] In another aspect, provided herein is a method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound provided herein.

[0009] In another aspect, provided herein is a method of inhibiting bile acid deconjugation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein.

[0010] 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.

[0011] In another aspect, 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 provided herein. In another aspect, 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancers of the digestive system; liver cancer; colon cancer). cancer); esophageal cancer; cancer of the stomach; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), 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), the method comprising administering a compound of Formulas (I)-(XVIII) to a subject in need thereof. The genetically modified microorganism or population thereof secretes cholic acid 7-sulfate.

[0012] In another aspect, provided is 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancer of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or and cancers of the urinary system) 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) in a subject in need thereof.

[0013] In another aspect, provided is a kit 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 some embodiments, the kit further comprises instructions for administration (e.g., human administration) and / or use.

[0014] Details of certain embodiments of the present invention are presented in the Detailed Description of Certain Embodiments, as set forth below. Other features, objects, and advantages of the present invention will be apparent from the definition, examples, figures, and claims. [Brief explanation of the drawings]

[0015] Brief description of the drawings This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] [Figure 1] Figures 1A and 1B demonstrate the chemical and biological effects of enterobacterial bile salt hydrolases (BSH). Figure 1A shows that BSH is the entry enzyme in the conversion of primary (host-produced) bile acids to secondary (bacterial-produced) bile acids. Removal or inhibition of BSH should result in a decrease in deconjugated primary and secondary bile acids. Figure 1B shows that certain primary and secondary bile acids are ligands for host nuclear hormone receptors (NhRs) and G protein-coupled receptors (GPCRs). By acting as agonists or antagonists on these receptors, these bile acids influence host processes, including metabolic regulation and immune response.

[0017] [Figure 2A-C] Figures 2A-2E demonstrate the rational design of small-molecule, broad-spectrum BSH inhibitors. Figure 2A shows the mechanism of enzymatic amide bond cleavage by BSH. Figure 2B shows the co-crystal structure of BSH from the gram-positive enterobacterium Clostridium perfringens with deconjugated tauro-deoxycholic acid (TDCA) (PDB 2BJF) guided by inhibitor design. Hydrophobic interactions orient the bile acid core in the active site (magenta residues), while the D-ring side chain and amino acids are exposed to solvent. Figure 2C shows a representative mechanism of BSH inhibition by rationally designed inhibitors. Attack of the catalytically relevant nucleophilic cysteine ​​residue in the BSH active site can result in covalent binding to the inhibitor. [Figure 2D]Figure 2D shows the library of inhibitors synthesized. Electrophilic warheads, which have been successfully incorporated into the design of kinase and protease inhibitors, were added to the chenodeoxycholic acid core to create a broad range of BSH inhibitors. [Figure 2E] FIG. 2E shows the most potent BSH inhibitors identified from the high-throughput screen, riboflavin and caffeic acid phenethyl ester (CAPE), which were also included in this study.

[0018] [Figure 3] Figures 3A-3B demonstrate that the screening identified inhibitor 7 as a potent and long-lasting inhibitor of recombinant BSH. Figure 3A shows the screening of the inhibitor library versus B. theta BSH, showing the % deconjugation at 2 and 21 hours. Figure 3B shows the screening of compounds 1, 7, and CAPE versus B. longum BSH, showing the % deconjugation at 2 and 21 hours. Inhibitors (100 μM) were incubated with 200 nM rBSH for 30 min, followed by the addition of taurine-conjugated bile acid substrates (TβMCA, TCA, TUDCA, and TDCA, 25 μM each). Substrate deconjugation was followed by UPLC-MS. Assays were performed in biological triplicate. Data are presented as mean ± SEM.

[0019] [Figure 4A-B]Figures 4A-4D show that compound 7 is a potent, non-toxic inhibitor of BSH in growing cultures of Gram-positive and Gram-negative enterobacteria. Figure 4A demonstrates that compound 7 inhibits BSH activity in live Gram-negative (B. theta 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 compound 7 or CAPE) and taurine-conjugated bile acid substrates (TβMCA, TCA, TUDCA, and TDCA, each at 25 μM) were added to bacterial cultures at an OD of 0.1. Bacterial cultures were grown to stationary phase, and percent deconjugation at 24 h was determined by UPLC-MS. Assays were performed in biological triplicate. Data are presented 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 is not presented for B. vulgatus because replicates had a standard error of zero. Figure 4B shows that compound 7 is not bactericidal. OD600 of bacterial cultures was measured at 24 h. CAPE inhibited the growth of the Gram-positive strains tested. Red downward arrows indicate the percentage reduction compared to the DMSO control. [Figure 4C] FIG. 4C shows the dose-response curve and calculated IC50 value for compound 7 incubated with growing cultures of B. theta (Gram-negative) and B. adolescentis (Gram-positive), demonstrating that compound 7 is a potent, broad-spectrum BSH inhibitor. [Figure 4D]Figure 4D shows representative UPLC-MS traces demonstrating that inhibitor structure determines BSH inhibitory activity against growing B. theta cultures. Compounds 1, 7, and 9 were tested at 1 and 10 μM concentrations. For simplicity, one substrate (GUDCA) was added to the bacterial cultures, and its deconjugation to UDCA was followed by UPLC-MS. Inhibitor 9, which contains a cholic acid (C12=OH) core and an α-FMK warhead, demonstrates significantly reduced activity in inhibiting B. theta BSH.

[0020] [Figure 5] Figures 5A-5C demonstrate that compound 7 covalently modifies B. theta BSH at the active site cysteine ​​residue. Figures 5A-5B show that mass spectrometry revealed that compound 7 monolabels B. theta BSH. Figure 5A shows the mass spectrum (left) and zero-charge mass spectrum (right, overlaid) of BSH treated with DMSO (top, red trace) or with 10-fold excess inhibitor compound 7 for 2 h (bottom, green trace). The mass shift of 388 Da is consistent with covalent modification of BSH with loss of HF. Figure 5B shows top-down MS of BSH treated with 10-fold excess inhibitor compound 7. Ions of types c and z are indicated by red and green glyphs, respectively. Ion c3 indicates that the modification is at the Cys2 residue on the N-terminus. Figure 5C shows the X-ray cocrystal structure of compound 7 bound to B. theta BSH, confirming that compound 7 was covalently linked to Cys2 rather than Cys67 in the active site and that the C25 fluorine had been removed. C3 of the steroid core was exposed to solvent, indicating that this site was amenable to modification.

[0021] [Figure 6]Figures 6A-6C demonstrate that compound 7 exhibits minimal off-target effects. Figure 6A shows that compound 7 is neither an agonist nor an antagonist of the farnesoid X receptor (FXR), as determined by an FXR coactivator recruitment assay. The FXR antagonist activity of compound 7 was assessed at its EC value (50 nM) in the presence of the known FXR agonist GW4064. n = 4 biological replicates per concentration. Data are presented as mean ± SEM. Figure 6B shows that compound 7 is neither an agonist nor antagonist of the G protein-coupled bile acid receptor (GPBAR1, also known as TGR5). Endogenous TGR5 agonist activity was measured by incubating Caco-2 cells overnight with varying concentrations of compound 7. Endogenous TGR5 antagonist activity was assessed in the presence of 10 μM of the TGR5 agonist LCA. n≧3 biological replicates per concentration. Data are presented as mean±SEM. One-way ANOVA followed by Dunnett's multiple comparison test. ns=not significant. Figure 6C shows that compound 7 was not toxic to Caco-2 cells up to concentrations of 50 μM. n≧3 biological replicates per concentration. Data are presented as mean±SEM. One-way ANOVA followed by Dunnett's multiple comparison test. *p<0.05.

[0022] [Figure 7A-C]Figures 7A-7F demonstrate that compound 7 inhibits BSH activity ex vivo and in vivo. Figure 7A shows the fecal BSH activity assay design. Freshly collected feces (1 mg / mL) from conventional mice were resuspended in PBS and incubated with 20 μM inhibitor (compounds 1, 7, or CAPE) for 30 min. Glycochenodeoxycholic acid-d4 (GCDCA-d4, 100 μM) was added as a substrate, and deconjugation was determined by UPLC-MS after 18 h. Figure 7B shows that compound 7 effectively inhibited BSH activity in fecal slurry, while CAPE exhibited minimal inhibitory activity. Consistent with the in vitro results, compound 1 exhibited moderate BSH inhibition. Assays were performed in biological triplicate. Data are presented as mean ± SEM. Figures 7C-7E show that treatment of conventional mice with a single dose of Compound 7 resulted in recoverable inhibition of BSH activity and a shift toward deconjugated bile acids. n = 4 mice per group; Welch's t-test, *p < 0.05, **p < 0.01, ns = not significant. Figure 7C shows the design of the in vivo BSH inhibition experiment. Conventional C57BL / 6 male mice were gavaged with a single dose of Compound 7 (10 mg / kg) or vehicle control. Feces were collected 1, 1.5, 2, and 2.5 days after gavage. Bile acid profiling was performed 1 day after gavage. [Figure 7D-F]Figure 7D shows that fecal BSH activity was significantly lower in the compound 7-treated group compared to the control group at 1 and 1.5 days after gavage, as determined by fecal BSH activity. BSH was regenerated 2 days after gavage. BSH activity was determined by resuspending fresh feces from inhibitor- or vehicle-treated groups with substrate (GCDCA-d4, 100 μM), incubating for 25 min, and quantitating deconjugation by UPLC-MS. Figure 7E shows the fecal bile acid composition 1 day after gavage. Deconjugated bile acids, including the secondary bile acid deoxycholic acid (DCA), were reduced in the inhibitor-treated group. Figure 7F shows that microbial biomass did not differ between the inhibitor- and vehicle-treated groups at 1 or 2.5 days after gavage. n = 4 mice per group, Mann-Whitney test.

[0023] [Figure 8A-C] Figures 8A-8D demonstrate that administration of 3-sulfated lithocholic acid-fluoromethylketone (3S-LCA-FMK), a gut-restricted derivative of compound 7, when fed in chow, resulted in a significant reduction in BSH activity over a 1-week period. Figure 8A shows the structure of 3-sulfated lithocholic acid-fluoromethylketone (3S-LCA-FMK). Figure 8B shows the design of the in vivo BSH inhibition experiment. Conventional C57Bl / 6 male mice were fed either regular chow or 3S-LCA-FMK (0.03% wt / wt) in chow ad libitum for 7 days. Feces were collected before the diet change and on days 3, 4, and 7 after the diet change. n = 5 mice per group. FIG. 8C shows that BSH activity was significantly reduced in the feces of mice fed 3S-LCA-FMK in chow. [Figure 8D] Figure 8D shows the concentrations of 3S-LCA-FMK measured in feces and cecal contents at the time of sacrifice. 3S-LCA-FMK was undetectable in circulating plasma on day 4, indicating that the compound was restricted to the intestine.

[0024] [Figure 9] Figure 9A shows that the primary reaction in the conversion of primary bile acids to secondary bile acids is hydrolysis (deconjugation) of the C24 amide bond of the conjugated primary bile acid. Figure 9B shows that although there is significant variation in BSH protein sequence across enteric strains, all BSHs possess a conserved active site, encompassing the catalytic cysteine ​​(Cys2). Figure 9C shows the co-crystal structure of Clostridium perfringens BSH with the substrate taurodeoxycholic acid, demonstrating that hydrophobic interactions engaged the bile acid core and oriented the amide toward Cys2 when the amino acid remained solvent exposed. Figure 9D shows a compound of the present disclosure.

[0025] [Figure 10] Figure 10 shows the screening of inhibitors versus B. theta BSH (Figure 10A) and B. longum BSH (Figure 10B), showing the percent deconjugation of tauro bile acids at 2 and 21 hours. Strains were incubated with 100 μM conjugated bile acids and plated at 21 hours to assess viability (Figure 10C). Compound 7 was not bactericidal (Figure 10D). CAPE reduced cell viability of the tested Gram-negative strains. Red downward arrows indicate fold reductions compared to the DMSO control. One-way ANOVA followed by Dunnett's multiple comparison test was used for (Figure 10C) and (Figure 10D). (Figure 10E) Compound 7 inhibited BSH activity in fecal slurry. All assays were performed in biological triplicate. Data are presented as mean ± SEM.

[0026] [Figure 11]Figure 11A shows the X-ray structure of compound 7 bound to B. theta BSH. BSH (turquoise) is shown in ribbon representation, and the side chain (turquoise, heteroatoms in CPK color) is indicated in stick representation. Figure 11B shows the co-crystal structure of B. theta BSH and compound 7 shown in ribbon (left, with electron density for the compound shown as a blue grid) and surface (right) representation. The A ring of 7, including the C3 hydroxyl group, is solvent exposed. Panels a and b were generated using PYMOL software (Schroedinger).

[0027] [Figure 12]Figure 12A shows the structure of 7-N3 (12), a "clickable" probe for on- and off-target studies. Figure 12B shows that 7-N3 exhibited significant BSH inhibition in conventional mouse feces, indicating that this probe retained its function as a BSH inhibitor. Figure 12C shows the 1-hour treatment of B. adolescentis L-32 cultures with 7-N3, followed by cell lysis, click reaction with Fluor 488-alkyne, and visualization of only one protein (~35 kDa in size, the mass of the annotated B. adolescentis BSH) using in-gel fluorescence revealed labeling. Figure 12D shows that lysates from B. adolescentis cultures treated with 7-N3 were reacted with desthiobiotin-alkyne, resolved by SDS-PAGE, and visualized by silver staining. The arrow indicates a band in the probe-treated sample at the predicted molecular weight of BSH (~35 kDa). Figure 12E shows that treatment of B. adolescentis cultures with decreasing concentrations of compound 7, followed by treatment with 10βM 7-N3 and click reaction with Fluor 488-alkyne, resulted in a dose-dependent increase in fluorescent labeling of annotated B. adolescentis BSH. Figure 12F shows that treatment of NCI-H716 intestinal cells with 7-N3 for 1 hour, followed by click reaction with Fluor 488-alkyne and visualization by in-gel fluorescence, did not result in significant labeling of the protein compared to control-treated cells. For (Figures 12B, 12C, 12D, and 12F), n = 3 biological replicates per condition. For (Figure 12B), data are presented as mean ± SEM.

[0028] [Figure 13]Figures 13A-13C show that conventional treatment of mice with a single dose of compound 7 resulted in reproducible inhibition of BSH activity and a shift toward conjugated bile acids. n = 4 mice per group, Student's t-test. Figure 13A shows the design of the in vivo BSH inhibition experiment. Adult male C57BL / 6 mice were gavaged with a single dose of compound 7 (10 mg / kg) or vehicle control. Figure 13B shows that BSH activity was measured in half-day increments starting 1 day after gavage. Resuspended fresh feces from inhibitor- or vehicle-treated groups were incubated with substrate (GCDCA-d4, 100 μM) for 25 min, and product formation was quantified by UPLC-MS. n = 4 mice per group, two-tailed Student's t-test. Figure 13C shows the fecal bile acid composition 1 day after gavage. Deconjugated bile acids, including the secondary bile acid deoxycholic acid (DCA), were reduced in the inhibitor-treated group. n = 4 mice per group, two-tailed Student's t-test. Figure 13D shows that bacterial OTUs (operational taxonomic units) were not different between the inhibitor-treated and vehicle-treated groups 1 day after gavage. n = 4 mice per group, one-way ANOVA followed by Tukey's multiple comparison test. Figure 13E shows the structure of gut-restricted compound 7 (GR-7, 13). Figure 13F shows the design of an in vivo proof-of-concept study using GR-7. Adult male C57BL / 6 mice were fed powdered chow containing 0.09% (w / w) GR-7 or powdered chow alone for 30 hours. Fecal pellets were collected 8 hours after the diet change. n = 10 mice per group. Figure 13G shows that resuspended fresh feces (20 mg / mL) from mice treated with inhibitors or controls were incubated with substrate (GCDCA-d4, 100 μM) for 25 min, and product formation was quantified by UPLC-MS. Significant inhibition of BSH activity was observed in feces from GR-7-treated mice compared with control-treated mice. Student's t-test. n = 10 mice per group, two-tailed Student's t-test. Figure 13H shows quantification of GR-7 in tissues and plasma.The inhibitor was detected in feces 8 hours after the diet change and in cecal contents at the time of sacrifice. GR-7 was not detected in liver or plasma. ND = not detected. n = 10 mice per group. All data are presented as mean ± SEM.

[0029] [Figure 14] Figure 14 shows the purification and kinetic characterization of BSH. (Figure 14A) SDS-PAGE of B. theta BSH purification. The experiment was repeated seven times with similar results. (Figure 14B) SDS-PAGE of B. longum BSH purification. Michaelis-Menten analysis of BSH kinetic data. Velocity vs. substrate concentration curves for B. theta BSH (Figure 14C) and B. longum BSH (Figure 14D).

[0030] [Figure 15] Figure 15 shows the identification of compound 7 as a potent, broad-spectrum BSH inhibitor. Figures 15A-B show the screening of inhibitors versus B. theta BSH (Figure 15A) and B. longum BSH (Figure 15B), showing the % deconjugation of tauro bile acids at 5 hours. Inhibitors (100 μM) were incubated with 200 nM rBSH for 30 min, followed by the addition of taurine-conjugated bile acid substrates (tauro-β-muricholic acid, TβMCA; tauro-cholic acid, TCA; tauro-ursodeoxycholic acid, TUDCA; and tauro-deoxycholic acid, TDCA, each at 25 μM). Substrate deconjugation was followed by UPLC-MS. Assays were performed in biological triplicate, and all data are presented as mean ± SEM.

[0031] [Figure 16]Figure 16 shows bile acid quantification to report % deconjugation of purified BSH protein. The concentrations of formed product (deconjugated bile acids) and unreacted starting material (SM) at each time point were determined using UPLC-MS for both B. theta BSH (Figure 16A) and B. longum BSH (Figure 16B). The % deconjugation for each sample was then determined using the following equation: % deconjugation = product concentration / (product concentration + starting material concentration) * 100.

[0032] [Figure 17] Figure 17 shows that compound structure affects BSH inhibitory activity against growing B. theta cultures. (Figure 17A) Compounds 8 and 9 are less potent inhibitors of B. theta BSH than compound 7. Inhibitors (10 μM of compound 7, 8, or 9) and 100 μM TUDCA were added to B. theta cultures at an OD of 0.1. (Figure 17B) Structural comparison of compounds 7, 8, and 9. Compound 8 lacks the α-FMK warhead, and compound 9 possesses a C=OH hydroxyl group.

[0033] [Figure 18] Figure 18 shows that compound 7 is a potent inhibitor of recombinant BSH. Dose-response curve and calculated IC value for compound 7. 200 nM recombinant B. theta BSH (Figure 18A, Gram-negative) or B. adolescentis BSH (Figure 18B, Gram-positive) were preincubated with varying concentrations of compound 7 for 60 min, followed by the addition of the conjugated bile acid substrates TUDCA and TDCA, respectively.

[0034] [Figure 19]Figure 19A shows the time required for complete inhibition of B. theta BSH. 100 μM Compound 7 and bile acids to be conjugated (25 μM each of tauro-β-muricholic acid, TβMCA; tauro-cholic acid, TCA; tauro-ursodeoxycholic acid, TUDCA; and tauro-deoxycholic acid, TDCA) were added simultaneously to 200 nM rBSH without a preincubation period. The formation of deconjugated bile acids was measured using a UPLC-MS-based assay and reported as % conversion. Figure 19B shows that in the presence of Compound 7, no increase in product formation was observed after 15 seconds, indicating that enzyme activity was inhibited.

[0035] [Figure 20] Figure 20 shows the quantification of bile acids to report % deconjugation of bacterial cultures. The concentrations of formed product (deconjugated bile acids) (Figure 20A) and unreacted starting material (SM) (Figure 20B) in each culture were determined using UPLC-MS. The % deconjugation for each sample was then determined using the following equation: % deconjugation = Concentration of product / (Concentration of product + Concentration of starting material) * 100.

[0036] [Figure 21]Figure 21 shows that compound 7 did not alter the bile acid pool when incubated with the B. theta BSH KO strain. (Figure 21A) A 100 μM pool of taurine-conjugated bile acids (TCA, TβMCA, TUDCA, and TDCA, 25 μM each) and 100 μM inhibitor (compound 7 or CAPE) or DMSO were added to growing B. theta. Cultures were incubated for 24 h, and then bile acid profiling was performed using UPLC-MS. No bile acids were detected in any of the cultures other than the starting materials (TCA, TβMCA, TUDCA, and TDCA). (Figure 21B) Colony-forming units (CFUs) were determined after 24 h from the assay in panel (Figure 21A). Compound 7 was not found to be bactericidal against B. theta lacking BSH, while CAPE was found to significantly affect the growth of this bacterium.

[0037] [Figure 22] Figure 22 shows that compound 7 is a potent BSH inhibitor in growing bacterial cultures. Dose-response curve and calculated IC value for compound 7. Pre-logarithmic phase cultures of B. theta (Figure 22A, Gram-negative) and B. adolescentis (Figure 22B, Gram-positive) were incubated with the conjugated substrate (TUDCA or TDCA) and grown anaerobically for 48 h and 24 h, respectively.

[0038] [Figure 23]Figure 23 shows that mass spectrometry revealed that compound 7 single-labels B. theta BSH. (Figure 23A) Mass spectrum (left) and zero-charge mass spectrum (right) of BSH treated for 2 h with DMSO (top, red trace) or a 10-fold excess of compound 7 (bottom, green trace). The mass shift of 388 Da is consistent with covalent modification of BSH with loss of HF. Two independent labeling reactions produced similar results. (Figure 23B) Top-down MS / MS of BSH treated with a 10-fold excess of compound 7. Ions of types c and z are indicated by red and green symbols, respectively. Ion c3 indicates that the modification is at the Cys2 residue on the N-terminus.

[0039] [Figure 24] Figure 24 shows the apo and co-crystal structures of B. theta BSH. The X-ray structure of the B. theta BSH apoprotein (Figure 24A) was superimposed onto the X-ray structure of B. theta BSH covalently bound to compound 7 (Figure 24B). BSH (apo in magenta and co-crystal in cyan) is shown in ribbon representation, and side chains (magenta or cyan, respectively, with heteroatoms in CPK colors) are indicated in stick representation. Compound 7 (green, with heteroatoms in CPK colors) is indicated in stick form. The box (dashed line) indicates the loop (residues 127-138) repositioned in the co-crystal structure. Panels were generated using PYMOL software (Schroedinger).

[0040] [Figure 25]Figure 25 shows that compound 7 is neither an agonist nor an antagonist of FXR or TGR5 and is not toxic to human cells. (Figure 25A) Compound 7 is not a farnesoid X receptor (FXR) agonist, as determined by an FXR coactivator recruitment assay. n = 4 biological replicates per concentration. (Figure 25B) The FXR antagonist activity of compound 7 was evaluated in the presence of the FXR agonist GW4064 at its EC50 value (50 nM, as determined in the corresponding agonist assay). n = 4 biological replicates per concentration. (Figure 25C) Compound 7 is not a G protein-coupled bile acid receptor (GPBAR1 / TGR5) agonist. Endogenous TGR5 agonist activity was measured by incubating Caco-2 cells overnight with varying concentrations of 7. 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 overnight incubation of Caco-2 cells with varying concentrations of compound 7 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 25E) Compound 7 was not toxic to Caco-2 or NCI-H716 cells up to concentrations of 50 μM and 100 μM, respectively. n = 5 and n = 3 biological replicates per concentration, respectively. One-way ANOVA followed by Dunnett's multiple comparison test. Data are presented as mean ± SEM.

[0041] [Figure 26] Figure 26 shows that neither Compound 7 nor GR-7 significantly affected the integrity of the epithelial barrier. Incubation of differentiated Caco-2 cells with Compound 7 or GR-7 for 6 and 12 hours did not impair the integrity of the epithelial monolayer, as measured by passive transport of 4 kDa FITC-dextran. n=2 biological replicates for the DMSO control and n=3 biological replicates for the inhibitor-treated conditions. All data are presented as mean ± SEM.

[0042] [Figure 27] Figure 27A shows that compound 7-N labels B. adolescentis BSH with minimal off-target reactivity. Figure 27B shows that the fluorescence intensity of the BSH band was quantified (two-tailed Student's t-test). Data are presented as mean ± SEM. Figure 27C shows the complete SDS-PAGE gel from the experiment described in Figure 4H. B. adolescentis cultures were treated with decreasing concentrations of compound 7 for 1 hour, followed by 0 μM compound 7-N for another hour. Dose-dependent labeling of BSH was observed with decreasing concentrations of compound 7. The experiment was repeated twice with similar results. Figure 27D shows a silver-stained gel from the experiment described in Figure 4G, performed in biological triplicate. Figure 27E shows BSH-derived tryptic peptides identified by LC-MS / MS analysis of in-gel digests performed on the bands indicated in Figure 27D. Amino acids highlighted in red map to tryptic peptides identified at ∼1% FDR.

[0043] [Figure 28] Figure 28 shows that 7-N3 exhibited minimal off-target labeling in mammalian cells. SDS-PAGE gel from the experiment described in Figure 4I performed in biological triplicate (i.e., treatment of NCI-H716 cells with 10 μM 7-N3 for 1 hour, followed by click reaction with Fluor 488-alkyne).

[0044] [Figure 29] Figure 29 shows that compound 7 did not significantly affect bacterial community composition or microbial biomass in vivo. (Figure 29A) Mean relative abundance of the microbiota at the phylum level by taxon-based analysis, n = 4 mouse groups. (Figure 29B) CFU / g did not differ between inhibitor- and vehicle-treated groups 0.5, 1, 1.5, 2, or 2.5 days after gavage. n = 4 mice per group, two-tailed Mann-Whitney test. All data are presented as mean ± SEM.

[0045] [Figure 30] Figure 30 shows the activity and in vivo effects of GR-7. (Figure 30A) GR-7 inhibited BSH activity in fecal slurry. Freshly collected feces from conventional mice were resuspended in PBS (1 mg / mL) and incubated with 20 μM or 60 μM GR-7 for 30 min. Glycochenodeoxycholic acid-d4 (GCDCA-d4, 100 μM) was added as a substrate, and product formation was determined 18 h later by UPLC-MS. Assays were performed in biological triplicate. (Figure 30B) 16S rDNA copies / g cecal contents 30 h after diet change. Microbial biomass did not differ between inhibitor- and vehicle-treated groups. Two-tailed Mann-Whitney test. n = 10 mice per group. All data are presented as mean ± SEM.

[0046] [Figure 31] FIG. 31 shows that 3S-LCA-FMK reduces food intake in conventional mice compared to vehicle-dosed mice (n=8 mice per group). DETAILED DESCRIPTION OF THE INVENTION

[0047] definition Chemistry Definition For convenience, the meanings of some terms and phrases used in this specification, examples, and appended claims are provided below. Unless otherwise stated or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to help describe specific embodiments, but 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. If there is an apparent discrepancy in usage between a term in the art and its definition provided herein, the definition provided herein shall take precedence.

[0048] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor & Francis Limited,2014(ISBN0815345305,9780815345305);Lewin's Genes XI,published by Jones & Bartlett Publishers,2014(ISBN-1449659055);Michael Richard Green and Joseph Sambrook,Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York,USA(2012)(ISBN044460149X);Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.)Elsevier,2013(ISBN0124199542);Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entirety.

[0049] Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are defined in accordance with the Handbook of Chemistry and Physics, 75 th The Periodic Table of the Elements, CAS Edition, Ed., inside cover, is identified, and specific functional groups are generally defined as described therein. In addition, the general rules of organic chemistry, as well as specific functional moieties and reactivities, are described in detail in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Michael B. Smith, March's Advanced Organic Chemistry, 7 thEdition, 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, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0050] The compounds described herein may contain one or more asymmetric centers, and therefore may exist in various stereoisomeric forms, such as enantiomers and / or diastereomers.For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers (including racemic mixtures and mixtures enriched with one or more stereoisomers).Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, 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 individual isomers substantially free of other isomers, or alternatively, as mixtures of various isomers.

[0051] The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0052] When substituents are specified by their conventional chemical formula written from left to right, they equally cover chemically identical substituents derived from the structure written from right to left (e.g., -CH2O- is equivalent to -OCH2-).

[0053] The term "alkyl," by itself or as part of another substituent, unless otherwise stated, refers to a linear (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and having the specified number of carbon atoms (i.e., C1 to C6). 10 (meaning 1 to 10 carbons) refers to a carbon chain as defined above, which can include monovalent, divalent, and polyvalent radicals. Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are those groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

[0054] The term "alkylene," by itself or as part of another substituent, unless otherwise stated, means a divalent radical derived from an alkyl, as exemplified, but not limited by, -CHCHCHCH-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, although those groups having 10 or fewer carbon atoms are preferred in the present invention. An alkylene is a non-closed ring chain. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means a divalent radical derived from an alkene, unless otherwise stated.

[0055] The term "heteroalkyl," by itself or in combination with another term, unless otherwise stated, means a stable, straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. A heteroalkyl is a non-closed ring chain. The heteroatom(s) O, N, P, S, B, As, and Si may be placed at any position within the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, the following: -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 may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0056] The term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkyl, as exemplified, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for groups linking alkylene and heteroalkylene, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. A heteroalkylene is a non-closed ring chain. As noted above, heteroalkyl groups, as used herein, include those groups attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When "heteroalkyl" is recited and followed by a list of specific heteroalkyl groups (such as -NR'R'', or the like), it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups (such as -NR'R'', or the like).

[0057] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl," respectively, unless otherwise stated. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. A cycloalkyl or heteroalkyl is not aromatic. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0058] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, 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.

[0059] The term "acyl," unless otherwise stated, 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.

[0060] The term "aryl," unless otherwise stated, refers to a polyunsaturated, aromatic, hydrocarbon substituent that can be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused aryl) or covalently linked together. Fused aryl refers to multiple rings fused together, where at least one of the fused rings 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, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" encompasses fused heteroaryl groups (i.e., multiple rings fused together, where at least one of the fused rings is a heteroaromatic ring). 5,6-fused heteroarylene refers to two rings fused together, one ring having 5 members and the other ring having 6 members, where at least one ring is a heteroaryl ring. Similarly, a 6,6-fused heteroarylene refers to two rings fused together, one having 6 members and the other having 6 members, wherein at least one ring is a heteroaryl ring. And a 6,5-fused heteroarylene refers to two rings fused together, one having 6 members and the other having 5 members, wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon atom or a heteroatom.Non-limiting examples of aryl and heteroaryl groups 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- Examples of aryl and heteroaryl ring systems include thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above described aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Heteroaryl group substituents may be -O-bonded to a nitrogen ring heteroatom.

[0061] A "fused aryl-heterocycloalkyl" is an aryl fused to a heterocycloalkyl. A "fused heteroaryl-heterocycloalkyl" is a heteroaryl fused to a heterocycloalkyl. A "fused heterocycloalkyl-cycloalkyl" is a heterocycloalkyl fused to a cycloalkyl. A "fused heterocycloalkyl-heterocycloalkyl" is a heterocycloalkyl fused to another heterocycloalkyl. A fused aryl-heterocycloalkyl, a fused heteroaryl-heterocycloalkyl, a fused heterocycloalkyl-cycloalkyl, or a fused heterocycloalkyl-heterocycloalkyl can each independently be unsubstituted or substituted with one or more of the substituents described herein. A fused aryl-heterocycloalkyl, a fused heteroaryl-heterocycloalkyl, a fused heterocycloalkyl-cycloalkyl, or a fused heterocycloalkyl-heterocycloalkyl can each independently be named according to the size of each fused ring. Thus, for example, a 6,5 ​​aryl-heterocycloalkyl fused ring describes a 6-membered aryl moiety fused to a 5-membered heterocycloalkyl. A spirocyclic ring is two or more rings, with adjacent rings attached through a single atom. The individual rings within a spirocyclic ring may be the same or different. The individual rings within a spirocyclic ring may be substituted or unsubstituted and may have different substituents than the other individual rings within a series of spirocyclic rings. The possible substituents for an individual ring within a spirocyclic ring are the possible substituents for the same ring when not part of a spirocyclic ring (e.g., the substituents for a cycloalkyl ring or heterocycloalkyl ring). A spirocyclic ring may be a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heterocycloalkylene, and the groups for the individual rings within a spirocyclic ring may be any of the groups listed immediately above, including cases where all rings are one type (e.g., all rings are substituted heterocycloalkylene, where each ring may be the same or different substituted heterocycloalkylene).When referring to a spirocyclic ring system, a heterocyclic spirocyclic ring means a spirocyclic ring in which at least one ring is heterocyclic, and each ring may be a different ring. When referring to a spirocyclic ring system, a substituted spirocyclic ring means that at least one ring is substituted, and each substituent may optionally be different.

[0062] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0063] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical.

[0064] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), boron (B), arsenic (As), and silicon (Si).

[0065] "Substituent," as used herein, means a group selected from the following moieties: (A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -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 (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from the following: (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -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 (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from the following: (a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SON2NH2, -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 (b) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from the following: oxo, halogen, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —S02Cl, —S03H, —S04H, —SON2NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, —NHC═(O)NH2, —NHS02H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0066] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and hence the same molecular weight, but differ with respect to the structural arrangement or configuration of the atoms.

[0067] The term "tautomer," as used herein, refers to one of two or more structural isomers, which exist in equilibrium and are readily converted from one isomeric form to another.

[0068] It will be apparent to one skilled in the art that certain compounds of the present invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.

[0069] The term "silyl ether," as used herein, generally refers to a silyl ether having the structure R w R x R y Si-OR z (In the formula R w , R x , R y , and R z refers to a chemical compound containing a silicon atom covalently bonded to an alkoxy group having, independently, an alkyl or aryl group.

[0070] The term "pharmaceutically acceptable salts" is intended to encompass salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When a compound of the present invention contains a relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of such a compound 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 salts, or similar salts. When a compound of the present invention contains a relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid (either neat or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine acid, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0071] As used herein, the term "salt" refers to an acid or base salt of a compound used in the method of the present invention. Illustrative examples of salts include salts of mineral acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and salts of quaternary ammonium (such as methyl iodide, ethyl iodide, etc.). The term salt also refers to the formation of a salt between two compounds.

[0072] The term "metabolic disorder" refers to any disorder involving changes in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof. Metabolic disorders are associated with either a deficiency or excess in metabolic pathways, resulting in an imbalance in the metabolism of nucleic acids, proteins, lipids, and / or carbohydrates. Factors that affect metabolism include, but are not limited to, endocrine (hormonal) control systems (e.g., insulin pathways, enteroendocrine hormones (including GLP-1, PYY), or the like), neural control systems (e.g., brain GLP-1), or 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.

[0073] The term "obesity" refers to excess body fat. Obesity can be determined by any measure accepted and utilized by those skilled in the art. Currently, an accepted measure of obesity is the body mass index (BMI), which measures weight in kilograms to the square of height in meters. Generally, for adults over the age of 20, a BMI between about 18.5 and 24.9 is considered normal, a BMI between about 25.0 and 29.9 is considered overweight, a BMI of about 30.0 or higher is considered obese, and a BMI of about 40 or higher is considered morbidly obese. (See, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.) These BMI ranges are based on the effect of body weight on increased risk for disease. Some common diseases associated with high BMI and obesity include cardiovascular disease, high blood pressure (i.e., hypertension), osteoarthritis, cancer, and diabetes. Although BMI correlates with body fat, the relationship between BMI and actual body fat varies with age and gender. For example, women are more likely to have a higher percentage of body fat than men with the same BMI. Furthermore, the BMI threshold separating normal, overweight, and obese may vary among other factors, such as, for example, age, gender, ethnicity, fitness, and body type. In some embodiments, obese subjects have a body mass index of at least about 25 kg / m prior to receiving treatment as described herein. 2 In some embodiments, an obese subject may have a body mass index of at least about 30 kg / m prior to administration of a treatment, compound, or agent as described herein. 2 may be subject to.

[0074] As used herein, the terms "inflammation" or "inflamed" or "inflammatory" refer to the activation or recruitment of the immune system or immune cells (e.g., T cells, B cells, macrophages). Inflamed tissues may be red, white, swollen, hot, painful, exhibit loss of function, or have a thin film or mucus. Methods for identifying inflammation are well known in the art. Inflammation generally occurs following microbial injury or 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; Shingella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus.

[0075] As used herein, the term "inflammatory disease" refers to any disease that affects the immune system.Inflammatory disease can cause at least one symptom of the disease.These symptoms can include, but are not limited to, diarrhea, vomiting, nausea, upset stomach, pain, joint swelling, fatigue, fever, weight loss, weight gain, attendance, changes in bowel movements or stool consistency or frequency, or any other symptoms associated with inflammatory disease in subjects.In some embodiments, the inflammatory disease is an autoimmune disease.

[0076] In some embodiments of any of the aspects, the inflammatory disease is selected from the group consisting of: infections; Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis, or any other inflammatory disease known in the art.

[0077] As used herein, the term "gastrointestinal disease" refers to any disease that affects the gastrointestinal tract or intestines. Gastrointestinal disease can cause at least one symptom of the disease. These symptoms can include, but are not limited to, diarrhea, vomiting, nausea, upset stomach, pain, swollen joints, fatigue, fever, weight loss, weight gain, attendance, changes in bowel movements or stool consistency or frequency, or any other symptoms associated with gastrointestinal disease in a subject. Non-limiting examples of gastrointestinal disease include gastrointestinal infection, inflammatory bowel disease (IBD), gastrointestinal damage, 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.

[0078] As used herein, the term "liver disease" refers to any disease that affects the liver.

[0079] Liver disease can cause at least one symptom of disease.These symptoms include, but are not limited to, bile acid dysbiosis, fatigue, weight loss, pain, yellowing of 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 of the liver.

[0080] As used herein, the term "cancer" refers to the excessive proliferation of cells that exhibits a loss of normal cellular control, resulting in unregulated growth, lack of differentiation, invasion of local tissues, and metastasis. Cancer may be a solid tumor, leukemia, lymphoma, or multiple myeloma. As used herein, the term "tumor" refers to an abnormal (e.g., malignant or benign) growth of cells or tissues. Non-limiting examples of cancer include cancer of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancer of the urinary system.

[0081] As used herein, "subject" refers to a human or an animal. Most often, animals are vertebrates, such as primates, rodents, domestic animals, or game animals. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, for example, rhesus monkeys. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, feline species (for example, domestic cats), canine species (for example, dogs, foxes, wolves), avian species (for example, chickens, emus, ostriches), and fish (for example, trout, catfish, and salmon). In some embodiments, the subject is a mammal, for example, a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0082] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment, where the objective is to reverse, alleviate, ameliorate, inhibit, slow down, or stop the progression or severity of a disease-related condition. The term "treating" encompasses reducing or alleviating at least one adverse effect or symptom of diabetes. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" encompasses not only improvement in symptoms or markers, but also a halt in, or at least a delay in, the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), whether detectable or undetectable, attenuation of the extent of the disease, stabilized (i.e., not worsening) disease, slowing or delaying disease progression, amelioration or palliation of the condition, remission (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0083] As used herein, the term "small molecule" refers to organic or inorganic molecules, either natural (i.e., found in nature) or non-natural (i.e., not found in nature), including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (including, by way of example, heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Examples of naturally occurring "small molecules" include, but are not limited to, taxol, dynemycin, and rapamycin. In other preferred embodiments, natural product-like small molecules are also utilized.

[0084] As used herein, "compound" refers to any chemical, test chemical, drug, new chemical entity (NCE), or other moiety. For example, a compound can be any exogenous chemical not normally present in a subject, such as a mammal (including humans). A compound can also be an endogenous chemical normally present and synthesized in a living system, such as a mammal (including humans). For example, a compound, such as a test compound, such as a drug, can reduce the deconjugation of primary and secondary bile acids as provided herein.

[0085] The term "derivative" as used herein refers to any chemical, conservative substitution, or structural modification of an agent. A derivative may improve the characteristics of an agent or small molecule, such as pharmacology, pharmacokinetics, absorption, distribution, delivery, targeting, or efficacy for a specific receptor. For example, for a small molecule, a derivative may consist essentially of at least one chemical modification to about 10 modifications. A derivative may also be the corresponding salt of an agent. A derivative may be a prodrug of a small molecule as provided herein.

[0086] As used herein, the term "bile acid" refers to a steroid acid that aids digestion as an emulsifier of fat and may also play a role in various systemic endocrine hormone-like functions. Mammalian bile acids are synthesized from cholesterol in the liver as primary bile acids and metabolized to secondary bile acids by specific mammalian intestinal microorganisms. Bile acids are stored in the gallbladder and released into the duodenum during food digestion, where they aid in the absorption of lipids and fat-soluble vitamins. Over 95% of bile acids are reabsorbed in the ileum and recycled to the liver. The remaining ~5% enter the colon, where the majority of intestinal bacteria reside. Intestinal bacteria then enzymatically modify the primary bile acids to produce a group of molecules called secondary bile acids.

[0087] Mammalian bile acids regulate metabolic pathways by activating G-protein-coupled receptors (GPCRs) such as farnesoid X receptor and TGR5. Through the activation of these diverse signaling pathways, bile acids can regulate their own enterohepatic circulation, but also the homeostasis of triglycerides, cholesterol, energy, and glucose. 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" can also refer to the salt forms of bile acids, sulfated bile acids, and other metabolites.

[0088] As used herein, "bile salt hydrolase" or "BSH" refers to an enzyme widely expressed by mammalian intestinal bacteria that converts primary bile acids produced by the host into bacterially modified secondary bile acids. Figure 1A provides an example of the deconjugation of primary and secondary bile acids by BSH and their conversion to secondary bile acids by bacterial bile acid-modifying enzymes. Numerous amino acid sequences for BSH from various bacterial species are known in the art (e.g., NCBI Accession Nos. Accession:ABC26911.1; Accession:ABC26910.1; Accession:ACL98203.1; Accession:AAS98803.1; Accession:AKI55714.1; Accession:AAP20760.1). BSH can refer to any bacterial BSH enzyme, without limitation. The keystone reaction in the conversion of primary bile acids to secondary bile acids is the hydrolysis of the C24-amide bond of the conjugated primary bile acid by enterobacterial BSH (Fig. 1A).

[0089] As used herein, a "suitable control" refers to an otherwise identical cell or population that has not been treated (e.g., a subject that has not received an agent provided herein, or a subject that has received only a portion of an agent provided herein, compared to non-control cells). As used herein, the term "pharmaceutical composition" can encompass any material or substance that, when combined with an active ingredient (e.g., compound 7 or a derivative thereof), enables the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers (e.g., phosphate-buffered saline solution), emulsions (e.g., oil / water emulsions), and various types of wetting agents. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0090] An "agent," as used herein, is a chemical molecule of synthetic or biological origin. In the context of the present invention, an agent is generally a molecule that can be used in a pharmaceutical composition.

[0091] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a target agent from one organ (or part of the body) to another organ (or part of the body). The term "pharmaceutically acceptable carrier" excludes 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 during 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 pharmaceutical carriers include particle- or polymer-based vehicles, such as nanoparticles, microparticles, polymeric microspheres, or polymer-drug conjugates.

[0092] As used herein, the term "limiting delivery of a composition to the gastrointestinal tract" refers to a viable form of formulation that permits or facilitates delivery of an agent or pharmaceutical composition described herein to the colon, large intestine, or small intestine. Enteric coatings or micro- or nano-particle formulations can facilitate delivery, as can, for example, buffers or other protective formulations.

[0093] The term "effective amount" is used interchangeably with the term "therapeutically effective amount" or "sufficient amount" and refers to the amount of at least one inhibitor of BSH (e.g., any one of Formulas (I)-(XVIII) or their derivatives) of a pharmaceutical composition for a period and at a dosage necessary to achieve a desired therapeutic result (e.g., "attenuating," reducing, or preventing at least one symptom of diabetes, obesity, or inflammatory disease). For example, an effective amount using the methods as disclosed herein would be considered an amount sufficient to reduce one or more symptoms of diabetes, obesity, or inflammatory disease by at least 10%. As used herein, an effective amount would also encompass an amount sufficient to prevent or delay the onset of such symptoms, alter the course of a disease symptom (e.g., but not limited to, slowing the progression of a disease symptom), or reverse a disease symptom in a subject suffering from diabetes, prediabetes, hyperglycemia, obesity, or inflammatory disease. Consequently, the term "effective amount" or "therapeutically effective amount," as used herein, refers to the amount of a therapeutic agent (e.g., a compound represented by Formulas (I)-(XVIII) or a derivative thereof) in a pharmaceutical composition that alleviates at least one symptom of a disease. In other words, a "therapeutically effective amount" of an inhibitor of BSH as disclosed herein is the amount of the agonist that exerts a beneficial effect on, for example, a symptom of 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 various factors, including the pharmacokinetic properties of the inhibitor, the route of administration, the condition and characteristics of the subject (sex, age, weight, health, size), the severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. The effective amount in each individual case can be determined empirically by one of ordinary skill in the art according to methods established in the art and without undue experimentation.In general, the phrases "therapeutically effective" and "effective in treating, preventing, or inhibiting" are intended to qualify agonists as disclosed herein that will achieve the goal of reducing the severity of, or symptoms associated with, diabetes, cancer, gastrointestinal disease, obesity, or inflammatory disease.

[0094] The term "co-administration" or the like, as used herein, is intended to encompass the administration of selected therapeutic agents to a single affected individual and is intended to encompass treatment regimens in which administration is by the same route or different routes, or at the same time or different times.

[0095] "Unit dosage form," as the term is used herein, refers to a dosage suitable for one administration. As an example, a unit dosage form can be the amount of a therapeutic agent placed in a delivery device (e.g., a syringe or an intravenous drip bag). In one embodiment of any of the aspects, the unit dosage form is administered in a single administration. In another embodiment, more than one unit dosage form can be administered simultaneously.

[0096] The terms "administered" and "provided" are used interchangeably in the context of treating a disease or disorder.

[0097] In jurisdictions that prohibit the patenting of methods practiced on the human body, the meaning of "administering" a composition to a human subject shall be limited to prescribing a controlled substance that the human subject would self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with the statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit the patenting of methods practiced on the human body, "administering" a composition shall encompass both methods practiced on the human body and the activities described above.

[0098] As used herein, the term "administering" 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 so that a desired effect occurs. The compounds or compositions described herein can be administered by any suitable route known in the art, including oral or parenteral routes, including, but not limited to, intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0099] The phrases "parenteral administration" and "parenterally administered," as used herein, refer to modes of administration other than enteral and topical administration, often by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and substernal injection, infusion, and other injection or infusion techniques. Without limitation, oral administration can be in the form of a solution, suspension, tablet, pill, capsule, sustained-release formulation, mouthwash, powder, etc.

[0100] As used herein, the term "modulate" refers to an effect that includes increasing or decreasing a given parameter (as those terms are defined herein).

[0101] As used herein, the term "contacting," when used in 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 permits physical contact between the cell and the agent, surface, hormone, etc., and the introduction of an element such as a genetic construct or vector that permits expression in the cell of an agent (such as a miRNA, polypeptide, or other expression product). It should be understood that a cell that has been genetically modified to express an agent is "contacted" with the agent, as are progeny of the cell that express the agent.

[0102] The terms "statistically significant" or "significant" refer to statistical significance, generally meaning a difference of two standard deviations (2SD) or greater.

[0103] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s), where said component is essential to the method or composition, but are open to encompass unspecified elements, whether essential or not.

[0104] As used herein, the term "consisting essentially of" refers to those elements required in a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0105] Detailed Description of Certain Embodiments of the Invention Human-associated bacteria play essential roles in health and disease. Microbial imbalances lead to a wide range of pathologies. Studies of germ-free mice colonized with single strains, multiple strains, or defined communities of bacteria have revealed the ability of gut bacteria to influence host processes, including metabolism, immune function, and neurological responses. Compounds that selectively alter the levels of specific bacterial metabolites and proteins can be useful for assessing how bacterial products affect host physiology in fully mature animals harboring complex microbial communities and can be used as therapeutics to treat diseases such as metabolic disorders (e.g., diabetes, obesity), gastrointestinal diseases, cancer (e.g., liver cancer), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).

[0106] The compositions and methods provided herein are directed, in part, to the discovery of several compounds that inhibit bile salt hydrolase (BSH) and modulate the deconjugation of primary and secondary bile acids in a subject.

[0107] The compounds provided herein selectively and potently inhibit BSH in a wide range of bacteria, have no off-target effects in the host, can allow restriction to the intestine, and modulate bile acids present in the host subject.

[0108] compound In one aspect, provided herein is a compound of formula (I): [ka] During the ceremony: 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, where each R18 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.

[0109] In some embodiments, the compound of formula (I) has formula (I'): [ka] During the ceremony: 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, -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, where each 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; or a pharmaceutically acceptable salt thereof.

[0110] In some embodiments, the compound of Formula (I) has the formula (Ia): [ka] During the ceremony: 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.

[0111] In some embodiments, the compound of formula (Ia) has the formula (I-a'): [ka] During the ceremony: 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.

[0112] In some embodiments, the compound of formula (I) has formula (Ib): [ka] During the ceremony: 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.

[0113] In some embodiments, the compound of formula (Ib) has the formula (I-b'): [ka] During the ceremony: 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.

[0114] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (Ic): [ka] or a pharmaceutically acceptable salt thereof.

[0115] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (Id): [ka] or a pharmaceutically acceptable salt thereof.

[0116] In some embodiments, the compound of Formula (I) or Formula (I') is of Formula (Ie): [ka] or a pharmaceutically acceptable salt thereof.

[0117] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (If): [ka] or a pharmaceutically acceptable salt thereof.

[0118] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (Ig): [ka] or a pharmaceutically acceptable salt thereof.

[0119] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (Ih): [ka] or a pharmaceutically acceptable salt thereof.

[0120] In some embodiments, the compound of Formula (I) or Formula (I') has Formula (Ii): [ka] or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (I-c'): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 3a , R 7a , and R 12a are independently -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 some embodiments, R 3a , R 7a , and R 12a are independently -OR 18 , —SO3H, —OSO3H, —PO3H2, —OPO3H2, —OSO2H, and —SO2NH2, wherein each R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a , R 7a , and R 12a are independently -OR 18 , —SO3H, and —OSO3H, where R 18 is H, or substituted or unsubstituted alkyl. In some embodiments, R 3a , R 7a , and R 12a is independently selected from the group consisting of —OH and —OSO3H.

[0122] In some embodiments, the compound of Formula (I) or Formula (I') has the formula (I-d'): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony:

[0123] R 3a and R 12a are independently -OR 18 , -SO3R18 , -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 some embodiments, R 3a and R 12a are independently -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a and R 12a is independently selected from the group consisting of -OH and -OSOH. In some embodiments, the compound of formula (I) or formula (I') has formula (I-d''): [ka] or a pharmaceutically acceptable salt thereof.

[0124] In some embodiments, the compound of formula (I) or formula (I′) has formula (I-e′): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 3a and R 7a are independently -OR 18 , -SO3R 18 , -OSO3R 18 , -PO3(R 18 )2, -OPO3(R 18 )2, -OSO2R 18 , and -SO2N(R 18 )2, wherein each R 18is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a and R 7a are independently -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a and R 7a is independently selected from the group consisting of -OH and -OSOH. 3a is -OSO3H, and R 7a -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a is -OH, and R 7a -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl.

[0125] In some embodiments, the compound of formula (Ie) has the formula (I-e″): [ka] or a pharmaceutically acceptable salt thereof.

[0126] In some embodiments, the compound of formula (I-e″) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0127] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (I-f′): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 3a -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 some embodiments, R 3a -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a is -OSOH. In some embodiments, R 3a is -OH.

[0128] In some embodiments, the compound of formula (I) or (I′) has the formula (I-f″): [ka] or a pharmaceutically acceptable salt thereof.

[0129] In some embodiments, the compound of formula (I-f′) has formula (I-f′″): [ka] or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the compound of formula (I-f''') has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0131] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (I-g′): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 3a , R 6a , and R 7a are independently -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 some embodiments, R 3a , R 6a , and R 7a are independently -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a , R 6a , and R 7a is independently selected from the group consisting of —OH and —OSO3H.

[0132] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (I-g″): [ka] or a pharmaceutically acceptable salt thereof.

[0133] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (I-h′): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 7a and R 12a are independently -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 some embodiments, R 7a and R 12a are independently -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 7a and R 12a is independently selected from the group consisting of —OH and —OSO3H.

[0134] In some embodiments, the compound of Formula (I) or Formula (I′) has the formula (I-h″): [ka] or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments, the compound of formula (I) or formula (I′) has formula (I-i′): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony: R 3a and R 6a are independently -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 some embodiments, R 3a and R 6a are independently -OR 18 , -SO3H, -OSO3H, -PO3H2, -OPO3H2, -OSO2H, and -SO2NH2, where R 18 is independently H or substituted or unsubstituted alkyl. In some embodiments, R 3a and R 6a is independently selected from the group consisting of —OH and —OSO3H.

[0136] In some embodiments, the compound of formula (I) or formula (I′) is of formula (I-i″): [ka] or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the compound of any one of formulas (I-c'), (I-d'), (I-e'), (I-f'), (I-g'), (I-h'), or (I-i') may comprise a substituent R 3a , R 6a , R 7a , or R 12a In some embodiments, R 3a , R 6a , R 7a , and R 12a are independently -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 some embodiments, R 3a , R 6a , R 7a , or R 12a Independently, -OR if at least one of 18 In some embodiments, R 3a , R 6a , R 7a , or R 12a In at least one of the cases, independently, -SO3R 18 In some embodiments, R 3a , R 6a , R 7a , or R 12a Independently, -OSO3R for at least one of 18 In some embodiments, R 3a , R 6a , R 7a , or R 12a In at least one of the cases, independently, -PO3(R 18 )2. In some embodiments, R 3a , R 6a , R 7a , or R 12a In at least one of the cases, independently, -OPO3(R 18)2. In some embodiments, R 3a , R 6a , R 7a , or R 12a Independently, -OSO2R for at least one of 18 In some embodiments, R 3a , R 6a , R 7a , or R 12a In at least one of the cases, independently, -SO2N(R 18 )2. In some embodiments, R 3a is -OH, and R 6a , R 7a , and R 12a is independently selected from the group consisting of -OH and -OSOH. 3a is -OSO3H, and R 6a , R 7a , and R 12a is independently selected from the group consisting of —OH and —OSO3H.

[0138] In some embodiments of the various aspects disclosed herein, the compound of formula (I) is represented by formulas (II)-(XV): [ka] [ka] The compound may be a compound represented by any one of the following formulas:

[0139] In compounds represented by formulas (I)-(XV), X is an electrophilic group. The terms "electrophile" and "electrophile" refer to a functional group susceptible to nucleophilic attack, i.e., a functional group susceptible to reaction with an incoming nucleophilic group (e.g., thiol, amine). Generally, an electrophilic group is a grouping of atoms, one or more of which are electron-deficient. Electrophilic groups often contain an electron-donating group. Examples of electron-donating groups include, but are not limited to, halo, nitro, cyano, ester, aldehyde, keto, sulfone, or amide groups. The electron-deficient atom(s) 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, acid halides, isothiocyanates, isocyanates, epoxy, and anhydride groups.

[0140] 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" refers to any group that is susceptible to nucleophilic attack by the lone electron pair on the sulfur atom of a thiol group or by a thiolate anion. Examples of thiol-reactive electrophilic groups include groups with good leaving groups. For example, α-halocarbonyl groups, isothiocyanate groups, isocyanate groups, alkyl groups, or alkoxy groups attached thereto, and electron-deficient vinyl groups, each of which has a halide. In some embodiments, X is an α-halocarbonyl group or an isothiocyanate group.

[0141] 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 is alkyl.

[0142] In some embodiments, X is —C(O)R 19 However, here R 19 is alkyl, haloalkyl, alkenyl, or alkynyl. In some embodiments, X is —C(O)R 19 However, here R 19 In some embodiments, X is -NCS. In some embodiments, X is -NHC(O)R 19 However, here R 19 is alkyl (e.g., Me, Et, Pr), haloalkyl (e.g., CHF), and in some embodiments, X is -CH=C(CN)COR 20 However, here R 20 is alkyl. In some embodiments, X is -CN.

[0143] In some embodiments, X is [ka] is an electrophilic group selected from the group consisting of:

[0144] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka] In some embodiments, X is [ka]

[0145] In some embodiments, X is —C(O)R 19 or -NCS, where R 19 is haloalkyl. For example, X is —C(O)CHF or —NCS.

[0146] In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently H. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently substituted or unsubstituted alkyl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently substituted or unsubstituted heteroalkyl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently substituted or unsubstituted cycloalkyl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17are independently substituted or unsubstituted heterocycloalkyl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently substituted or unsubstituted aryl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently substituted or unsubstituted heteroaryl. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently OR 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently N(R 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 independently, SR 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently halogen. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently CN. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R17 are independently -CHO. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently —COH. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 is independently -CO2R 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -NO. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently -ONO. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently —SO2Cl. In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -SO3 - In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 is independently -OSO3 - In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17are independently -NR 18 SO3 - In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 is independently -PO3 2- In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -OPO3 2- In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 independently, -OSO2R 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -SO2N(R 18 In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -OSON(R 18 In some 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 some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -SO2N(R 18In some embodiments, R1, R2, R3, R4, R6, R7, R 11 , R 12 , R 15 , R 16 and R 17 are independently -NHNH. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently -ONH. In some embodiments, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 and R 17 are independently —NHC(O)NHNH2.

[0147] In some embodiments, R 18 is H. In some embodiments, R 18 is substituted or unsubstituted alkyl. In some embodiments, R 18 is substituted or unsubstituted heteroalkyl. In some embodiments, R 18 is substituted or unsubstituted cycloalkyl. In some embodiments, R 18 is a substituted or unsubstituted heterocycloalkyl. In some embodiments, R 18 is substituted or unsubstituted aryl. In some embodiments, R 18 is a substituted or unsubstituted heteroaryl.

[0148] In the compound represented by formula (I), R1, R2, R4, R6, R 11 , R 15 , and R 16 At least one of R, R, R, R, R can be H. For example, R, R, R 11 , R 15 , and R 16 Of the various aspects disclosed herein, 1, 2, 3, 4, 5, 6, or all 7 can be H. In some embodiments of the various aspects disclosed herein, R, R, R, R, R 11 , R15 , and R 16 All of them are H.

[0149] In some compounds of formula (I), R3, R7 and R 12 At least one of the is -OR 18 For example, R3, R7, and R 12 One, two or all three of the 18 Accordingly, in some embodiments of the various aspects disclosed herein, R3 can be -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 -OR 18 In some embodiments, R3 and R7 are -OR 18 In some embodiments, R and R 12 -OR 18 In some embodiments, R and R 12 -OR 18 In some embodiments, R, R, and R 12 All of the -OR 18 It could be.

[0150] In some embodiments of the various aspects disclosed herein, at least one of R3 and R7 is -OR 18 and R 12 is H or OR 18 For example, at least one of R3 and R7 is -OH, and R 12 is H or -OH.

[0151] In some additional embodiments of the various aspects disclosed herein, R3 and R7 are -OR 18 and R 12 is H or -OR 18 For example, R3 and R7 are -OH, and R 12 is H or -OH.

[0152] In some compounds of formula (I), R3, R6, R7, and R 12 At least one of the is -OSO3 - , -NR 18 SO3 - , or -OPO3 2- In some further embodiments thereof, R3, R6, R7, and R 12 At least one of the is -OSO3 - In some specific embodiments, R3 is -OSO3 - is.

[0153] Example 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 is H.

[0154] In the compound of formula (I), R 17 can be C1-C6 alkyl. For example, R 17 can be methyl, ethyl, propyl, isopropyl, butyl, pentyl. In some embodiments of the various aspects disclosed herein, R 17 is methyl.

[0155] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0156] In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

[0157] In compounds of formula (I), n can be 1 or 2. In some exemplary compounds of formulas (I)-(XVIII), n is 2.

[0158] In compounds of formula (I), m can be 1, 2, or 3. In some exemplary compounds of formulas (I)-(XVIII), m is 1.

[0159] In some embodiments of the various aspects described herein, the compound of Formula (I) has Formula (XVI): [ka] where R 19 is haloalkyl; R, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 , R 17 , n, and m are as defined for Formula (I). In some exemplary compounds of Formula (XVI), m is 1 or 2; n is 1 or 2; R, R, R, R, 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, for example, —CHF. In some other exemplary compounds of formula (XVI), m is 1 or 2; n is 1 or 2; R, R, R, R, 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, for example, —CHF.

[0160] In some embodiments of the various aspects described herein, the compound of Formula (I) has the formula (XVII): [ka] where R 19 is haloalkyl; R, R, R, R, R, R, R 11 , R 12 , R 15 , R 16 , R 17 , n, and m are as defined for Formula (I). In some exemplary compounds of Formula (XVII), m is 1 or 2; n is 1 or 2; R, R, R, R, 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 In some other exemplary compounds of formula (XVII), m is 1 or 2; n is 1 or 2; R, R, R, R, 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, for example, —CHF.

[0161] In some embodiments of the various aspects described herein, the compound of Formula (I) has Formula (XVIII): [ka] where R 19 is haloalkyl; R, R, R, R, R, R, R 11 , R 12 , R15 , R 16 , R 17 , n, and m are as defined for Formula (I). In some exemplary compounds of Formula (XVIII), m is 1 or 2; n is 1 or 2; R, R, R, R, 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, for example, —CHF. In some other exemplary compounds of formula (XVIII), m is 1 or 2; n is 1 or 2; R, R, R, R, 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, for example, —CHF.

[0162] In some embodiments, the compound of formula (XVIII) has formula (XVIII-a): [ka] During the ceremony: 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 R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 18 is H, and R 19is haloalkyl (e.g., —CHF).

[0163] In some embodiments, the compound of formula (XVIII-a) has formula (XVIII-a′): [ka] During the ceremony: R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 19 is haloalkyl (e.g., —CHF).

[0164] In some embodiments, the compound of formula (XVIII) has formula (XVIII-b): [ka] During the ceremony: 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 R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 18 is H, and R 19 is haloalkyl (e.g., —CHF).

[0165] In some embodiments, the compound of formula (XVIII-b) has formula (XVIII-b'): [ka] During the ceremony: R 19is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 19 is haloalkyl (e.g., —CHF).

[0166] In some embodiments, the compound of formula (XVIII) has formula (XVIII-c): [ka] During the ceremony: 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 R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 18 is H, and R 19 is haloalkyl (e.g., —CHF).

[0167] In some embodiments, the compound of formula (XVIII-c) has formula (XVIII-c'): [ka] During the ceremony: R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 19 is haloalkyl (e.g., —CHF).

[0168] In some embodiments, the compound of formula (XVIII) has formula (XVIII-d): [ka] During the ceremony: 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 R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 18 is H, and R 19 is haloalkyl (e.g., —CHF).

[0169] In some embodiments, the compound of formula (XVIII-d) has formula (XVIII-d'): [ka] During the ceremony: R 19 is alkyl, haloalkyl, alkenyl, or alkynyl; or a pharmaceutically acceptable salt thereof. In some embodiments, R 19 is haloalkyl (e.g., —CHF).

[0170] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF, —NCS, —C(O)CH═CH, —C(O)C═CH, —NHC(O)CH═CH, —CN, —CH═C(CN)COEt, or —C(O)CH; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R3 is -OH or -OSO3 - R7 is -OH; and R 12is H or -OH.

[0171] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF, —NCS, —C(O)CH═CH, —C(O)C≡CH, —NHC(O)CH═CH, —CN, —CH═C(CN)COEt, or —C(O)CH; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R is -OH or -OSOH; R is -OH; and R 12 is H or -OH.

[0172] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF or —NCS; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R3 is -OH or -OSO3 - R7 is -OH; and R 12 is H or -OH.

[0173] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF or —NCS; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R is -OH or -OSOH; R is -OH; and R 12 is H or -OH.

[0174] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF; R, R, R, R 16 , R 11 , R 15 and R 16is H; R3 is -OH or -OSO3 - R7 is -OH; and R 12 is H or -OH.

[0175] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R is -OH or -OSOH; R is -OH; and R 12 is H or -OH.

[0176] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R3 is -OH and R7 is OH; and R 12 is H or -OH.

[0177] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R3 is -OSO3 - and R7 is -OH; and R 12 is H.

[0178] In some embodiments of the various aspects disclosed herein, m is 1; n is 2; X is —C(O)CHF; R, R, R, R 16 , R 11 , R 15 and R 16 is H; R is -OSOH; and R is -OH; and R 12is H.

[0179] In embodiments of the various aspects disclosed herein, the compounds of formula (I) do not modulate the activity of TGR5, in other words, the compounds of formula (I) are neither agonists nor antagonists of TGR5.

[0180] Substituents for alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —COR′, —CONR′, in numbers ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in the radical. and n is 0 or 1. The substituents may be one or more of a variety of groups selected from the group consisting of —R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSO2R′, —NR′NR″R′″, —ONR′R″, —NR′C═(O)NR″NR′″R′″, —CN, —NO2, —NR′S02R″, —NR′C═(O)R″, —NR′C(O)—OR″, and —NR′OR″. R', R', R'', R''', and R'''' each preferably independently represent hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1 to 3 halogens), a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkyl group, an alkoxy group, or a thioalkoxy group, or an arylalkyl group. When a compound of the invention includes more than one R group, e.g., each of the R groups is independently selected, similarly to each R', R'', R''', and R'''' group, when more than one of these groups is present. 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.From the above discussion of substituents, one of ordinary skill in the art will understand that the term "alkyl" is intended to include groups that include a carbon atom bonded to a group other than a hydrogen group, such as haloalkyl (e.g., -CF and -CHCF) and acyl (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0181] Similar to the substituents recited for the alkyl radical, the substituents on the aryl and heteroaryl groups vary in number from zero to the total number of open valences on the aromatic ring system, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R'''', -NR''C(O)R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR''R'')-NR'''', -S(O)R', -S(O)R', -S(O)NR'R'', -NRSOR', -NR'NR'' 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, each of the R groups is independently selected, as are each R', R'', R''', and R'''' groups when more than one of these groups is present.

[0182] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be drawn as a substituent on the ring rather than on a specific atom of the ring (commonly referred to as a floating substituent). In such cases, the substituent may be attached to any of the ring atoms (subject to the rules of chemical valence). And, in the case of a fused or spirocyclic ring, a substituent drawn as being associated with one member of the fused or spirocyclic ring (a floating substituent on a single ring) may be a substituent on either the fused or spirocyclic ring (a floating substituent on a polycyclic ring). When a substituent is attached to a ring rather than to a specific atom (a floating substituent), and the subscript associated with the substituent is an integer greater than 1, multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, or different spirocyclic rings, and each substituent may optionally be different. When the point of attachment of the ring to the rest of the molecule is not limited to a single atom (flowing substituent), the point of attachment may be any atom of the ring, and in the case of a fused or spirocyclic ring, it may be any atom of either the fused or spirocyclic ring, subject to the rules of chemical valence. When a monocyclic, fused, or spirocyclic ring contains one or more ring heteroatoms, and when a monocyclic, fused, or spirocyclic ring is shown with more than one flowing substituent (including, but not limited to, points of attachment to the rest of the molecule), the flowing substituent may be bonded to a heteroatom. When a ring heteroatom is shown in a structure or formula with a flowing substituent as being bonded to one or more hydrogens (e.g., a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen), it will be understood that when the heteroatom is bonded to the flowing substituent, the substituent replaces the hydrogen, subject to the rules of chemical valence.

[0183] Two or more substituents may optionally be joined to form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group. Such ring-forming substituents, as they are called, are typically, but not necessarily, found attached to a cyclic platform structure. In some embodiments of any of the aspects, the ring-forming substituents are attached to adjacent members of the platform structure. For example, two ring-forming substituents attached to adjacent members of a cyclic platform structure create a fused ring structure. In another embodiment of any of the aspects, the ring-forming substituents are attached to a single member of the platform structure. For example, two ring-forming substituents attached to a single member of a cyclic platform structure create a spirocyclic structure. In another embodiment, the ring-forming substituents are attached to non-adjacent members of the platform structure.

[0184] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CRR') q Optionally, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may form a ring represented by the formula -A-(CH) r A and B may be optionally replaced by a substituent represented by the formula -B-, where 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 in the new ring so formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent represented by the formula -(CRR') s -X'-(C''R''R'') d-, where 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.

[0185] In some embodiments, the compound of formula (I) has the formula: [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0186] In some embodiments, the compound of formula (I) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0187] In some embodiments, the compound of formula (I) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0188] In some embodiments, the compound of formula (I) has the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0189] In some embodiments, the compound of formula (I) has the formula: [ka] It is not expressed in

[0190] In some embodiments, the compound of formula (I) has the formula: [ka] It is not expressed in

[0191] In some embodiments, each substituent described in the compounds herein is substituted with at least one substituent. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one substituent that is limited in size. In other embodiments, at least one or all of these groups are substituted with at least one lower-rank substituent.

[0192] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 cycloalkyl, and / or each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-8 membered heterocycloalkyl. 20 alkylene, wherein 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.

[0193] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, and / or each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, and / or each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-7 membered heterocycloalkylene.

[0194] 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, and individual isomers, sometimes defined in terms of absolute stereochemistry as (R)- or (S)- for amino acids, or (D)- or (L)-, are encompassed within the scope of the present invention. Compounds of the present invention do not include those known in the art to be too unstable to synthesize and / or isolate. The present invention is intended to encompass compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When compounds described herein contain olefinic bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to encompass both E and Z geometric isomers.

[0195] Thus, the compounds of the present invention may exist as salts with pharmaceutically acceptable acids, etc. The present invention encompasses such salts. Examples of such salts include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or a mixture thereof, including a racemic mixture), succinate, benzoate, and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.

[0196] 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.

[0197] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that easily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0198] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may also exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0199] Unless otherwise stated, structures depicted herein are also intended to encompass all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Accordingly, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0200] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of hydrogen by deuterium or tritium, or 13 C or 14 Compounds having the present structure, except for the replacement of a carbon with a C-enriched carbon, are within the scope of this invention.

[0201] 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 contain isotopes of, for example, 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.

[0202] Pharmaceutical Compositions, Kits, and Administration In yet another aspect, provided herein is a pharmaceutical composition comprising a compound of Formulas (I)-(XVIII) and a pharmaceutically acceptable carrier or excipient.

[0203] In some embodiments of any of the aspects, the agent or compound as provided herein is formulated in a pharmaceutical composition. In another embodiment of any of the aspects, the pharmaceutical composition is used to treat diseases (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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and and urinary system 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).

[0204] In another aspect of any of the embodiments, provided herein is a composition comprising an agent that inhibits bile salt hydrolase (BSH) in a subject.

[0205] In another embodiment of any of the aspects, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0206] The present disclosure provides pharmaceutical compositions comprising a compound represented by Formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and, optionally, a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions described herein comprise a compound represented by Formula (I)-(XVIII), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0207] In some embodiments, the pharmaceutical composition is in a liquid or solid dosage form. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the compound of any one of Formulas (I) to (XVIII), the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0208] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound of any one of Formulas (I)-(XVIII) is combined 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, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders, such as paraffin. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.

[0209] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0210] The compounds of any of Formulas (I)-(XVIII) may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulation art. In such solid dosage forms, the compounds of any of Formulas (I)-(XVIII) may be admixed with at least one inert diluent (such as sucrose, lactose, and starch). Such dosage forms may also contain, as is customary, additional substances other than inert diluents, such as tableting lubricants and other tableting aids (such as magnesium stearate and microcrystalline cellulose). In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0211] 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 the addition of sulfate groups or polar groups to the compounds.

[0212] In some embodiments, the carrier or excipient is an enteric coating or enteric-coated drug delivery device.As used herein, the term "enteric coating" or "enteric-coated drug delivery device" refers to any drug delivery method that can be administered orally but is not degraded or activated until the device enters the intestine.For example, such a method can utilize a coating or encapsulation that is degraded using a pH-dependent means, allowing the delivery device and agent to be administered or implanted to be protected throughout the gastrointestinal tract until the device reaches the alkaline pH of the intestine (for example, the cecum or colon).

[0213] Enteric coatings can control the location in the digestive system where an agent is released. Thus, enteric coatings can be used so that the pharmaceutical composition does not dissolve and release the agent in the stomach, but rather flows to the intestine, where it dissolves and releases the agent in the most beneficial environment for inhibiting BSH (e.g., targeting the intestines located in the cecum, ileum, large intestine, or colon). Enteric coatings can be stable at low pH (such as in the stomach) and dissolve at higher pH (e.g., 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 by reference in their entireties. The enteric coating may be an aqueous enteric coating. Examples of polymers that can be used in enteric coatings include, for example, shellac (trade names EmCoat 120 N, Marcoat 125); cellulose acetate phthalate (trade names AQUACOAT®, AQUACOAT ECD®, SEPIFILM®, KLUCEL®, and METOLOSE®); polyvinyl acetate phthalate (trade name SURETERIC®); and methacrylic acid (trade names EUDRAGIT®, EUDRAGIT L 100-55®, from Evonik Industries, Germany).

[0214] Another example of a method known in the art that allows for the restriction of a pharmaceutical composition to the intestine involves enteric magnesium micromotors (EMgM), which have been described in the art, for example, in Li et al., ACS NANO, (2016).

[0215] Pharmaceutical compositions include formulations suitable for oral administration which may be presented as discrete units such as tablets, capsules, cachets, syrups, elixirs, prepared foods, microemulsions, solutions, suspensions, lozenges, or gel-coated ampoules, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.

[0216] Accordingly, formulations suitable for rectal administration include, and may be used, gels, creams, lotions, aqueous or oily suspensions, dispersible powders or granules, emulsions, dissolvable solid materials, douches, and the like. The formulations are preferably provided as unit-dose suppositories, comprising the active ingredient in one or more solid carriers (e.g., cocoa butter) forming the suppository base. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycols, alcohols, and combinations thereof. Alternatively, colon cleansing with the rapid recolonization / deployment agent of the present disclosure can be formulated for colonic or rectal administration.

[0217] In some embodiments, the compound or pharmaceutical composition is a solid. In some embodiments, the compound or pharmaceutical composition is a powder. In some embodiments, the compound or pharmaceutical composition can be dissolved in a liquid to form a solution. In some embodiments, the compound or pharmaceutical composition can be dissolved in water to form an aqueous solution. In some embodiments, the pharmaceutical composition is a liquid for parenteral injection. In some embodiments, the pharmaceutical composition is a liquid for oral administration (e.g., ingestion). In some embodiments, the pharmaceutical composition is a liquid for intravenous injection (e.g., an aqueous solution). In some embodiments, the pharmaceutical composition is a liquid for subcutaneous injection (e.g., an aqueous solution).

[0218] After formulation with suitable pharmaceutically acceptable excipients in the desired dosage, the pharmaceutical compositions of the present disclosure can be administered to humans and other animals orally, parenterally, intracisternally, intraperitoneally, topically, bucally, or the like, depending on the disease or condition being treated.

[0219] In some embodiments, pharmaceutical compositions comprising compounds of Formulae (I)-(XVIII) are administered orally or parenterally at dosage levels of each pharmaceutical composition sufficient to deliver from about 0.001 mg / kg to about 200 mg / kg in one or more doses (depending on the mode of administration) over one or more days. In certain embodiments, the effective amount per dose ranges from about 0.001 mg / kg to about 200 mg / kg, from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg of subject body weight per day, one or more times per day, to obtain the desired therapeutic and / or prophylactic effect. In certain embodiments, the compounds described herein may be at a dosage level sufficient to deliver from about 0.001 mg / kg to about 200 mg / kg, from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 40 mg / kg, preferably from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, and more preferably from about 1 mg / kg to about 25 mg / kg of subject body weight per day, one or more times per day, to obtain the desired therapeutic and / or prophylactic effect. The desired dosage may 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 some embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). In some embodiments, the compositions described herein are administered at a dose below the dose at which the compound or agent causes non-specific effects.

[0220] In some embodiments, the pharmaceutical composition is administered at a dose of about 0.001 mg to about 1000 mg per unit dose. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 200 mg per unit dose. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 100 mg per unit dose. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 50 mg per unit dose. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg to about 10 mg per unit dose. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.1 mg to about 10 mg per unit dose.

[0221] The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology. In general, such methods include the step of bringing into association a composition comprising a compound of Formulas (I)-(XVIII) with the carrier and / or one or more other accessory ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0222] Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0223] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, 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, as well as the route by which the composition is to be administered. As an example, the composition may contain between 0.1% and 100% (w / w) active ingredient.

[0224] Pharmaceutically acceptable excipients used in the preparation of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and perfuming agents may also be present in the compositions.

[0225] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0226] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, 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), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0227] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, 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 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, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor™), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (Brij30), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, Poloxamer-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0228] 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., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, isapol husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0229] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0230] 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.

[0231] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., edetate sodium, edetate disodium, edetate trisodium, edetate calcium disodium, edetate dipotassium, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malate and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0232] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0233] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0234] 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.

[0235] Other preservatives include tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, 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.

[0236] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0237] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, 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.

[0238] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor oil, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, loofah, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon. 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.

[0239] 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 ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, and mixtures thereof, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan.In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavoring agents, and fragrances. In certain embodiments for parenteral administration, the agents of the present invention are mixed with a solubilizing agent such as CREMOPHOR EL® (polyethoxylated castor oil), alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

[0240] Injectable preparations, for example, sterile injectable aqueous or oily suspensions can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents.Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol.Among acceptable vehicles and solvents, water, Ringer's solution (USP), and isotonic sodium chloride solution can be used.In addition, sterile fixed oils are conventionally used as solvents or suspending media.For this purpose, any non-irritating fixed oil, including synthetic mono- or diglycerides, can be used.In addition, fatty acids such as oleic acid are used in injectable preparations.

[0241] Injectable formulations can be sterilized, for example, by filtration through a bacterial-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 prior to use.

[0242] 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, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders, such as paraffin. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0243] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0244] The active agent may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings known in the pharmaceutical formulation art. In such solid dosage forms, the active agent may be admixed with at least one inert diluent (such as sucrose, lactose, and starch). These dosage forms may also contain, as is customary, additional substances other than inert diluents, such as tableting lubricants and other tableting aids (such as magnesium stearate and microcrystalline cellulose). In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents, and may also be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0245] Formulations suitable for topical administration include liquid or semi-liquid preparations, such as liniments, lotions, gels, applicants, oil-in-water or water-in-oil emulsions (e.g., 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 lotions, creams, ointments, or soaps. Useful carriers are capable of forming a thin film or layer on the skin to localize application and inhibit 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, hydroxypropyl cellulose or fibrinogen / thrombin solutions can be advantageously used. Alternatively, tissue-coating solutions, such as pectin-containing formulations, can be used. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of an agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the correct medium. Absorption enhancers can also be used to increase the flux of the agent across the skin. The rate can be controlled by providing either a rate-controlling membrane or a dispersing agent in a polymer matrix or gel.

[0246] In addition, carriers for topical formulations can be in the form of hydroalcoholic systems (e.g., quids 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 viscosities, including thin lotions (which may also be suitable for spray or aerosol delivery), creamy lotions, light creams, heavy creams, and the like. Emulsions can also include microemulsion systems. Other suitable topical carriers include anhydrous solids and semisolids (such as gels and sticks); and aqueous-based mousse systems.

[0247] Also encompassed by the present disclosure are kits (e.g., pharmaceutical packs). The provided kits may include a pharmaceutical composition or compound described herein and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the provided kits may optionally further include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical compositions or compounds described herein provided in the first and second containers are combined to form a single-unit dosage form.

[0248] Thus, in one aspect, provided is a kit comprising a first container containing a compound or pharmaceutical composition represented by Formulae (I)-(XVIII) described herein. In some embodiments, the kit is 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., urinary tract infections (e.g., urinary tract infections (U.S ... For example, they are useful for treating cancers of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), inflammatory diseases (for example, 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 some embodiments, the kits are used to treat diseases (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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancer of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; The kit is useful for preventing cancers (e.g., cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), inflammatory diseases (e.g., 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 some embodiments, the kit is useful for reducing the risk of developing a disease (e.g., metabolic disorders (e.g., diabetes, obesity), gastrointestinal disease, cancer, inflammatory diseases (e.g., 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.

[0249] In some embodiments, the kits described herein further include instructions for using the kit. The kits described herein may also include information from regulatory authorities, such as the U.S. Food and Drug Administration (FDA), as needed. In some embodiments, the information included in the kit is prescribing information. In some embodiments, the kits and instructions provide treatment of a disease (e.g., metabolic disorders (e.g., diabetes, obesity), inflammatory diseases (e.g., 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 some embodiments, the kits and instructions provide treatment of 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 of the liver; gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancer of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer) and cancers of the urinary system), 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) in a subject in need thereof.In certain embodiments, the kits and instructions provide for a reduced 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 also include one or more additional pharmaceutical compounds described herein in separate compositions.

[0250] Treatment method In one aspect, provided herein is a method for modulating bile acids in a subject. In another aspect, provided herein is a method for inhibiting deconjugation of bile acids in a subject. In another aspect, provided herein is a method for promoting conjugation of bile acids in a subject.

[0251] In one aspect of any of the embodiments, 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 represented by any one of Formulas I-XVIII, a derivative thereof, or a pharmaceutical composition provided herein.

[0252] In some embodiments of any of the aspects, the agent is an inhibitor of BSH. In some embodiments of any of the aspects, the agent is an inhibitor of BSH of bacteria present in the host subject.

[0253] In another embodiment of any of the aspects, the agent or inhibitor is a compound of Formula (I)-(XVIII) or a derivative thereof; Compounds 1-9 or a derivative thereof; riboflavin; or caffeic acid phenethyl ester (CAPE). Compounds 1-9 are also shown in Figure 2D.

[0254] In another embodiment of any of the aspects, the inhibitor is selected from the group consisting of a small molecule, an antibody, a peptide, a genome editing system, an antisense oligonucleotide, an shRNA, and an siRNA.

[0255] In some embodiments of any aspect, the agent that inhibits BSH is RNAi, siRNA, or shRNA.The term "RNAi" or "siRNA" or "shRNA" as used herein refers to interfering RNA or RNA interference.RNAi refers to a method of selective post-transcriptional gene silencing by binding to mRNA and inhibiting its processing (for example, inhibiting mRNA translation) or destroying specific mRNA by molecules that cause mRNA degradation.As used herein, the term "RNAi" refers to any type of interfering RNA, including but not limited to siRNA, shRNA, endogenous microRNA, and artificial microRNA.For example, it includes the sequence previously identified as siRNA, regardless of the mechanism of downstream processing of RNA.

[0256] In some embodiments of any of the aspects, the agent that inhibits BSH is an antisense oligonucleotide. As used herein, "antisense oligonucleotide" refers to a synthetic nucleic acid sequence complementary to a DNA or mRNA sequence, such as a microRNA sequence. Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and silencing expression at the transcription, translation, or splicing level. As described herein, antisense oligonucleotides are complementary nucleic acid sequences designed to hybridize with a gene under cellular conditions. Thus, oligonucleotides are selected that are sufficiently complementary to the target, i.e., sufficiently hybridize to exert the desired effect with sufficient specificity in the context of the cellular environment. For example, antisense oligonucleotides that directly or indirectly inhibit BSH levels or activity may contain at least 5, 10, 15, 20, 25, 30, or more bases complementary to a portion of the coding sequence of bacterial BSH. Furthermore, antisense oligonucleotides can target transcription factors that regulate bacterial BSH expression.

[0257] 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 predetermined antigen. The antibody reagent may include an antibody or a polypeptide that includes the antigen-binding domain of an antibody. In some embodiments of any of the aspects, the antibody reagent may include a monoclonal antibody or a polypeptide that includes the antigen-binding domain of a monoclonal antibody. For example, the 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 antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996;26(3):629-39, incorporated by reference herein in its entirety)) as well as antigen-binding fragments of complete antibodies. Antibodies may have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as subtypes and combinations thereof). Antibodies may be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) antibodies, as well as primatized antibodies. Antibodies also encompass broadly neutralizing antibodies, minibodies, nanobodies, humanized antibodies, chimeric antibodies, and the like.

[0258] In other embodiments, the agent that inhibits BSH is a polypeptide. As used herein, the term "polypeptide" is intended to encompass the singular form "polypeptide" as well as the plural form "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 encompassed within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term further encompasses polypeptides that have undergone one or more post-translational modification(s), 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 exists in the art for polynucleotide and polypeptide structures. For example, one-letter and three-letter abbreviations are widely employed to describe the amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine ​​(C; Cys), glutamine (Q; Gln), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val), and lysine (K; Lys). The amino acid residues provided herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue while retaining the desired properties of the polypeptide.

[0259] In another embodiment of any aspect, BSH is inhibited in bacterial cell genome using a genome editing system, including but not limited to zinc finger nucleases, TALEN, meganucleases, and CRISPR / Cas systems.In some embodiments of any aspect, the genome editing system used to integrate nucleic acids encoding one or more guide RNAs into the genome of a cell is not a CRISPR / Cas system; this can prevent undesired cell death in cells that retain small amounts of Cas enzyme / protein.It is also contemplated herein that either the Cas enzyme or sgRNA can be expressed under the control of different inducible promoters, thereby allowing each temporal expression to prevent such interference.The gene editing system can directly or indirectly modulate the level or activity or expression of BSH.

[0260] 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.

[0261] In some embodiments of any of the aspects, the agent is an inhibitor of bile salt hydrolase (BSH). In other embodiments of any of the aspects, the agent is a compound represented by any one of Formulas I-XVIII or 3-sulfated-lithocholic acid-fluoromethylketone (3S-LCA-FMK). In other embodiments of any of the aspects, the agent is a derivative represented by any one of Formulas I-XVIII or 3-sulfated-lithocholic acid-fluoromethylketone (3S-LCA-FMK). In other embodiments of any of the aspects, the agent is a bile acid or a derivative thereof. In other embodiments of any of the aspects, the agent is chenodeoxycholic acid (CDCA) or a derivative thereof.

[0262] In some embodiments, inhibition of BSH results in a reduction of secondary bile acids. In other embodiments, inhibition of BSH promotes the conjugation of bile acids. In another embodiment, inhibition of BSH reduces the deconjugation of bile acids. The activity of BSH can be determined by the presence or absence of deconjugated bile acids.

[0263] In some embodiments of any of the aspects, 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 relative to a suitable control.

[0264] Imbalances in bile acid homeostasis may play a causal role in the pathophysiology of diseases including hypercholesterolemia, obesity, diabetes, cancer, gastrointestinal disease, and gallstone formation, further highlighting the biological importance of these metabolites.

[0265] In some embodiments of any of the aspects, the subject is at risk for 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcer; infectious colitis; irritable bowel syndrome; leaky gut; and cancer).

[0266] In some embodiments of any of the aspects, the disease is a gastrointestinal disease. In some embodiments, the gastrointestinal disease is a gastrointestinal infection. In some embodiments, the gastrointestinal infection is an infection caused by a bacterium selected from the group consisting of Staphylococcus; Helicobacter pylori; Escherichia coli; Salmonella; Campylobacter; Yersinia enterocolitica; Shingella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus. In some embodiments, the gastrointestinal disease is inflammatory bowel disease (IBD). In some embodiments, the gastrointestinal disease is appendicitis. In some embodiments, the gastrointestinal disease is Crohn's disease (CD). In some embodiments, the gastrointestinal disease is ulcerative colitis (UC). In some embodiments, the gastrointestinal disease is gastritis. In some embodiments, the gastrointestinal disease is enteritis. In some embodiments, the gastrointestinal disease is esophagitis. In some embodiments, the gastrointestinal disease is pancreatitis. In some embodiments, the gastrointestinal disease is diabetes. In some embodiments, the gastrointestinal disease is hepatitis. In some embodiments, the gastrointestinal disease is liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis of the liver). In some embodiments, the gastrointestinal disease is gastroesophageal reflux disease (GERD). In some embodiments, the gastrointestinal disease is celiac disease. In some embodiments, the gastrointestinal disease is diverticulitis. In some embodiments, the gastrointestinal disease is food intolerance. In some embodiments, the gastrointestinal disease is an ulcer. In some embodiments, the gastrointestinal disease is infectious colitis. In some embodiments, the gastrointestinal disease is irritable bowel syndrome. In some embodiments, the gastrointestinal disease is leaky gut. In some embodiments, the gastrointestinal disease is cancer.

[0267] In another embodiment of any of the aspects, the gastrointestinal disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the liver disease is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is hepatitis A. In some embodiments, the liver disease is hepatitis B. In some embodiments, the liver disease is hepatitis C. In some embodiments, the liver disease is autoimmune hepatitis. In some embodiments, the liver disease is cirrhosis of the liver.

[0268] In another embodiment of any of the aspects, the subject is at risk of having or has obesity. As used herein, the term "obesity" refers to excess fat in the body.

[0269] In some embodiments of any of the aspects, the obese subject has a body mass index of at least about 25 kg / m prior to administration of a treatment as described herein. 2 In some embodiments, an obese subject may have a body mass index of at least about 30 kg / m prior to administration of a treatment, compound, or agent as described herein. 2 may be subject to.

[0270] In another embodiment of any of the aspects, the subject is at risk for or has an inflammatory disease (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, liver disease, biliary atresia, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, rheumatoid arthritis).

[0271] In one aspect, provided herein is a method of treating diabetes in a subject.

[0272] In some embodiments, the diabetes is type I diabetes, type II diabetes, neonatal diabetes, maturity-onset diabetes of the young, or gestational diabetes.

[0273] In some embodiments, the diabetes is caused by obesity. In one aspect, provided herein is a method of treating obesity in a subject.

[0274] In some embodiments, the disease is cancer. In some embodiments, the cancer is a cancer of the digestive system. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is kidney or renal cancer. In some embodiments, the cancer is oral cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is basal cell carcinoma. In some embodiments, the cancer is biliary tract cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is a cancer of the urinary system.

[0275] 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, 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.

[0276] In some embodiments, the subject being treated is an animal. The animal may be of either sex and at any stage of development. In some embodiments, the subject is a mammal. In some embodiments, the subject being treated is a human. In some embodiments, the subject is a domestic animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In some embodiments, the subject is a companion animal, such as a dog or cat. In some embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In some embodiments, the subject is a zoo animal. In other embodiments, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate animal. In some embodiments, the animal is a genetically modified animal. In some embodiments, the animal is a transgenic animal.

[0277] In another embodiment of any of the aspects, the subject is at risk of or has cancer. The conversion of primary bile acids to secondary bile acids can lead to a decrease in tumor suppressors in the liver. It is contemplated 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.

[0278] The methods and compositions provided herein can also be applied to treat or prevent prediabetes in subjects.The subject can also be suffering from or at risk of developing diabetes or prediabetic condition.The cause of diabetes can be due to gene mutation, hereditary diabetes, obesity, lifestyle, or idiopathic.

[0279] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds is preferably administered at an ED level with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of use or administration utilized.

[0280] An effective dose can be initially estimated from cell culture assays. The dose can be formulated in animals. Generally, the compositions contain a compound disclosed herein in a range of 1 μg / kg to 1000 mg / kg; 1 μg / kg to 500 mg / kg; 1 μg / kg to 150 mg / kg, 1 μg / kg to 100 mg / kg, 1 μg / kg to 50 mg / kg, 1 μg / kg to 20 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 50 mg / kg, 100 μg / kg to 10 ... The doses used or given may be administered at a dose of 100µg / kg to 20mg / kg, 100µg / kg to 10mg / kg, 100µg / kg to 1mg / kg, 1mg / kg to 100mg / kg, 1mg / kg to 50mg / kg, 1mg / kg to 20mg / kg, 1mg / kg to 10mg / kg, 10mg / kg to 100mg / kg, 10mg / kg to 50mg / kg, or 10mg / kg to 20mg / kg. It is to be understood that the ranges given herein encompass all intermediate ranges, for example, the range 1 mg / kg to 10 mg / kg encompasses 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. Also contemplated are doses (either as a bolus or continuous infusion) 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. It should further be understood that ranges intermediate to the ranges given above, e.g., in the range 1 mg / kg to 10 mg / kg, e.g., 2 mg / kg to 8 mg / kg, 3 mg / kg to 7 mg / kg, 4 mg / kg to 6 mg / kg, etc., of use or dosage ranges are also within the scope of this disclosure.

[0281] The compounds described herein can be administered at once or divided into several smaller doses and administered at intervals. It is understood that the exact dosage and duration of treatment will depend on the carrier and other variables, which can be determined using known testing protocols or by extrapolation from in vivo or in vitro test data, where the composition is administered parenterally. It should be noted that concentration and dosage values ​​may also vary with the age of the individual being treated. It should further be understood that for any particular subject, a particular dosage regimen may need to be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the formulation. Thus, the concentration ranges described herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.

[0282] In one embodiment of any of the aspects, the agent, compound, or composition is administered continuously (e.g., at a constant level over a period of time). Continuous administration of the agent or compound can be achieved, for example, by an epidermal patch, a continuous release formulation, or on-body injectors.

[0283] The compound may be administered as a single bolus or multiple boluses, as a continuous infusion, or a combination thereof. For example, the compound may be administered initially as a single bolus, followed by a bolus followed by a continuous infusion. The infusion rate may be any desired rate. Some contemplated infusion rates include 1 μg / kg / min to 100 mg / kg / min, or 1 μg / kg / hr to 1000 mg / kg / hr. The infusion rate may range from 0.2 to 1.5 mg / kg / min, or more specifically, from 0.25 to 1 mg / kg / min, or even more specifically, from 0.25 to 0.5 mg / kg / min. It will be appreciated that the infusion rate may be determined based on the dose required to maintain an effective plasma concentration and elimination rate of the compound, such that the compound is administered via infusion at a rate sufficient to safely maintain a sufficient effective plasma concentration of the compound in the bloodstream.

[0284] The dosage of the agent or compound as described herein can be determined by a physician and adjusted as necessary to suit the observed effect of treatment.Regarding the duration and frequency of treatment, it is typical for a skilled clinician to monitor the subject to determine when treatment provides therapeutic benefit, and to determine whether to administer additional agents, discontinue treatment, resume treatment, or otherwise modify the treatment plan.Dosage should not be so high as to cause harmful side effects such as cytokine release syndrome.In general, dosage will vary with the age, condition, and sex of the affected individual, and can be determined by those skilled in the art.Dosage can also be adjusted by an individual physician if complications occur.

[0285] In one embodiment of any of the aspects, the agents, compounds, or compositions described herein are used as monotherapy. In another embodiment of any of the aspects, the agents or compounds described herein can be used in combination with other known agents and treatments for diabetes. Administered "in combination," as used herein, means that two (or more) different treatments are delivered to a subject during the course of the subject's illness, e.g., two or more treatments are delivered after the subject is diagnosed with an illness (e.g., diabetes) and before the illness is cured or eliminated, or before the treatments are stopped for other reasons. In some embodiments, one treatment is still being delivered when the second delivery begins, so as to overlap in terms of administration. This is sometimes referred to herein as "simultaneous delivery" or "concurrent delivery."

[0286] In other embodiments, delivery of one treatment is completed before delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective (e.g., the effect is equal to the effect in the absence of the second treatment) than the effect seen when the second treatment is administered in the absence of the first treatment (or the effect seen in a similar situation with the first treatment), or the second treatment significantly reduces symptoms. In some embodiments, delivery is performed so that the reduction in symptoms or other parameters related to the disorder is greater than that observed when one treatment is delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive. Delivery is performed so that the effect of the first treatment delivered is still detectable when the second treatment is delivered. The compounds and agents described herein and at least one additional therapeutic agent can be administered simultaneously, in the same composition or in separate compositions, or sequentially. For sequential administration, an agent described herein may be administered first and an additional agent may be administered second, or the order of administration may be reversed. The agent and / or other therapeutic agents, procedures, or modalities may be administered during a period of active disorder or during a period of remission or less active disease. The agent may be administered before, in parallel with, after, or during remission of another treatment.

[0287] Currently, therapies used to treat or prevent gastrointestinal diseases, inflammatory diseases, 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., canagliflozin), bile acid sequestrants (e.g., colesevelam), 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., aminosalicylates, norfloxacin, penicillins, cephalosporins), antivirals (e.g., zanamivir, oseltamivir), vaccines, corticosteroids (e.g., hydrocortisone, prednisone, prednisolone, budesonide), analgesics (e.g., acetaminophen, ibroprofen), nonsteroidal anti-inflammatory drugs (e.g., mesalamine), anti-inflammatory drugs (e.g., sulfasalazine), immunosuppressants (e.g., infliximab, azathioprine, adalimumab, mercaptopurine), nutritional supplements (e.g., iron), surgery (e.g., colostomy, ileostomy, colectomy, proctocolectomy) , gastric bypass), ursodeoxycholic acid (UDCA, also known as ursodiol, INN, NAN, AAN, or USAN), cholestyramine, stanozolol, naltrexone, rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinol esters, vitamin A, liver dialysis or liver transplant, IV fluids, enemas, and other treatments known in the art.

[0288] In addition to the treatment for the above disease, chemotherapeutic agents can also be administered.Non-limiting examples of the treatment for cancer (for example, liver cancer) include nucleoside analogues (for example, tegafur), antifolates, anthracyclines, podophyllotoxins, taxanes, alkaloids, alkylating agents, platinum compounds, antibodies, retinoids, histone deacetylase inhibitors, arsenic trioxide, kinase inhibitors (for example, sorafenib), surgery, or any other chemotherapeutic agents known in the art. Those skilled in the art can readily identify chemotherapeutic agents to use (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in 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).

[0289] In addition to the treatment for the above disease, chemotherapeutic agents can also be administered.Non-limiting examples of the treatment for cancer (for example, liver cancer) include nucleoside analogues (for example, tegafur), antifolates, anthracyclines, podophyllotoxins, taxanes, alkaloids, alkylating agents, platinum compounds, antibodies, retinoids, histone deacetylase inhibitors, arsenic trioxide, kinase inhibitors (for example, sorafenib), surgery, or any other chemotherapeutic agents known in the art. Those skilled in the art can readily identify chemotherapeutic agents to use (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in 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).

[0290] When administered in combination, the agent or composition and the additional agent (e.g., a second or third agent), or all, can be administered in amounts or doses greater than, less than, or the same as the amount or dosage of each agent used individually (e.g., as monotherapy). In some embodiments, the amount or dosage of the administered agent, additional agent (e.g., a second or third agent), or all, is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually. In other embodiments, the amount or dosage of the agent, additional agent (e.g., a second or third agent), or all, that produces a desired effect (e.g., treatment of diabetes) is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less) than the amount or dosage of each agent required individually to achieve the same therapeutic effect.

[0291] Administration In some embodiments of any of the aspects, the agent is administered by direct injection, subcutaneous injection, intramuscular injection, oral administration, or intranasal administration. In some embodiments, administering an agent or pharmaceutical composition provided herein reduces glucose levels in the serum of the subject.

[0292] Exemplary modes of administration include, but are not limited to, injection, infusion, infusion, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and substernal injection and infusion. In certain preferred embodiments, the composition is administered orally. In some embodiments, the agent or composition provided herein is injected directly into the portal vein. For example, injection into the portal vein can limit systemic side effects of the agent or pharmaceutical composition. In some embodiments, the composition provided herein is implanted into the portal vein for sustained release. In some embodiments, the composition is administered via an injection port.

[0293] 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 to be dissolved or suspended in a pharmaceutically acceptable injection vehicle, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.

[0294] Suitable vehicles that can be used to provide 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; glucose 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, ethyl alcohol, 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.

[0295] In some embodiments of any of the aspects, described herein are agents or pharmaceutical compositions administered to a subject by controlled-release or delayed-release means. Ideally, the use of optimally designed controlled-release preparations in medical treatments is characterized by the minimum amount of active ingredient employed to cure or control a disease within the shortest time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosing frequency; 3) increased patient compliance; 4) utilization of a smaller total drug dose; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved efficacy of treatment; 9) reduced potentiation or loss of drug activity; and 10) improved speed of disease or disorder control. (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 compounds of Formula (I). In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that maximum efficacy of the agent is achieved while minimizing potential adverse effects and safety concerns, which can arise from both underdosing (i.e., below the minimum therapeutic level) and exceeding toxic levels of the drug.

[0296] Various known controlled release or extended release dosage forms, formulations and devices can be adapted 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; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 (each of which is incorporated herein by reference in its entirety). These dosage forms can be used to provide delayed or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif., USA)), multilayer coatings, microparticles, liposomes, or microspheres, or combinations thereof, which provide the desired release profile in various proportions. In addition, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds, thereby resulting in controlled delivery of the drug. Examples of specific ion exchangers include, but are not limited to, DUOLITE® A568 and DUOLITE® AP143 (Rohm & Haas, Spring House, Pa., USA).

[0297] efficacy The efficacy of an agent described herein, e.g., for the treatment of a disease, can be determined by one skilled in the art. However, as the term is used herein, a treatment is considered "effective" if it beneficially alters one or more signs or symptoms of diabetes, obesity, gastrointestinal disease, cancer, or inflammatory disease, improves or even reverses other clinically observed symptoms, or induces a desired response, e.g., by at least 10%, after treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a disease treated according to the methods described herein, or any other suitable measurable parameter (e.g., glucose level or glucose tolerance). Efficacy can also be measured by the failure of an individual who has deteriorated (i.e., progressed symptoms), as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein.

[0298] Efficacy can optionally be assessed in an animal model of a disease described herein (e.g., a mouse model of a disease provided herein or a suitable animal model). When using an experimental animal model, efficacy of a treatment is evident when a statistically significant change in a marker (e.g., reduced blood glucose levels in a model of diabetes) is observed.

[0299] It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. 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.

[0300] In certain aspects, provided herein are therapeutic agents for 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer. (e.g., cancers of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof.

[0301] In certain aspects, provided herein are therapeutic agents for 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer. (e.g., cancers of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; stomach cancer; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis) in a subject in need thereof.

[0302] The present disclosure also provides therapeutic agents for 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancers of the digestive system; liver cancer; Also provided are compounds of Formulas (I)-(XVIII), or pharmaceutically acceptable salts thereof, for use in treating cancers of the urinary tract, such as urinary tract cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, kidney or renal cancer, oral cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, basal cell carcinoma, biliary tract cancer, lung cancer, bladder cancer, cervical cancer, endometrial cancer, uterine cancer, and cancers of the urinary system, or inflammatory diseases (e.g., Crohn's disease, inflammatory bowel disease, ulcerative colitis, pancreatitis, hepatitis, appendicitis, gastritis, diverticulitis, celiac disease, food intolerance, enteritis, ulcers, gastroesophageal reflux disease (GERD), psoriatic arthritis, psoriasis, and rheumatoid arthritis).

[0303] The present disclosure also provides therapeutic approaches to the treatment of 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 of the liver); gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcers; infectious colitis; irritable bowel syndrome; leaky gut; and cancer), cancer (e.g., cancers of the digestive system; liver cancer; colon cancer; Also provided is a compound of Formulas (I)-(XVIII), or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of cancer of the esophagus; cancer of the stomach; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma, biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancers of the urinary system), or inflammatory diseases (e.g., 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).

[0304] In some embodiments, the disease is a metabolic disorder. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the diabetes is type I diabetes. In some embodiments, the diabetes is type II diabetes. In some embodiments, the metabolic disorder is obesity.

[0305] In some embodiments, the disease is an inflammatory disease. 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 or 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, ulcers, or 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.

[0306] In certain embodiments, the methods of the present disclosure include administering to a subject an effective amount of a compound represented by 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.

[0307] Certain methods described herein may include administering one or more additional pharmaceutical agents in combination with a compound described herein. The additional pharmaceutical agent(s) may be administered at the same time as the compound of Formulae (I)-(XVIII) or at a different time than the compound of Formulae (I)-(XVII). For example, the compound of Formulae (I)-(XVIII) and any additional pharmaceutical agent(s) may be on the same or different dosing schedules. All or some doses of the compound of Formulae (I)-(XVIII) may be administered before all or some doses of the additional pharmaceutical agent, after all or some doses of the additional pharmaceutical agent, within the dosing schedule of the additional pharmaceutical agent, or a combination thereof. The timing of administration of the compound of Formulae (I)-(XVIII) and the additional pharmaceutical agent may vary for different additional pharmaceutical agents.

[0308] example Assay Protocol Bacterial culture. All strains were grown in Cullen-Haiser Gut (CHG) medium (which consists of Brain Heart Infusion Medium (Bacto™ BHI, BD) supplemented with 1% BBL Vitamin K1-hemin solution (BD), 1% trace minerals 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 in BHI. + All strains were grown at 37°C in Bacto™ BHI (BD, supplemented with 5 mg / L hemin and 2.5 uL / L vitamin K1). All strains were grown under anaerobic conditions in an anaerobic chamber (Coy Lab Products Airlock) with a 5% hydrogen and 20% carbon dioxide / nitrogen gas mix. Escherichia coli were grown aerobically at 37°C in LB medium supplemented with ampicillin to select for the pET21b plasmid.

[0309] UPLC-MS analysis. Bile acid profiling by UPLC-MS was performed using published methods 16 Correction factors for extraction efficiency were used and determined by extraction of known concentrations of relevant bile acids from buffer or bacterial culture medium and comparison to a standard curve. Detection limits for individual bile acids were determined using commercial standards / synthetic compounds solubilized in 1:1 MeOH / water. The detection limits are as follows: βMCA, 0.03 pmoles / μL; TβMCA, 0.01 pmoles / μL; CA, 0.04 pmoles / μL; TCA, 0.01 pmoles / μL; UDCA, 0.04 pmoles / μL; TUDCA, 0.01 pmoles / μL; DCA, 0.04 pmoles / μL; TDCA, 0.05 pmoles / μL; GCDCA-d4, 0.1 pmoles / μL; CDCA-d4, 0.1 pmoles / μL; 7-oxo-CA, 0.5 pmoles / μL; 7, 1.0 pmoles / μL; GR-7, 0.05 pmoles / μL.

[0310] Protein expression and purification.

[0311] B. thetaiotaomicron rBSH. The gene encoding BT_2086 (without leader sequence) was codon-optimized for E. coli and cloned into the pET-21b(+) vector containing a C-terminal His6 tag (see Table 2 for primers). The expression plasmid was then transformed into BL21(DE3)pLysS Escherichia coli (New England Biolabs) cells under ampicillin selection. An overnight culture grown in LB medium with ampicillin (50 μg / mL) was diluted 1:1000 into fresh LB medium with ampicillin and grown at 37°C. Expression was confirmed at an OD of 0.6-0.7. 600 The cells were induced at RT by the addition of 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and further incubated overnight at 18°C. The cells were pelleted by centrifugation at 7,000 g for 20 minutes at 4°C. The pelleted cells were then resuspended in PBS buffer (with 5% glycerol) containing 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The resuspended cells were sonicated and pelleted by centrifugation at 16,000 g for 20 minutes at 4°C. The supernatant was then mixed with preformed Ni-NTA for 45 minutes at 4°C. Nickel-binding proteins were eluted with increasing concentrations of imidazole in PBS buffer (with 0.25 mM TCEP and 5% glycerol). Collected fractions were examined for purity by SDS-PAGE. Pure fractions were combined and concentrated, followed by dialysis using storage buffer (PBS at pH 7.5 with 0.25 mM TCEP and 5% glycerol).

[0312] For crystallization purposes, the protein was further purified using an S200 size-exclusion column (from GE) on a BioRad FPLC in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 7.5 with 300 mM NaCl, 0.25 mM TCEP, and 5% glycerol.

[0313] B. longum rBSH. Recombinant BSH from B. 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.

[0314] Enzyme kinetics. The enzyme was characterized using a modified BSH activity assay 26 To 144.8 μL of PBS buffer (containing 10 mM TCEP and 5% glycerol), 35.2 μL of rBSH was added to give final concentrations of 6.2 μM and 7.0 μM for B. theta BSH and B. longum BSH, respectively. This solution was preheated to 37°C in a water bath. 20 μL of bile acids conjugated at the appropriate concentration in DMSO 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 cloudy solution was centrifuged at 4,200 g for 15 minutes. Ten microliters of the supernatant was added to 190 μL of ninhydrin mix (15 mL of 0.5 M sodium citrate (pH 5.5), 36 mL of glycerol, and 6 mL of 1% [wt / vol] ninhydrin in 0.5 M sodium citrate buffer (pH 5.5)), and the mixture was heated to 100°C for 18 minutes in a BioRad thermocycler. The resulting solution was cooled at 4°C for 20 minutes, and the absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices).

[0315] Inhibitor screening using rBSH. 200 nM rBSH was incubated with 100 μM inhibitor in 3 mL of PBS buffer containing 0.25 mM TCEP and 5% glycerol at pH 7.5 for 30 minutes at 37°C. The bile acid pool (100 μM) was added to the above solution and incubated at 37°C. Between these times, 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 spectrometer (mass spec) vial. Samples were analyzed as described in "UPLC-MS Analysis." The resulting bile acid concentrations were used to determine % deconjugation.

[0316] Equation for calculating % deconjugation.

[0317] % deconjugation = concentration of detected deconjugated bile acids / (concentration of detected deconjugated bile acids + concentration of detected conjugated bile acids)*100.

[0318] Kinetic studies of compound 7. Assays were performed in PBS buffer (containing 0.25 mM TCEP and 5% glycerol), and all reactions were incubated at 37°C prior to initiation of the reaction. B. theta BSH (200 nM) was added to a 100 μM bile acid pool and a 100 μM 7 pool. 500 μL aliquots were removed at the indicated time points and flash-frozen in liquid nitrogen. After thawing, the solution was acidified to pH = 1 using 6 M HCl and then processed as described in "Screening of Inhibitors Using rBSH." The procedure was repeated with 8.2 mM TUDCA.

[0319] IC of compound 7 against recombinant protein 50 Determining values. 200 nM rBSH was incubated with increasing concentrations of 7 in 1 mL PBS buffer (containing 0.25 mM TCEP and 5% glycerol at pH 7.5) for 1 h at 37°C. 100 μM bile acid (TUDCA for B. theta BSH and TDCA for B. longum BSH) was added to the above solution and incubated for 2 h at 37°C. The solution was acidified to pH = 1 using 6 M HCl and then treated as described in "Screening of inhibitors using rBSH."

[0320] Screening for inhibitors in bacteria. Bacterial cultures were incubated in 4 mL of BHI + OD of 0.1 in 100 μM taurine-conjugated bile acid pool containing 100 μM inhibitor 600 These cultures were then grown anaerobically at 37°C. After 21 h, the serial dilutions were diluted in BHI + Cell viability (CFU / mL) was determined by plating on agar. 1 mL of the total bacterial culture was acidified to pH = 1 using 6 M HCl, followed by the addition of 2 mL ethyl acetate and vortexing. Better separation was achieved by spinning down the culture in a centrifuge at 2,500 g for 5 minutes. The organic layer was then removed, and the aqueous layer was extracted again using 2 mL ethyl acetate. The dried organic extract was resuspended in 1:1 methanol:water, transferred to mass spectrometer vials, and analyzed as described in "UPLC-MS Analysis." The resulting bile acid concentrate was used to determine % deconjugation.

[0321] IC of compound 7 in bacterial cultures 50 Determining values. Note that due to the slow growth of B. longum, B. adolescentis was used for studies of developing bacteria. Overnight cultures of B. theta and B. adolescentis were cultured at an OD of 0.1 in 2 mL of fresh CHG medium (see "Cultivating Bacteria") containing 100 μM TUDCA or TDCA and increasing concentrations of inhibitor 7. 600B. theta and B. adolescentis maximally deconjugated either of the conjugated substrates, TUDCA and TDCA, respectively, in the bacterial assay inhibitor screening. Therefore, IC was determined using these substrates. 50 The values ​​were determined. Cultures were then grown anaerobically at 37°C for 24 h (B. adolescentis) or 48 h (B. theta). B. theta required a longer incubation time because significant BSH activity was only observed during the stationary phase of this bacterium. Cultures were extracted and analyzed as described in "Screening of Inhibitors in Bacteria."

[0322] Conventional screening of inhibitors in mouse feces. BSH activity in fecal pellets was quantified using a modified version of a published method 45 Fecal pellets (approximately 10-20 mg) were ground to 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 min. 100 μM glycochenodeoxycholic acid-d4 (GCDCA-d4) was added to the mixture and incubated at 37°C for 18 h. The tubes were then frozen in dry ice for 5 min and, upon thawing, diluted with an equal volume of methanol. The slurry was centrifuged at 12,500 g for 10 min. The supernatant was removed and placed in a clean Eppendorf tube and centrifuged again. The supernatant was transferred to MS vials, and the samples were analyzed as described in "UPLC-MS Analysis." The concentrations of the products detected from these assays were reported directly.

[0323] Crystallization, data collection, and structure determination. Crystals of BSH and BSH complexed with 7 were grown in hanging drops at room temperature in a 24-well format. BSH crystals (5.0 mg / mL) were grown from microseeding after 3 days in 42% tacimate, 100 mM Tris (pH 7.4). Crystals of the BSH-7 complex (5.0 mg / mL) were grown after 5 days in 21% PEG 3350 and 100 mM X sodium citrate tribasic dihydrate (pH 5.0). Crystals were cryoprotected by supplementing the mother liquor with 10% 2-methyl-2,4-pentanediol (v / v). Data collection was performed at 100 K using a wavelength of 0.979 Å at the Advanced Photon Source NE-CAT beamline 24 ID-C. Diffraction images were processed and scaled using XDS. To obtain the phases associated with the apoBSH structure, molecular replacement was performed using Phaser with 3HBC as the search model. 46 Iterative model building and reciprocal space refinement were performed in Phenix with COOT and phenix.refine, respectively. 47 The BSH-7 structure was phased using molecular replacement with apoBSH as the search model. Iterative model building and refinement for BSH-7 grouped atomic B-factors and used the adaptive twin rule kh-l. Model quality for both structures was assessed using composite omit density maps. In the final cycle of model building, NCS restraints were removed. Final model quality was assessed using MolProbity 48For 6UFY, 97% of residues were in the favored regions of the Ramachandran plot, 3% in the accepted regions, and none in the outlier regions; for 6UH4, 89.3% of residues were in the favored regions, 10.3% in the accepted regions, and 0.4% were outliers. All crystallographic data processing, refinement, and analysis software was provided by the SBGrid Consortium. 49 The figure was created using Pymol (Schroedinger).

[0324] Mass spectrometry to identify labeled residues on BSH. BSH protein was incubated with DMSO or a 10-fold molar excess of inhibitor 7 for 2 h at room temperature. Reactions were then analyzed by LC-MS using a Shimadzu LC and autosampler system (Shimadzu, Marlborough, MA) interfaced to an LTQ ion trap mass spectrometer (ThermoFisher Scientific, San Jose, CA).

[0325] To determine the modification site, proteins modified with compound 7 were analyzed as described above, except that the LC system was interfaced to an Orbitrap Lumos Mass Spectrometer (ThermoFisher Scientific). The mass spectrometer was programmed to perform successive cycles of 1 MS scan (m / z 300-2000, profile mode, electron multiplication detection) followed by an ETD MS / MS scan targeting the +41 charge state precursor of the protein modified with compound 7 (ETD reagent target = 200 ms, imaging current detection at 60K resolution, target value = 2E6, ETD reaction time = 100 ms or 200 ms). Ion assignments were calculated using mzStudio software. 50 It was carried out using.

[0326] 7 effects on FXR. The effect of 7 on FXR was tested using the LanthaScreen TR-FRET Coactivator Assay (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The known FXR agonist GW4064 (Sigma, G5172) was used as a positive control (agonism assay) or its EC 50 (50.3 nM, measured in this assay) was added (antagonism assay). After 1 h incubation at room temperature, the 520 / 495 TR-FRET ratio was measured on a PerkinElmer Envision fluorescent plate reader using the following filter sets: excitation 340 nm, emission 495 nm, and emission 520 nm. A 100 μsec delay time, followed by a 200 μsec integration time, was used to collect the time-resolved signal.

[0327] Cell culture. Caco-2 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 Salt, 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 a 5% CO atmosphere.

[0328] Plasmids and transient transfections. For luciferase reporter assays, vectors expressing human reporter constructs were used. To study TGR5 activation, pGL4.29[luc2P / CRE / Hygro] plasmids (Promega Corporation) were transiently transfected into Caco-2 cells at a concentration of 2 μg / ml of medium. pGL4.74[hRluc / CMV] plasmids (Promega Corporation) were used as a transfection efficiency control at a concentration of 0.05 μg / ml of medium. All plasmids were transfected using Opti-MEM (Gibco) and Lipofectamine 2000 (Invitrogen, Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. Plasmid transfections were performed in antibiotic-free MEM medium (with 10% FBS). After overnight incubation, 7 and / or bile acids were added to complete medium. 7 and / or bile acids were diluted in DMSO, keeping the DMSO concentration constant. To study TGR5 antagonism, 10 μM LCA was added together with 7 and incubated overnight. The next day, cells were harvested for luciferase assay.

[0329] Luciferase reporter assay. Luminescence was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's instructions. Cells were gently washed with PBS and lysed in PLB from the kit. Luminescence was measured using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA) at the ICCB-Longwood Screening Facility at HMS. Luminescence was normalized to Renilla luciferase activity, and the percentage of relative luminescence was calculated compared to the DMSO control.

[0330] Cell viability assay. Caco-2 cells and NCI-H716 cells were treated with the indicated compounds, diluted in DMSO in complete MEM medium and complete RPMI medium, respectively. The concentration of DMSO was kept constant and used as a negative control. Cells were incubated with compounds overnight at 37°C in a 5% CO2 atmosphere. The next day, cells were treated with 0.25% trypsin in HBSS (GenClone) for 10 minutes at 37°C. Cell viability was measured in a Countess II automated cell counter (Invitrogen). The percentage of relative viability was calculated compared to the DMSO control.

[0331] Epithelial permeability assay. Undifferentiated Caco-2 cells were seeded at 200,000 cells per transwell in 24-well plate transwells (0.4 μM pore size, Costar). The medium was changed on days 4, 8, 12, 16, and 18 to allow Caco-2 cells to differentiate in vitro. 51 On day 21, fully differentiated and polarized cells were used for FITC-dextran permeability assays. Briefly, 7 and GR-7 were added at the indicated concentrations in PBS to the apical chamber of transwells containing differentiated Caco-2 cells and incubated for 6 or 12 h. The apical chamber of the transwell contained a 100 μL volume of PBS with compound or DMSO control, while the basolateral chamber contained 500 μL of PBS. Caco-2 epithelial integrity was assayed by measuring the passive diffusion of 4 kDa FITC-dextran (Sigma-Aldrich) added to the apical chamber at a concentration of 5 μM. Diffusion from the apical to basolateral side was measured by reading fluorescence in PBS on the basolateral side of the transwell system using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA) at the ICCB-Longwood Screening Facility at HMS. Fluorescence readings were normalized to the DMSO control.

[0332] Target validation and off-target profiling using 7-N3 in B. adolescentis. Preliminary studies with 7-N3 were performed using B. adolescentis (Gram-positive) and B. theta (Gram-negative). We chose to use B. adolescentis due to its stronger total fluorescent signal as detected by in-gel fluorescence. B. adolescentis cultures were cultured at an OD of 0.1 in 6 mL of fresh CHG medium containing 100 μM taurine-conjugated bile acid pool. 600 The cultures were allowed to grow anaerobically at 37°C for 21 h. Then, 10 μM 7-N3 (10 mM stock in DMSO) or 6 μL DMSO (to the control tube) was added to the cultures and incubated anaerobically at 37°C for 1 h. The cultures were centrifuged at 2,500 g for 15 min at 4°C. The medium was discarded, and the cells were resuspended in PBS containing 1 mM TCEP and 1 mM PMSF and centrifuged at 4,200 rpm for 15 min at 4°C. The buffer was discarded, and the cells were suspended in 300 μL of fresh buffer and transferred to homogenizing tubes (Precellys lysing kit tough micro-organism lysing VK05 tubes) along with ceramic beads. The suspension was then homogenized (5000 speed for 90 s*2, 6500 speed for 60 s) and spun down at 15,000 g for 20 min 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 click reaction as described in "Click chemistry for in-gel fluorescence imaging" for fluorescence imaging and "Click chemistry for MS / MS on bacterial lysate" for mass spectrophotometer-based quantification and identification.

[0333] Dose-dependent labeling of BSH in B. adolescentis via competition between 7 and 7-N3. B. adolescentis cultures were added to 6 mL of fresh CHG containing 100 μM taurine-conjugated bile acid pool at an OD of 0.1.600 The cultures were allowed to grow anaerobically at 37°C for 21 h. Diluting concentrations of 7 were added to the various tubes, and the cultures were incubated anaerobically at 37°C for 1 h. 10 μM 7-N3 was then added to the cultures, which were then incubated anaerobically at 37°C for an additional hour. The cultures were further processed as reported in "Target validation and off-target profiling using 7-N3 in B. adolescentis" and "Click chemistry for in-gel fluorescence imaging."

[0334] Off-target profiling using 7-N3 in mammalian cells. The human epithelial cell line NCI-H716 was used to study interactions with mammalian proteins. 10 μM 7-N3 (10 mM stock in DMSO) or 1 μL DMSO (for control) was added to ~8 × 10 cells in 1 ml DPBS (HiMedia). 6 The solution was added to the cells and incubated for 1 hour. The cells were collected in a 15 ml Falcon tube and washed twice in 15 ml DBPS by centrifugation at 500 g for 5 minutes. A third wash by centrifugation was performed in a 1 mM solution of cOmplete™ Protease Inhibitor Cocktail (Roche, Switzerland) in DPBS. The cells were resuspended in 250 μl of DPBS with 1 mM cOmplete™ Protease Inhibitor Cocktail and sonicated at 50% amplitude for 2 seconds, followed by three cycles of 30 seconds on ice. 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 lysates were then subjected to click reactions as per "Click Chemistry for In-Gel Fluorescence Imaging" for in-gel fluorescence and "Click Chemistry for MS / MS on Mammalian Lysates" for mass spectrophotometer-based quantification and identification.

[0335] Click chemistry for in-gel fluorescence imaging. Click reactions were performed in 25 μL volumes. Lysates pretreated with 10 μM compound 7-N3 (normalized to 1.5 mg / mL for both bacterial 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) in the dark at 37°C for 1 h. 10 μL of 2x Laemmli buffer (containing 5% β-mercaptoethanol) was added to the reaction, and the tube was heated at 95°C for 10 min. 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 for 30 minutes using 40% methanol, 50% acetic acid, 10% water and visualized using a Bio-Rad ChemiDoc MP Imaging System. The gel was stained in Coomassie blue for 20 minutes and destained for 2 hours prior to imaging.

[0336] Click chemistry for MS / MS on bacterial lysates. Click reactions were performed in 100 μL volumes. Lysates pretreated with 10 μM compound 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) at 37° C. for 1 h. Samples were then processed for further analysis as described in "Proteomic Analysis of Click-Tagged Proteins."

[0337] Click chemistry for MS / MS on mammalian lysates. Click reactions were performed in 100 μL volumes. Lysates (1.5 mg / mL for mammalian cells) pretreated with 10 μM compound 7-N3 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) at 37 °C for 1 h. The samples were then processed for further analysis as described in "Proteomic Analysis of Click-Tagged Proteins."

[0338] Proteomic analysis of click-tagged proteins. Pull-down and on-bead digestion of desthiobiotinylated proteins was performed according to previously described protocols. 52 After resuspension of tryptic peptides in 5% acetonitrile (with 0.1% formic acid), peptides were analyzed by nanoflow LC-MS / MS as described. 53 The raw data was multiplied 54 The sequences were converted to .mgf using and searched using Mascot 2.6.2 against a forward-reversed database (uniprot) of either human proteins or Bifidobacterium adolescentis proteins. Search results were downloaded from Mascot, converted to xls, and filtered to 1% FDR using the multiplierz script. Normalized spectral abundance factors were derived as described. 55Data were filtered for proteins with spectral counts greater than 5 (averaged across biological triplicates) for 7-N3-treated samples. In separate experiments, clicked 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. Indicated bands were excised and subjected to in-gel digestion, and extracted peptides were analyzed by nanoflow LC-MS / MS as described. 53 analyzed.

[0339] animal research. C57BL / 6 mice obtained from Jackson Laboratories were maintained under a strict 12 h / 12 ​​h light / dark cycle and constant temperature (21 ± 1°C) and humidity (55–65%). All experiments were performed on 8–9 week-old male mice.

[0340] 7 single gavage. Based on the potency of 7 at 10 μM to 100 μM in in vitro assays, Our in vivo target concentration of 7 was ~50 uM: (0.00005 M) × (~10 mL volume / 1 mouse GI tract) × (1 mmol compound 7 / 408 mg) = 0.2 mg / mouse × (1 mouse / ~0.02 kg) = 10 mg / kg.

[0341] Mice were maintained on a standard chow diet (LabDiet, catalog no. 5053) throughout 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). For fecal pellet collection, each mouse was temporarily transferred to a cardboard cage for several minutes until the mouse defecated.

[0342] GR-7 in solid feed (Chow) for 1 day. Mice were fed a powdered standard chow diet (LabDiet, catalog no. 5053) throughout the experimental period. Mice were divided into two groups of 10 mice each and maintained on powdered chow (control group) or powdered chow containing 0.09% (w / w) GR-7 (experimental group). Feces from these mice were collected for 8 h as described above. Mice were euthanized using carbon dioxide 30 h after accessing powdered chow with or without GR-7. Blood samples were collected by cardiac puncture and placed in EDTA-coated tubes on ice. Liver and cecal contents were then collected from each mouse, snap-frozen in liquid nitrogen, and stored at -80°C until further analysis. Blood samples were then centrifuged at 2500 g for 15 minutes at 4°C. The resulting supernatant (plasma) was collected and stored at -80°C until analysis.

[0343] 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 buffer (10% PBS, 90% sodium acetate, pH 5.2) containing 100 μM GCDCA-d4 to obtain a concentration of 20 mg / mL. The fecal pellets were crushed into fine particles, and the mixture was incubated at 37°C for 25 minutes. Samples were processed and analyzed as described in "Conventional Screening of Inhibitors in Mouse Feces." The concentrations of the products detected from these assays were reported directly.

[0344] Quantification of bile acids in tissues and plasma. Bile acids from tissues and plasma collected from mouse studies were extracted using previously published methods. 16 .

[0345] Determination of microbial biomass by seeding. 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 onto CHG agar plates (see "Bacterial Culture") and incubated at 37°C.

[0346] Isolation and 16S rRNA gene sequencing analysis of fecal bacterial microbiota. Mouse fecal microbiota DNA was isolated using the ZymoBIOMICS 96 DNA Kit (ZymoBIOMICS™) according to the manufacturer's instructions. Variable region 4 of the 16S rRNA gene was amplified using the forward primer 5'-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3'.

[0347] Reverse 5'-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3'. PCR products were quantified using the Quant-IT dsDNA High Sensitivity Assay (Invitrogen) according to the manufacturer's instructions. Successful PCR amplification was checked using gel electrophoresis. The concentration of the PCR products was measured by the Quant-IT dsDNA High Sensitivity Assay. Approximately 120 ng of DNA from each PCR product was pooled together to generate an aggregated library for downstream processing. PCR DNA amplicons between 300 and 500 bp were selected from the aggregated library on a targeted size-selection platform (Pippin Prep 1.5% agarose cassettes from Sage Sciences) according to the manufacturer's instructions. DNA amplicon size was characterized on an Agilent Technologies 2100 bioanalyzer trace. The DNA concentration of the aggregated library was measured by the Quant-IT dsDNA High Sensitivity Assay. The DNA in the library was denatured with NaOH and diluted to 7.5 pM with the HT buffer provided in the Illumina kit. 600 ul of the denatured and diluted library with a 20% phiX spike-in (120 ul, 7.5 pM phiX) was loaded onto a MiSeq V2 reagent cartridge (Illumina) and sequenced with paired-end 250 bp reads using the custom primers described above. After MiSeq, demultiplexed fastq files were generated using the Illumina MiSeq control software with default parameters, and quality control was performed using the Massachusetts Host-Microbiome Center pipeline. The resulting fastq sequences were then analyzed using the following QIIME_mothur_DADA2 56~59The quality of the analysis was filtered by

[0000] . Operational taxonomic units (OTUs) were curated at 97% sequence similarity. The phylogenetic affiliation of each OTU was aligned against the Greengenes reference database and 99% ID.

[0348] Quantification of bacterial 16S rDNA copy numbers. 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 7 Flex Real-Time PCR System according to the provided qPCR protocol. The cycle threshold of each sample was compared to a standard curve obtained from serial dilutions of B. theta genomic DNA. 60 .

[0349] Example 1. Development of broad-spectrum covalent inhibitors of enterobacterial bile salt hydrolases Development of broad-spectrum covalent inhibitors of enterobacterial bile salt hydrolases Described herein is the development of broad-spectrum, covalent inhibitors of enterobacterial BSH. A rational design strategy was used to generate a small library of potential BSH inhibitors. By testing these compounds against purified enterobacterial BSH protein and growing cultures, a lead inhibitor bearing an alpha-fluoromethylketone warhead was identified. Another BSH inhibitor, caffeic acid phenethyl ester (CAPE), was determined to inhibit the growth of Gram-negative enterobacteria but lacked the same broad-spectrum activity as the other BSH inhibitors described herein. Mass spectrometry and X-ray crystallography confirmed the covalent single labeling of the protein at the catalytic cysteine ​​residue by the inhibitor. 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 exhibited a loss of fecal BSH activity and a decrease in deconjugated bile acids. Overall, these studies demonstrate the potential of covalent BSH inhibitors to act as chemical tools to modulate bile acid composition in vivo.

[0350] introduction Human-associated bacteria play essential roles in health and disease. Microbial imbalances are a key driver of inflammatory bowel disease. 1 ,cancer 2 ,autism 3 , and obesity 4 However, how bacterial guests affect their human hosts at the molecular level remains poorly understood. Studies of germ-free mice colonized with single strains, multiple strains, or defined communities of bacteria have revealed alterations in host processes (metabolism, 5 , immune function 6,7 , and neurological response 8 Although germ-free mice are a useful tool, they have a unique way of processing food for energy compared to conventional animals. 9 , defects in immune cell balance (especially in the intestine) 10,11, and altered stress response behavior 12 These differences may complicate determining whether effects observed in germ-free animals can be extrapolated to conventional animals and humans. Chemical tools that selectively alter the levels of specific bacterial metabolites and proteins may enable researchers to investigate how these bacterial products affect host physiology in fully mature animals harboring complex microbial communities. The use of small molecules as chemical tools may also present therapeutic opportunities. Indeed, small molecule inhibitors of enterobacterial beta-glucuronidase have been shown to reduce dose-limiting diarrhea in mice caused by the colon cancer chemotherapy drug CPT-11. 13 Recent studies have shown that small molecule inhibitors of the gut bacterial enzyme cutC reduce levels of the prothrombotic metabolite trimethylamine N-oxide (TMAO) in vivo. 14 These studies demonstrate the power of non-bactericidal agents that target specific bacterial enzymes to beneficially alter host physiology.

[0351] Bacteria in the GI tract are flooded with molecules from the host, including both feed compounds and host metabolic products. The bacteria then chemically modify these compounds to produce new classes of metabolites, which can then act as signaling molecules between the bacteria and the host. 15 An important example of a class of signaling molecules produced by the host and modified by bacteria is bile acids. 16Primary bile acids are produced from cholesterol in the liver and conjugated to taurine or glycine to produce conjugated primary bile acids (Figure 1A). These molecules are then stored in the gallbladder and released into the duodenum upon food ingestion, where they aid in the absorption of lipids and fat-soluble vitamins. Over 95% of bile acids are reabsorbed in the ileum and recycled to the liver. The remaining ~5% enter the colon, where the majority of gut bacteria reside. Gut bacteria then enzymatically modify these primary bile acids to produce a group of molecules called secondary bile acids (Figure 1A). Approximately 50 secondary bile acids have been detected in human feces. Due to the high concentration of bile acids released into the small intestine, the resulting concentrations of these molecules in the lower gut remain in the low millimolar range. 17 As a result, even the less abundant secondary bile acids are present in physiologically relevant concentrations.

[0352] Bile acids were initially studied due to their detergent properties, but 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 affect host processing by acting as either 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 In particular, bile acids, by engaging with host receptors, regulate energy expenditure and glucose and lipid homeostasis. 18,23 , as well as the host's immune response (including both innate and adaptive immunity). 24,25 In addition, bile acids tightly regulate their own biosynthesis through a negative feedback loop controlled by FXR.23 Imbalances in bile acid homeostasis contribute to hypercholesterolemia, obesity, diabetes, cancer, gastrointestinal disease, and gallstone formation. 18,26,27 These metabolites are believed to play a causal role in the pathophysiology of diseases involving cerebrospinal fluid (CSF), further highlighting the biological importance of these metabolites.

[0353] Importantly, individual primary and secondary bile acids possess distinct binding affinities for host receptors, suggesting that the specific composition of the in vivo bile acid pool determines downstream host signaling events. 18,28 The key reaction in the conversion of primary bile acids 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 the enterobacterial bile salt hydrolase (BSH) enzyme. 16 BSH (EC 3.5.1.24) is found in a wide range of human gut bacteria. Recent studies have identified BSH in gut species from 117 genera and 12 phyla, including the two dominant gut phyla, Bacteroidetes and Firmicutes, as well as Actinobacteria and Proteobacteria. 29 Furthermore, this study identified BSH in human microbiomes from 11 different populations across six continents, including the indigenous population of Tanzania. These results suggest that BSH activity is a conserved function of the human gut metagenome. Thus, broad-spectrum, nontoxic, small-molecule inhibitors of enterobacterial BSH may limit BSH activity across a variety of Gram-negative and Gram-positive strains without significantly affecting the growth of these bacteria. Furthermore, the in vivo use of such inhibitors can result in a shift in the bile acid pool toward conjugated bile acids and away from deconjugated and secondary bile acids (Figure 1A). These compounds, as described herein, can be used to study how secondary bile acids produced by bacteria affect the physiology of fully colonized hosts.

[0354] Described herein is the development of broad-spectrum covalent inhibitors of bacterial BSH, determined using a rational design approach. Importantly, the compounds described herein can significantly inhibit BSH activity in conventional mouse feces, demonstrating their activity as broad-spectrum inhibitors of BSH.

[0355] Experimental results Rational Design and Synthesis of Covalent Small Molecule Inhibitors of Bile Salt Hydrolase To achieve the goal of generating potent and long-lasting inhibitors of BSH, covalent inhibitors of these enterobacterial enzymes have been developed and described herein. Covalent inhibitors have gained widespread interest in the field of drug discovery due to their ability to inactivate their protein targets with high potency and selectivity, even in the presence of high concentrations of the native substrate. 30 Conjugated bile acids, the substrates of BSH, are found in high concentrations in the colon (1-10 mM). 17 This suggests that covalent inhibition may be an effective strategy for targeting these enzymes. In addition, recently developed inhibitors of bacterial cutC are irreversible and block the in vivo production of trimethylamine while exhibiting minimal off-target effects. 14 This study demonstrates that covalent inhibitors of bacterial enzymes can be effective in the intestine, thus further validating this approach.

[0356] Although there is significant diversity in BSH protein sequences across enteric strains, all BSHs possess a conserved active site composed of five amino acids: cysteine ​​2 (Cys2), arginine 18 (Arg18), aspartic acid 21 (Asp21), asparagine 175 (Asn175), and arginine 228 (Arg228). 16,29Cys2 performs a nucleophilic attack on the substrate carbonyl, resulting in cleavage of the amide bond (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 in the design strategy. The co-crystal structure of C. perfringens BSH with the substrate taurodeoxycholic acid (TDCA) showed that hydrophobic interactions hold the bile acid core in place and orient the amide bond toward the conserved cysteine, exposing that amino acid to solvent (Figure 2B). 31 Furthermore, purified C. perfringens BSH tolerates a wide variety of amino acid side chains, including longer chain conjugates. 32 These results suggest that the bile acid D-ring side chain was a likely site for incorporation of electrophilic groups into the inhibitors.

[0357] Next, we designed a small library of potential inhibitors containing both a bile acid core motif to selectively target BSH and a pendant electrophilic warhead to irreversibly bind the inhibitor to the enzyme (Figure 2C). Although previous literature suggested that BSH hydrolyzes the amide bond cleavage of all conjugated bile acids, regardless of the steroid core, 16,26 It has recently been determined that species from the abundant gram-negative enterobacteria phylum Bacteroidetes cleave primary bile acids at C12=H but not C12=OH (Figure 1A). 33 As the goal was to develop BSH inhibitors that target both Gram-negative and Gram-positive strains, the steroid moiety of the human primary bile acid chenodeoxycholic acid (CDCA, C═H) was used as the scaffold for the inhibitor described here (Figure 2C).

[0358] Electrophilically trapping groups have been successfully deployed in the development of selective and potent inhibitors of proteases and kinases.34,35 , which are isothiocyanates (1) 36~38 , cyanoacrylate (2) 39,40 , α,β-unsaturated systems (3 and 4) 41 , acrylamide (5) 42 , and nitrile (6) 43,44 Among the libraries, inhibitors with an α-fluoromethylketone warhead (FMK) (7) were selected. Covalent inhibitors with this warhead have been shown to exhibit high potency and selectivity. 45~47 In contrast to the more electrophilic α-iodo-, α-bromo-, and α-chloromethyl ketone warheads, the weak leaving group ability of fluorine makes the FMK warhead less reactive and therefore more selective. 45,47,48 As a result, FMK-based inhibitors have been shown to elicit minimal off-target effects. 45,49 .

[0359] All of the compounds in the library were accessible in 3–9 steps from the commercially available bile acid chenodeoxycholic acid (CDCA, 12) (Scheme 1). A one-pot modified Curtius rearrangement of isothiocyanate (1) and acrylamide (5) from CDCA incorporating a C23-substituted primary amine was performed. 50 The synthesis of cyanoacrylate (2), α,β-unsaturated systems (3 and 4), and nitrile (6) compounds proceeded rapidly in 2–3 steps from bis-methoxymethyl ether (MOM) protected C24-aldehyde CDCA via either Grignard addition or condensation (Scheme S1). To access compound 7, bis-MOM protected CDCA (13) was coupled with magnesium benzyl fluoromalonate to provide the β-keto-α-fluorobenzyl ester product 14 in 66% yield. 51 Hydrogenation followed by deprotection provided the target compound 7. [ka] Scheme 1: Synthesis of compound 7 containing an α-fluoromethyl ketone warhead on a chenodeoxycholic acid core. Abbreviations: SOCl, thionyl chloride; DIPEA, N,N-diisopropylethylamine; MOMCl, methoxymethyl chloride; THF, tetrahydrofuran; CDI, 1,1'-carbonyldiimidazole; Pd / C, palladium on carbon; R = methoxymethyl ether.

[0360] Biochemical characterization of BSH With inhibitors 1-9 in hand, the next goal was to biochemically evaluate the activity of these compounds against both Gram-negative and Gram-positive BSHs. In particular, these compounds were tested against the selective Bacteroides BSH, although it is speculated that the more restricted substrate range of this enzyme may make targeting more difficult. To date, biochemical characterization has focused on the genus Lactobacillus 53 , Bifidobacterium 54 , Clostridium 31 , and Enterococcus 55 was largely limited to BSH from Gram-positive bacteria, including 16,26,52 Among Gram-negative bacteria, only BSH from Bacteroides vulgatus and Bacteroides fragilisave have been biochemically characterized, but the corresponding genes were not identified. 56,57 Moreover, these strains do not possess the selective BSH selectivity. 33 Recently, BT2086 was identified as a gene responsible for selective BSH activity in the enterobacterium Bacteroides thetaiotaomicron VPI-5482 (B. theta). 33 To test compounds against this selective BSH, the heterologously expressed purified enzyme encoded by BT2086 was molecularly cloned. Because this enzyme has not been previously characterized, kinetic parameters were analyzed using a ninhydrin-based assay. 58Using this enzyme, we established the hydrolysis of conjugated primary and secondary bile acids (primary, taurocholic acid, TCA, and taurochenodeoxycholic acid, TCDCA; secondary, tauroursodeoxycholic acid, TUDCA, and taurodeoxycholic acid, TDCA). Glyco-bile acids are almost absent in mice, but taurine conjugates are present in both mice and humans. 28 Consistent with previous results from B. theta cultures, purified B. theta BSH demonstrated a preference for deconjugation of TDCA, but not TCA (Table 1). 33 These results suggest that the enzyme selectivity observed in whole B. theta cell cultures was due to intrinsic biochemical properties of BSH, rather than differences in transport or substrate availability to the enzyme.

[0361] To test the efficacy of inhibitors against Gram-positive BSH, we used a known strain of Bifidobacterium longum SBT2928 BSH. 54 was cloned and expressed, and the kinetic parameters of this enzyme were determined using the same panel of taurine-conjugated bile acid substrates (Table 1). Notably, the K for all of the recognized substrates was m The values ​​are in the low millimolar range, which is the approximate intestinal concentration of these bile acids. m The values ​​are higher than those previously reported 54 This difference may be a result of the conditions under which the assays were performed, i.e., physiological pH (7.5) in this study versus activity-optimized pH (6) in the previous study. Overall, both enzymes exhibited similar kinetic parameters to those of BSH previously characterized. 53,54,56 The kinetic parameters were comparable to those of the hydroxylase. Table 1: Kinetic characterization of Gram-negative Bactereroides thetaiotaomicron (B. theta) BSH and Gram-positive Bifidobacterium longum (B. longum) BSH. [Table 1] a Characterization was performed using ninhydrin reagent and experiments were carried out in PBS buffer at pH 7.5 and 37°C. b The conjugated primary and secondary bile acids used as substrates were taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA).

[0362] Biochemical evaluation identifies α-FMK compound 7 as a lead inhibitor Next, the compounds in the library were evaluated for their ability to inhibit B. theta and B. longum BSH. Two additional compounds were included in the assay: riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11) (Figure 2E). These molecules had previously been identified as BSH inhibitors through a high-throughput screen against BSH from the chicken intestinal isolate Lactobacillus salivarius. 59 To determine the BSH inhibitory activity of these compounds, B. theta BSH was incubated with each inhibitor (100 μM) for 30 min, followed by the addition of equimolar amounts of four conjugated bile acids (TβMCA, TCA, TUDCA, and TDCA, all 100 μM).

[0363] The conversion of conjugated to deconjugated bile acids was monitored by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) over a total period of 21 h (Figure 3). Of the synthetic inhibitors, the isothiocyanate (1) exhibited moderate inhibition over the course of the experiment. Other compounds containing a Michael acceptor warhead (inhibitors 2–6) did not inhibit deconjugation (Figure 3A). In contrast, incubation with the α-fluoromethyl ketone-based inhibitor 7 resulted in almost complete inhibition of B. theta BSH activity over 21 h (>98%, Figure 3A). To verify that the inhibitory activity of compound 7 was due to the presence of fluorine as a leaving group, a methyl ketone analog lacking the fluorine atom was synthesized (8). 49 This analog did not exhibit BSH inhibition, indicating that the α-fluorine group was required for activity. Riboflavin, a previously identified BSH inhibitor, did not exhibit any inhibitory activity, while CAPE provided only moderate inhibition of B. theta BSH.

[0364] Next, the activity of the two most potent inhibitors against B. theta BSH was evaluated. Compounds 1 and 7, as well as CAPE, were tested against BSH from the Gram-positive species B. longum (Figure 3B). These compounds exhibited the same differential efficacy against B. longum BSH as observed against B. theta BSH. Compound 7 was the most potent inhibitor at the 2-, 5-, and 21-h time points, while compound 1 exhibited moderate inhibition, and CAPE was ineffective at inhibiting deconjugation by B. longum BSH at all time points. These data indicate that compound 7 is a potent inhibitor of purified BSH protein from both Gram-negative and Gram-positive strains. Additionally, since the activity of CAPE and riboflavin against genera other than Lactobacillus has not been determined, 59 These results suggest that these molecules may not be broadly effective inhibitors.

[0365] Compound 7 inhibits BSH activity in growing cultures of enterobacteria. Given that compound 7 demonstrated activity against purified BSH from B. theta and B. longum, the efficacy of this inhibitor in growing bacterial cultures was evaluated. To test the range of BSH inhibition, three Gram-negative and three Gram-positive strains of human intestinal bacteria known to possess BSH activity (Gram-negative, B. theta, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482; Gram-positive, Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32) were tested in this screen. 16,33 .

[0366] Bacterial cultures were diluted to pre-log phase, and both the inhibitor (100 μM) and a mixture of conjugated bile acids (final concentration 100 μM; TCA, TβMCA, TDCA, and TUDCA) were added simultaneously. Deconjugation was monitored over 24 hours using UPLC-MS. Strikingly, while all six strains deconjugated bile acids in the presence of vehicle control, little detectable deconjugation was observed in any of the cultures grown in the presence of compound 7. These results suggest that compound 7 exhibits potent BSH inhibition of both Gram-negative and Gram-positive bacteria (Figure 4A). Compound 7 did not significantly affect the growth of any of the strains tested (Figure 4B), indicating that the observed BSH inhibition was not due to bacteriostatic activity. To quantify the efficacy of compound 7, the IC of this inhibitor against the Gram-negative strain B. theta and the Gram-positive strain B. adolescentis was measured. 50 The values ​​were determined to be 913 nM and 227 nM, respectively (FIG. 4C). Taken together, these results indicate that compound 7 is a potent, broad-spectrum inhibitor of BSH.

[0367] In contrast, no inhibition of deconjugation was observed over 21 h in five of the six strains grown in the presence of CAPE (100 μM) (Figure 4A). Although CAPE was found to inhibit deconjugation in L. plantarum, this result is consistent with the hypothesis that this compound inhibits BSH from Lactobacilli but is not a broad-spectrum BSH inhibitor. Moreover, in contrast to inhibitor 7, CAPE inhibited the growth of all three Gram-negative strains tested (Figure 4B). These results suggest that the dominant effect of CAPE on Gram-negative bacteria is growth inhibition rather than BSH inhibitory activity.

[0368] To assess the hypothesis that C12=OH compounds are effective but not broad-spectrum inhibitors, because they would not inhibit B. theta BSH activity, we synthesized an inhibitor by adding the α-fluoromethylketone warhead from the most potent inhibitor, compound 7, to the cholic acid of the C12=OH bile acid core (compound 9, Figure 2D). Next, growing cultures of B. theta were incubated with compound 9 (1 μM or 10 μM) and the conjugated bile acid substrate (GUDCA, 100 μM), and deconjugation was monitored using UPLC-MS. While incubation with 10 μM compound 7 resulted in nearly complete inhibition of deconjugation, significant deconjugation was observed in the presence of the same concentration of compound 9 (Figure 4D). These results support the hypothesis that the bile acid core structure, specifically the C12 substitution, affects the ability of the probe to act as a broad-spectrum inhibitor. Additionally, these results suggest that the α-fluoromethyl ketone warhead is not broadly reactive but rather requires favorable positioning within the active site, which can be further tested using mass spectrometry and crystallography studies.

[0369] Compound 7 covalently binds to the catalytic cysteine ​​residue of BSH. Having determined the potency of compound 7, we investigated its mechanism of inhibition. Mass spectrometry experiments were performed to confirm that compound 7 is a covalent inhibitor, modifying the catalytic cysteine ​​residue, Cys2. B. theta BSH contains two cysteine ​​residues, Cys2 and Cys67. Analysis of the apo crystal structure of the enzyme revealed that both cysteine ​​residues face the active site, indicating that either residue could be a potential binding site for compound 7 (PDB 3HBC). We found that reincubation of B. theta BSH with compound 7 resulted in a shift of the intact bulk protein by 388 mass units. This mass shift is consistent with the addition of a single equivalent of inhibitor to the protein (Figure 5A). While digestion with trypsin or Lys-C did not identify any labeled peptides, a top-down approach revealed Cys2 as the modified residue, as indicated by the c3 ion (Figure 5B).

[0370] To understand the spatial arrangement of the inhibitor in the binding pocket and guide further inhibitor design, we determined the cocrystal structure of B. theta BSH covalently bound to compound 7 at 3.4 Å resolution. Consistent with the mass spectrometry data, the cocrystal structure revealed that Cys2 was attached to the C25-methylene of the bile acid structure and that the fluorine atom was removed. Collectively, these data indicate that compound 7 selectively labels B. theta BSH at the nucleophilic cysteine ​​residue in the active site of the protein. Furthermore, the cocrystal structure revealed that the C3-hydroxyl group was solvent-exposed, suggesting that this site may be amenable to further modification.

[0371] Compound 7 exhibits minimal off-target effects While covalent inhibitors have been shown to be highly potent, concerns have been raised that the nonspecific reactivity of these compounds may result in acute toxicity. 30The inhibitors described herein were 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 It is possible that the lead inhibitor may then bind to these receptors and induce off-target effects in the host. In particular, the binding of certain bile acids to FXR and GPBAR1 / TGR5 affects core host metabolic and immune processes. 18 To determine whether compound 7 can act as a ligand for FXR, an in vitro coactivator recruitment assay was performed (Figure 6A). 28 This 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 time-resolved fluorescence resonance energy transfer (TR-FRET) signal. While the known FXR agonist GW4064 showed a clear dose-dependent increase in the binding of SRC2-2 to FXR (EC 50 = 50 nM), the binding of SRC2-2 to FXR was not increased in the presence of compound 7, suggesting that this inhibitor does not activate FXR. 50 In the presence of FXR at concentrations above 100 μM, compound 7 did not exhibit a dose-dependent curve, indicating that compound 7 does not possess 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 over the concentration range tested. In addition, compound 7 did not antagonize TGR5 in the presence of the known TGR5 agonist LCA (10 μM) (Figure 6B). These results suggest that inhibitor 7 is unlikely to induce off-target effects through binding to either of these essential host receptors.

[0372] In addition to their effects on host receptors, bile acids are known to be toxic to cells due to their detergent properties. 16,60 Because the predicted in vivo area of ​​action of inhibitor 7 is in the lower intestine, we tested the toxicity of this compound against human intestinal cells (Caco-2). When these cells were incubated with up to 50 μM of compound 7, no resulting toxicity was observed (Figure 6C). The IC of compound 7 against bacterial BSH was 50 With values ​​ranging from 227 nM to 913 nM, these results suggest that it should be possible to obtain an effective in vivo dose at a concentration that would not result in toxicity to enterocytes. Collectively, these results suggest that inhibitor 7 is non-toxic and selective for bacterial BSH over potential host targets.

[0373] Compound 7 inhibits BSH activity in conventional mouse feces While the results demonstrate the efficacy of Inhibitor 7 against growing cultures of six different strains of gut bacteria, there are hundreds of bacterial species present in the human gut. 61 Previous studies have reported significant BSH activity in mouse feces. 62 To further strengthen the finding that compound 7 is a broad-spectrum BSH inhibitor, we tested the activity of compound 7 in resuspended feces from conventional (i.e., fully colonized) mice. Compounds 1, 7, and CAPE (20 μM) were added to fecal suspensions in buffer. After 30 min, the deuterated substrate GCDCA-d4 was added, and deconjugation was determined by quantifying the formation of CDCA-d4 after 18 h using UPLC-MS (Figure 7A). Strikingly, incubation with compound 1 resulted in reduced deconjugation, whereas incubation with compound 7 completely inhibited BSH activity in feces (Figure 7B). Consistent with the in vitro results, CAPE did not provide any inhibition of BSH in conventional mouse feces. These results further demonstrate that the lead inhibitor, compound 7, is a potent, broad-spectrum inhibitor of enterobacterial BSH activity.

[0374] A single dose of compound 7 inhibits BSH activity in conventional mice 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 either a single dose of compound 7 (10 mg / kg) or vehicle control, and BSH activity was monitored in half-daily increments up to 2.5 days after gavage (FIG. 7C). Without being bound by any particular theory, it was contemplated that if compound 7 were active in vivo, a decrease in BSH activity would be observed first, followed by restoration of BSH activity. This expected effect was observed.

[0375] At 1 and 1.5 days after gavage, we noticed a significant decrease in fecal BSH activity, while a regeneration of activity was observed at subsequent time points (2 and 2.5 days after gavage) (Figure 7D). Based on the initial hypothesis (Figure 1A), and without being bound by any particular theory, we contemplated that changes in the bile acid pool should be observed after BSH inhibition. At 1 day after gavage, we observed a significant decrease in conjugated bile acids and a significant increase in deconjugated bile acids. Notably, a decrease in the deconjugated secondary bile acid deoxycholic acid (DCA) was observed at this time point (Figure 7E).

[0376] The results of the bacterial culture indicated that compound 7 did not significantly inhibit bacterial growth. Consistent with this result, no significant decrease in bacterial biomass was observed at any time point after the first gavage (Figure 7F). Collectively, these results suggest that compound 7 inhibited the BSH activity of gut bacteria in the mouse GI tract in vivo, but did not significantly inhibit the overall growth of the gut bacterial community.

[0377] We generated a derivative of compound 7, 3-sulfated lithocholic acid-fluoromethylketone (3S-LCA-FMK), that restricts the delivery of the BSH inhibitor to the intestine (Figure 8A). Conventional C57Bl / 6 male mice were fed ad libitum with either normal chow or 3S-LCA-FMK (0.03% w / w) in chow for 7 days. Feces were collected before the diet change and on days 3, 4, and 7 after gavage. n = 5 mice per group (Figure 8B). We found that BSH activity was significantly reduced in the feces of mice fed 3S-LCA-FMK in chow, and 3S-LCA-FMK was undetectable in circulating plasma on day 4 (Figures 8C-8D). Collectively, these results confirm that the 3S-LCA-FMK compound is restricted to the intestine and maintains its inhibition of bile acid deconjugation in an animal model. 3S-LCA-FMK was also shown to reduce food intake in conventional mice compared with vehicle-administered mice (n = 8 mice per group). Mice administered with 3S-LCA-FMK exhibited inhibited BSH activity and a significant decrease in food consumption (Figure 31).

[0378] overview Described herein is the development of such a chemical tool: a potent, selective, and broad-spectrum inhibitor of enterobacterial BSH. We identified a lead inhibitor, compound 7, that effectively inhibits deconjugation by purified BSH protein, growing cultures of both 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 shifted bile acid pools in vivo, as expected. Importantly, compound 7 does not significantly affect the growth of these bacteria.

[0379] These results suggest that compound 7 or its derivatives can be used as a tool to study the biological effects of primary and secondary bile acids in fully colonized animals. For example, previous studies have suggested that bacterial BSH activity affects host metabolism. However, there are conflicting reports on how altering BSH activity in vivo affects host metabolic responses.

[0380] One study found that increasing BSH activity in conventional mice through the introduction of an E. coli strain engineered to express L. salivarius BSH resulted in reduced weight gain and lower serum and liver lipid levels. 63 The introduction of exogenous strains (overexpressing proteins from different bacterial sources) into the intestine significantly disrupts the natural ecosystem, complicating the interpretation of how BSH functions in the native system. Another study found that treating conventional mice with the antioxidant compound TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) resulted in a decrease in Lactobacillus BSH activity and reduced weight gain. 62 However, TEMPOL has not been shown to act directly as a BSH inhibitor, and it may exert its metabolic effects through BSH-independent mechanisms.

[0381] Furthermore, a recent study showed that deletion of the BSH-encoding gene from the Gram-negative intestinal commensal strain B. theta resulted in reduced weight gain, lower liver and blood lipid levels, and a reduced respiratory exchange rate in mice colonized with this bacterium compared to wild-type B. theta strain. 33However, these experiments were performed in monocolonized germ-free mice and do not clarify how limiting the activity of all BSHs affects the metabolism of conventional animals. Without being bound by any particular theory, we hypothesize that the reduced weight gain phenotype in B. theta BSH knockout (KO) mice is due to reduced food intake. Administration of chemical inhibitors, such as compound 7, to mice in metabolic cages may determine the origin of the metabolic effect of inhibiting both individual BSHs in monocolonized mice and all BSHs in conventional mice.

[0382] In addition to facilitating the study of the effects of bile acids on host metabolism, selective BSH inhibitors may also enable investigation of how primary and secondary bile acids influence the host immune response, specifically in the context of liver cancer. Recent studies suggest a causal relationship between bacterial bile acid metabolism (particularly the conversion of primary bile acids to secondary bile acids) and the reduction of the tumor-suppressive environment in the liver. 64 Through bile acid feeding, antibiotic treatment, and colonization of mice with bile acid-metabolizing bacteria, these researchers gathered support for a model in which secondary bile acids reverse the beneficial NKT cell recruitment and liver tumor growth inhibition promoted by primary bile acids. The use of BSH inhibitors in mouse models of liver cancer could further test this hypothesis by shifting the endogenous in vivo bile acid pool toward primary bile acids without significantly perturbing the enterohepatic system and microbial community. If such a shift in the bile acid pool limits liver tumor growth, bacterial BSH inhibitors could be developed as novel cancer therapeutics.

[0383] Finally, while developing BSH inhibitors, two molecules, riboflavin and CAPE, previously identified as inhibitors of BSH through high-throughput screening from the chicken intestinal isolate Lactobacillus salivarius, were also evaluated. 59In contrast to compound 7, neither riboflavin nor CAPE exhibited significant inhibitory activity against any of the Gram-negative strains of Enterobacteriaceae, nor against only one of three Gram-positive strains of Enterobacteriaceae (also from the genus Lactobacillus). Additionally, compound 7 (20 μM) almost completely inhibited BSH activity in resuspended mouse feces, whereas CAPE did not significantly reduce deconjugation in this assay at either 20 μM or 100 μM concentrations. CAPE significantly inhibited the growth of the Gram-negative Enterobacteriaceae strains tested. Studies have been reported on the use of CAPE to inhibit BSH in mice, and how the resulting shift toward a more FXR-antagonistic bile acid pool affects host metabolism (particularly hepatic gluconeogenesis). 65 In light of these results, especially the finding that CAPE possesses antibiotic properties, conclusions from previous in vivo results obtained using CAPE should be reconsidered 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.

[0384] Covalent inhibitors can inactivate their protein targets with a high degree of potency and selectivity, even in the presence of large concentrations of native substrates. 11 Conjugated bile acids, the substrates of BSH, are found in high concentrations in the colon (1-10 mM). 4 In addition, recent studies demonstrate that irreversible inhibitors of bacterial enzymes can be effective in the intestine. 12 .

[0385] Although there is significant diversity in BSH protein sequences across enteric strains, all BSHs possess a conserved active site encompassing the catalytic cysteine ​​(Cys2) (Fig. 9b). 1,10Thus, compounds targeting this conserved residue may be effective pan-BSH inhibitors. The co-crystal structure of Clostridium perfringens BSH with the substrate taurodeoxycholic acid showed that hydrophobic interactions hold the bile acid core in place and orient the amide toward Cys2, exposing that amino acid to solvent (Figure 9c). 13 Furthermore, C. perfringens BSH tolerates a wide variety of amino acid side chains, including longer chain conjugates. 14 .

[0386] We developed a small library of potential inhibitors containing both a bile acid core motif and a pendant electrophilic warhead (Figure 9d). While not wishing to be bound by any particular theory, previous literature suggests that the identity of the conjugated amino acid can significantly drive BSH specificity, while 1 indicated that the sterol core configuration also influences the reactivity of BSH. 15 In addition, some Bacteroidetes species cleave primary bile acids with C12=H but not C12=OH (Figure 9a). 16 .

[0387] Although several electrophilically capturing groups were selected, 17 , which are isothiocyanates (1) 18 , cyanoacrylate (2) 19 , α,β-unsaturated systems (3 and 4) 20 , acrylamide (5) 21 , and nitrile (6) 22 Inhibitors bearing the α-fluoromethyl ketone warhead (FMK) (7) were also synthesized. In contrast to the more electrophilic α-iodo-, α-bromo-, and α-chloromethyl ketone warheads, the weak leaving group ability of fluorine makes the FMK warhead less reactive and therefore more selective. 23,24 FMK-based inhibitors have been shown to have minimal off-target effects. 23,25 .

[0388] Example 2. Biochemical characterization of BSH The activity of inhibitors 1-9 against both Gram-negative and Gram-positive BSH was then evaluated using a selective Bacteroides BSH for inhibitor optimization. Consequently, a selective BSH (BT_2086) was heterologously expressed and purified (Table 2 and Figure 14). 16 . Table 2. Primers for BSH gene amplification. [Table 2]

[0389] Kinetic parameters were determined using a ninhydrin-based assay. 26 Purified B. theta BSH was shown to preferentially deconjugate tauro-ursodeoxycholic acid (TUDCA), but not tauro-cholic acid (TCA) (Table 3 and Figure 14). 16 . Table 3: Kinetic parameters of BSH from Bactereroides thetaiotaomicron (B. theta) and Bifidobacterium longum (B. longum). [Table 3] a Characterization was performed using ninhydrin reagent and experiments were carried out in PBS buffer at pH 7.5 and 37°C. b The conjugated primary and secondary bile acids used as substrates were taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA). c B. theta did not deconjugate TCA. n=3 biological replicates per condition. All data are presented as mean ± SEM.

[0390] Gram-positive strain Bifidobacterium lonbum SBT2928 BSH 27 BSH from was also cloned and expressed, and the kinetic parameters were determined (Table 3 and Figure 14). m The values ​​are in the low millimolar range, which is the approximate intestinal concentration of these bile acids. cat Values ​​are based on the k reported for Lactobacillus salivarius BSH. cat While lower, the K m Values ​​are similar to those for previously characterized BSH 27~29 .

[0391] Example 3. α-FMK compound 7 as a lead inhibitor inhibits recombinant BSH The ability of compounds in our library to inhibit B. theta BSH and B. longum BSH was also evaluated. Compounds previously identified in a high-throughput screen for the inhibition of BSH from the chicken intestinal isolate Lactobacillus salivarius, riboflavin (10) and caffeic acid phenethyl ester (CAPE, 11), were also tested (Figure 14). 30 BSH inhibitory activity was determined by preincubating B. theta BSH with each inhibitor (100 μM) for 30 min, followed by the addition of a mixture of conjugated bile acids (final concentration 100 μM). Because BSH exhibits variable reactivity to various conjugated bile acids, equimolar combinations of two primary and two secondary conjugated bile acids predominant in the gallbladder and small intestine of conventional mice were used as our substrate mixture (tauro-β-muricholic acid (TβMC), TCA, TUDCA, and tauro-deoxycholic acid (TDCA)). 31Bile acid deconjugation was monitored by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) over a 21-h period. Among the synthetic inhibitors, the isothiocyanate (1) exhibited moderate inhibition. Other compounds containing Michael acceptor groups (2–6) did not inhibit deconjugation. In contrast, incubation with the α-FMK-based 7 resulted in almost complete inhibition of B. theta BSH activity over a 21-h period (>98%, Figures 10a, 15, 16, and Table 4). Table 4. Percent deconjugation of each bile acid determined in experiments with a pool of four tauroconjugated bile acids. [Table 4-1] Table 4. (continued) [Table 4-2]

[0392] To verify that the inhibitory activity of 7 was due to the presence of a fluorine atom as a leaving group, the methyl ketone analogue (8) was synthesized. 25 This analog did not exhibit BSH inhibition against either the recombinant protein or B. theta cultures, indicating that the α-fluoro group was required for activity (Figure 10a, Figure 17, and Table 4). Riboflavin did not exhibit any inhibitory activity, while CAPE provided only moderate inhibition of B. theta BSH.

[0393] The activity of compounds 1, 7, and CAPE against BSH from B. longum was also evaluated. Compound 7 was again the most active inhibitor, while CAPE was ineffective at inhibiting B. longum BSH at all time points (Figures 10b, 15-16, and Table 3). Compound 7 inhibited both B. theta BSH and B. longum BSH in a dose-dependent manner (IC 50The values ​​were 427 nM and 108 nM, respectively (Figure 18). Taken together, these data indicate that compound 7 is a potent inhibitor of purified BSH protein from both Gram-negative and Gram-positive bacterial strains.

[0394] Compound 7 completely inhibited B. theta BSH, the more catalytically efficient of the two enzymes (Table 2), within 15 seconds at equimolar concentrations with the substrate without any preincubation of the inhibitor with the enzyme (Figure 19). In the presence of a large excess (~80-fold) of substrate, 7 completely inhibited B. theta BSH activity within 15 minutes, the earliest measurable time point for product formation under these conditions. These results indicate that 7 is a kinetically efficient inhibitor of BSH activity.

[0395] Example 4. Compound 7 inhibits BSH in cultures of enterobacteria The efficacy of 7 in growing bacterial cultures was also evaluated. To test the range of BSH inhibition, three Gram-negative and three Gram-positive strains of BSH-containing human intestinal bacteria (Gram-negative, B. theta, Bacteroides fragilis ATCC 25285, and Bacteroides vulgatus ATCC 8482; Gram-positive, Lactobacillus plantarum WCFS1, Clostridium perfringens ATCC 13124, and Bifidobacterium adolescentis L2-32) were used. 1,16 .

[0396] Bacterial cultures were diluted to pre-log phase, and both the inhibitor (100 μM) and a mixture of conjugated bile acids (final concentration 100 μM) were added simultaneously. Deconjugation was monitored over a 21-hour period using UPLC-MS. Strikingly, while all six strains deconjugated bile acids in the presence of the vehicle control, almost no deconjugation was observed in any of the cultures grown in the presence of 7 (Figures 10c, 20, and Table 3). Isogenic BSH-deficient B. theta strains were then added.16 was incubated with either DMSO, 7, or CAPE. Under all three conditions, bile acids conjugated to taurine were regenerated without being metabolized (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-utilizing processes. Compound 7 did not significantly affect cell viability of the majority of strains tested (Figure 10d), indicating that the observed inhibition of BSH was not due to bacteriostatic activity. The IC of this inhibitor against B. theta and B. adolescentis was 50 The values ​​were determined to be 1070 nM and 237 nM, respectively (Figure 22). These results indicate that 7 is a potent, broad-spectrum inhibitor of BSH.

[0397] No BSH inhibition was observed in five of the six strains grown in the presence of CAPE (Fig. 10c). Moreover, CAPE inhibited cell viability of all three Gram-negative strains tested (Fig. 10d). These results suggest that the predominant effect of CAPE on Gram-negative bacteria is growth inhibition rather than BSH inhibitory activity.

[0398] Finally, to assess whether C12=OH compounds might be effective broad-spectrum inhibitors, we synthesized a compound in which the α-FMK warhead was appended to the C12=OH bile acid core, cholic acid (compound 9, Figure 9d). Compared to 7, compound 9 exhibited significantly reduced inhibitory activity against BSH deconjugation in B. theta cultures (Figure 17). Thus, the bile acid core structure, specifically the C12 substitution, influences the BSH-selective inhibitory activity of our probe. Additionally, these results suggest that the α-FMK warhead is not broadly reactive but rather is required for favorable positioning within the active site.

[0399] Example 5. Compound 7 inhibits BSH activity in mouse feces Previous literature has reported significant BSH activity in mouse feces. 32To further assess whether 7 is a pan-inhibitor of BSH, we tested its activity in resuspended feces from conventional mice. This fecal slurry should contain BSH from nearly all bacterial communities in the distal colon. Compounds 1, 7, and CAPE (20 μM) were added to the fecal suspension in buffer. After 30 min, the deuterated substrate glycochenodeoxycholic acid-d4 (GCDCA-d4) was added, and the formation of the deconjugated product was quantified after 18 h using UPLC-MS. Strikingly, incubation with 7 completely inhibited fecal BSH activity (Figure 10e). CAPE did not provide any inhibitory activity of fecal BSH. These results demonstrate that 7 is a potent pan-inhibitor of BSH activity.

[0400] Example 6. Compound 7 covalently modifies the catalytic Cys2 residue. 7 Inhibitory mechanism. B. theta BSH contains two cysteine ​​residues, Cys2 and Cys67, which were 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 that modifies Cys2, purified B. theta BSH was incubated with an excess of this molecule. Mass spectrometry analysis revealed a mass shift consistent with the addition of a single molecule of 7, confirming the formation of a covalent bond (Figure 23). Subsequent top-down mass spectrometry identified Cys2 as the modified residue (Figure 23).

[0401] The structures of B. theta BSH were then determined, first in its apo form (at ∼2.7 A resolution) and then in its covalently bound form to 7 (at ∼3.5 A resolution) (Table 5) (PDB 6UFY and 6UH4, respectively). Table 5. Data collection and refinement statistics (molecule replacement) [Table 5-1] [Table 5-2] *Highest-resolution shell is shown in parentheses. Each data set was collected using a single crystal.

[0402] The structure of the BSH-inhibitor complex contains four copies of the protein in the asymmetric unit. The electron density map shows the best resolution for two of the four subunits, and the electron density clearly visualizes the inhibitor in one of these subunits covalently attached to Cys2 (Figures 3a and 3b). Comparison with the apo structure also suggests that the loop (residues 127–138) is repositioned to harbor the inhibitor in the active site in a solvent-exposed channel (Figure 24).

[0403] These data indicate that 7 selectively labels B. theta BSH at Cys2. Furthermore, the co-crystal structure revealed that the C3-hydroxyl group was solvent accessible, suggesting that this site may be amenable to further modification (Figure 11b).

[0404] Example 7. Compound 7 exhibits minimal off-target effects Concerns have been raised that nonspecific reactivity of covalent inhibitors may result in acute toxicity 11 Bile acids are ligands for the farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 5 (TGR5). 2 In vitro coactivator recruitment assays demonstrated 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 across the concentration range tested (Figure 25). These results suggest that 7 does not induce off-target effects on either of these essential host receptors.

[0405] Bile acids are also known to be toxic due to their detergent properties. 1,33 The toxicity of this compound to human intestinal cells (Caco-2 and NCI-H716) was also tested. When these cells were incubated with up to 50 μM or 100 μM of compound 7, respectively, no resulting toxicity was observed (FIG. 25). The IC 50 With values ​​ranging from 237 nM to 1070 nM, these results suggest that it should be possible to achieve an effective, non-toxic in vivo dose. To test the effect of compound 7 on epithelial integrity, Caco-2 cells were differentiated in transwell inserts to form polarized monolayers with tight cell-cell junctions. 34 Compound 7 was incubated in the apical chamber of the transwell, and epithelial integrity was measured by passive diffusion of 4 kDa FITC-dextran. No significant increase in fluorescence was observed in cells treated with 7 compared with control-treated cells, indicating that 7 did not impair the integrity of the epithelial monolayer (Figure 26).

[0406] It is important to understand the proteome-wide reactivity of 7 35 To assess the target engagement and off-target interactions of compound 7, a "clickable" version of this inhibitor was synthesized by incorporating an α-azide moiety. 36We synthesized 7-N3 (compound 12, Figure 26a) by adding 7 to 7 at the solvent-exposed C3 position. Similar to 7, 7-N3 potently inhibited BSH activity in mouse feces (Figure 26b). These results demonstrate that the azide substitution did not significantly disrupt the BSH inhibitory activity of this molecule. To study on- and off-target effects in bacterial cells, cultures of B. adolescentis L2-32 were tested with 10 μM 7-N3 (the concentration at which 7 inhibited BSH in bacterial culture) for 1 h (Figure 17). Lysed bacterial supernatants were then reacted with Fluor 488-alkyne under copper-catalyzed azide-alkyne cycloaddition conditions, and the protein was visualized by in-gel fluorescence. Only one fluorescent band was visualized at a mass of ~35 kDa (the predicted mass of the annotated B. adolescentis BSH) (Figures 12c and 27). To identify this protein, clarified lysates were clicked with desthiobiotin-alkyne and streptavidin pulldown was performed. Bound proteins from control and treated samples were resolved by SDS-PAGE and visualized by silver staining (Figures 12d and 27). A single silver-stained band was observed at the predicted molecular weight of BSH (~35 kDa). This band, along with the corresponding region from the control lane, was excised, and both were digested with trypsin and subjected to LC-MS / MS. BSH was identified with high confidence in the gel band, and semiquantitative analysis of these data indicated a 4.5-fold enrichment of 7-N3 relative to vehicle-treated bacterial cultures.

[0407] To assess off-target binding across the bacterial proteome, streptavidin bead-bound proteins isolated from treated and control bacterial cultures were digested. Label-free LC-MS / MS analysis identified BSH, which was 3.6-fold enriched in probe-treated cultures. Across biological triplicates, no other proteins exceeded the 2-fold enrichment threshold. Competition of 7 with 7-N3 demonstrated dose-dependent labeling of annotated B. adolescentis BSH (Figures 12e and 27), further confirming the on-target activity of 7.

[0408] We profiled the off-target effects of compound 7 in mammalian intestinal cells (NCI-H716). These cells were also treated with 7-N3 in the same manner as bacterial cells. Click reactions with Fluor 488-alkyne did not show any enrichment of any bands by in-gel fluorescence (Figures 12f and 28). No proteins in probe-treated lysates were enriched (≥2-fold) based on label-free LC-MS / MS analysis. Together, our data demonstrate on-target BSH binding and limited off-target activity of 7 against other bacterial or mammalian proteins in intestinal cells.

[0409] Example 8. A single dose of 7 inhibits BSH activity in vivo C57BL / 6 mice were gavaged with either a single dose of 7 (10 mg / kg; see Online Methods for dose calculation) or vehicle control, and fecal BSH activity was monitored over time on a half-day basis (Fig. 13a). A significant decrease in fecal BSH activity was observed 1 and 1.5 days after gavage, while BSH activity recovered at subsequent time points (Fig. 13b). A significant increase in conjugated bile acids and a decrease in deconjugated bile acids were observed in feces 1 day after gavage (Fig. 13c). 16S rDNA sequencing and inoculation of fecal samples from these mice indicated that compound 7 did not significantly affect gut bacterial OTUs, biomass, or community composition (Figs. 13d and 28). Taken together, our results indicate that a single dose of 7 can inhibit gut bacterial BSH activity and modulate bile acid pools in vivo, while not significantly affecting the gut bacterial community.

[0410] Example 9.7 Proof of Concept for Bowel Restriction To further minimize the possibility that 7 might induce off-target effects, ideally this molecule would be confined to the GI tract. A 3-sulfated variant (gut-restricted 7 or GR-7, compound 13, Figures 11b and 13e) was synthesized.

[0411] Evaluation of GR-7 in mouse feces revealed that GR-7 possesses a potent pan-BSH inhibitor (Figure 29). C57BL / 6 mice were fed either powdered chow containing 0.09% GR-7 (w / w) or powdered chow alone per day (Figure 13f). Significant BSH inhibitory activity was observed in the feces of inhibitor-treated mice 8 hours after sample change (Figure 13g). GR-7 was detected in feces collected at 8 h, demonstrating that the inhibitor was being excreted at a rate consistent with the mouse colonic transit time. 38We detected this compound (~20 μM) in cecal contents, 20 pmol / mg wet mass (mean, Figure 13h). This concentration was effective for BSH inhibition in mouse fecal assays and was lower than the 100 μM toxicity threshold for 7 (Figures 24 and 29). Furthermore, GR-7 (60 μM) did not affect the integrity of the epithelial barrier of Caco-2 cells, suggesting that this compound is relatively nontoxic (Figure 25). GR-7 also did not affect microbial biomass (Figure 29). GR-7 was not detected in the serum or liver of inhibitor-treated mice (Figure 5h). 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 chow.

[0412] Example 10. Compound synthesis General: All anhydrous reactions were carried out under a positive pressure of argon or nitrogen. Anhydrous methylene chloride (DCM) and tetrahydrofuran (THF) were purchased from Sigma-Aldrich. Silica gel column chromatography was performed using 60A silica gel (230-400 mesh). NMR spectra recorded in CDCl3 used residual chloroform or TMS as the internal standard. [ka] Scheme 2. Synthesis of compounds 1 and 5. compound 1 Step 1. To a solution of chenodeoxycholic acid (0.5 g, 1.27 mmol), sodium azide (0.29 g, 4.44 mmol), tetrabutylammonium bromide (61.0 mg, 0.19 mmol), and zinc trifluoromethanesulfonate (18.0 mg, 0.05 mmol) in 4.3 mL of anhydrous THF at 40 °C, di-tert-butyl dicarbonate (0.3 g, 1.40 mmol) was added, ...

Claims

1. Formula (I): 【Chemistry 1】 During the ceremony: 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; R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 15 , R 16 and R 17 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, OR 18 , N(R 18 ) 2 , S.R. 18 , halogen, CN, -CHO, -CO 2 H, -CO 2 R 18 , -NO 2 , -ONO 2 , -SO 2 Cl, -SO 3 - , -OSO 3 - , -NR 18 SO 3 - , -PO 3 2- , -OPO 3 2- , -OSO 2 R 18 , -SO 2 N(R 18 ) 2 , -OSO 2 N(R 18 ) 2 , -NR 18 SO 2 R 18 , -SO 2 N(R 18 ) 2 , -NHNH 2 , -ONH 2 , or -NHC(O)NHNH 2 is; 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. 2. The compound of claim 1, wherein X is a thiol-reactive electrophilic group.

3. X is -C(O)R 19 , -NCS, -NHC(O)R 19 , -CH=C(CN)CO 2 R 20 or -CN, where R 19 is haloalkyl, alkenyl, or alkynyl, and R 20 is alkyl.

4. X is -C(O)R 19 or -NCS, where R 19 The compound of any one of claims 1 to 3, wherein is haloalkyl.

5. X is -C(O)CH 2 5. The compound of claim 1, wherein the compound is F or -NCS.

6. R 3 But, OR 18 or -OSO 3 - R 7 But, OR 18 and R 12 But H or OR 18 The compound according to any one of claims 1 to 5, wherein

7. R 3 and R 7 is OH, and R 12 The compound of any one of claims 1 to 6, wherein is H or OH.

8. R 3 But, -OSO 3 - R 7 is OH; and R 12 The compound of any one of claims 1 to 6, wherein is H.

9. R 1 , R 2 , R 4 , R 16 , R 11 , R 15 and R 16 The compound of any one of claims 1 to 8, wherein all of are H.

10. 10. The compound of claim 1, wherein m is 1.

11. The compound of any one of claims 1 to 10, wherein n is 2.

12. 3. The compound of formula (XVI): 【Chemistry 2】 R in the formula 19 is haloalkyl, The compound according to any one of claims 1 to 11, represented by:

13. 3. The compound of formula (XVII): 【Transformation 3】 During the ceremony: R 19 is haloalkyl, The compound according to any one of claims 1 to 12, represented by:

14. The compound has the formula (XVIII): 【Chemistry 4】 R in the formula 19 is haloalkyl, The compound according to any one of claims 1 to 13, wherein

15. m is 1; n is 2; X is -C(O)CH 2 F, -NCS, -C(O)CH=CH 2 , -C(O)C=CH, -NHC(O)CH=CH 2 , -CN, -CH=C(CN)CO 2 Et, or -C(O)CH 3 R 1 , R 2 , R 4 , R 16 , R 11 , R 15 and R 16 is H;R 3 and R 7 is OH; and R 12 is H or OH.

16. X is -C(O)CH 2 16. The compound of claim 15, which is F or -NCS.

17. Formula (Ia): 【Transformation 5】 wherein X is an electrophilic group. or a pharmaceutically acceptable salt thereof.

18. X is -C(O)R 19 , -NCS, -NHC(O)R 19 , -CH=C(CN)CO 2 R 20 or -CN, where R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, and R 20 18. The compound of claim 17, wherein is alkyl.

19. X is -C(O)R 19 where R 19 19. The compound of claim 17 or 18, wherein is alkyl, haloalkyl, alkenyl, or alkynyl.

20. R 19 20. The compound of claim 18 or 19, wherein is alkyl.

21. R 19 The compound of any one of claims 18 to 20, wherein is haloalkyl.

22. R 19 But CH 2 21. The compound of any one of claims 18 to 20, wherein F.

23. formula: 【Transformation 6】 【Transformation 7】 10. The compound of any one of the preceding claims, represented by: or a pharmaceutically acceptable salt thereof.

24. formula: 【Transformation 8】 10. The compound of any one of the preceding claims, represented by: or a pharmaceutically acceptable salt thereof.

25. The compound has the formula (I-e'): 【Chemistry 9】 During the ceremony: R 3a and R 7a are independently -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 R 18 , and -SO 2 N(R 18 ) 2 where each R 18 are independently H or substituted or unsubstituted alkyl; The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein

26. Formula (I-e″): 【Chemistry 10】 26. The compound of claim 25, represented by: or a pharmaceutically acceptable salt thereof.

27. formula: 【Chemistry 11】 28. The compound of claim 26 or 27, wherein:

28. The compound has the formula (I-f'): 【Chemistry 12】 During the ceremony: R 3a -OR 18 , -SO 3 R 18 , -OSO 3 R 18 , -PO 3 H 2 , -OPO 3 H 2 , -OSO 2 R 18 , and -SO 2 N(R 18 ) 2 where each R 18 are independently H or substituted or unsubstituted alkyl; The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein

29. Formula (I-f''): 【Chemistry 13】 29. The compound of claim 28, represented by: or a pharmaceutically acceptable salt thereof.

30. Formula (I-f'''): 【Chemistry 14】 29. The compound of claim 28, represented by: or a pharmaceutically acceptable salt thereof.

31. X is -C(O)R 19 , -NCS, -NHC(O)R 19 , -CH=C(CN)CO 2 R 20 or -CN, where R 19 is alkyl, haloalkyl, alkenyl, or alkynyl, and R 20 The compound of any one of claims 25, 26 or 28-30, wherein is alkyl.

32. X is -C(O)R 19 where R 19 32. The compound of any one of claims 25, 26, or 28-31, wherein is alkyl, haloalkyl, alkenyl, or alkynyl.

33. X is, 【Chemistry 15】 32. The compound of any one of claims 1, 6-15, 17, 25 or 27-31, wherein the electrophilic group is selected from the group consisting of:

34. 1. The compound of formula: 【Chemistry 16】 The compound according to any one of claims 28 to 33, or a pharmaceutically acceptable salt thereof, wherein

35. A pharmaceutical composition comprising a compound according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier or excipient.

36. 36. The pharmaceutical composition of claim 35, wherein the carrier or excipient limits delivery of the compound to the gastrointestinal tract.

37. 37. A method for inhibiting bile salt hydrolase (BSH), the method comprising contacting BSH with a compound according to any one of claims 1 to 34 or a pharmaceutical composition according to claim 35 or 36.

38. 38. The method of claim 37, wherein the contacting is in vitro or in vivo.

39. 39. The method of any one of claims 36 to 38, wherein said contacting is of interest.

40. 37. A method for inhibiting bile acid deconjugation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 34 or a pharmaceutical composition of claim 35 or 36.

41. 37. A method for promoting bile acid conjugation in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 34 or a pharmaceutical composition according to claim 35 or 36.

42. 42. The method of any one of claims 37 to 41, wherein the subject is in need of treatment for cancer, a gastrointestinal disorder, obesity, or an inflammatory disorder.

43. 37. 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 claims 1 to 34 or a pharmaceutical composition of claim 35 or 36.

44. The method of any one of claims 37 to 43, wherein the subject is a mammal.

45. The method of any one of claims 37 to 44, wherein the subject is a human.

46. 46. ​​The method of any one of claims 37 to 45, wherein the subject is at risk of having or has cancer.

47. 47. The method of any one of claims 37 to 46, wherein the subject is at risk of having or has a gastrointestinal disorder.

48. 48. The method of any one of claims 37 to 47, wherein the subject is at risk of having obesity or has obesity.

49. 46. ​​The method of any one of claims 35 to 45, wherein the subject is at risk of having or has an inflammatory disease.

50. 45. The method of claim 44, wherein the cancer is selected from the group consisting of: cancer of the digestive system; liver cancer; liver cancer; colon cancer; esophageal cancer; cancer of the stomach; liver cancer; kidney or renal cancer; oral cancer; pancreatic cancer; prostate cancer; rectal cancer; stomach cancer; basal cell carcinoma; biliary tract cancer; lung cancer; bladder cancer; cervical cancer; endometrial cancer; uterine cancer; and cancer of the urinary system.

51. 49. The method of claim 44 or 48, wherein the cancer is liver cancer.

52. 48. The method of claim 47, wherein the gastrointestinal disease is selected from the group consisting of: infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease; gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcer; infectious colitis; irritable bowel syndrome; leaky gut; and cancer.

53. 50. The method of claim 49, wherein the inflammatory disease is selected from the group consisting of: infection; Crohn's disease; inflammatory bowel disease; ulcerative colitis; pancreatitis; hepatitis; liver disease; biliary atresia; appendicitis; gastritis; diverticulitis; celiac disease; food intolerance; enteritis; ulcers; gastroesophageal reflux disease (GERD); psoriatic arthritis; psoriasis; and rheumatoid arthritis.

54. 53. The method of claim 52, wherein the infection is caused by a bacterium selected from the group consisting of: Staphylococcus; Helicobacter pylori; Escherichia coli; Salmonella; Campylobacter; Yersinia enterocolitica; Shingella; Clostridium; Bacteroides; Lactobacillus; Parabacteroides; Bifidobacterium; Listeria; and Streptococcus.

55. 54. The method of claim 52 or 53, wherein the liver disease is selected from the group consisting of: nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis of the liver.

56. A compound according to any one of claims 1 to 34, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 35 or 36; and Instructions for using the compound, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition Includes a kit.

57. 57. The kit of claim 56, wherein the instructions are for treating liver cancer in a subject in need thereof.

58. 57. The kit of claim 56, wherein the instructions are for treating a gastrointestinal disease selected from infection; inflammatory bowel disease (IBD); appendicitis; Crohn's disease (CD); ulcerative colitis (UC); gastritis; enteritis; esophagitis; pancreatitis; diabetes; hepatitis; liver disease; gastroesophageal reflux disease (GERD); celiac disease; diverticulitis; food intolerance; ulcer; infectious colitis; irritable bowel syndrome; leaky gut; and cancer.

59. 57. The kit of claim 56, wherein the instructions are for treating an inflammatory disease selected from the group consisting of: 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; and rheumatoid arthritis.

60. 57. The kit of claim 56, wherein the instructions are for treating a liver disease selected from the group consisting of: nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH); hepatitis A; hepatitis B; hepatitis C; autoimmune hepatitis; and cirrhosis of the liver.

61. 57. The kit of claim 56, wherein the instructions are for a method of inhibiting BSH activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 33 to 34.

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