Rifampicin, dexamethasone, forskolin (FSK), Diprotin A, 3-isobutyl-1-methylxanthine (IBMX), chenodeoxycholic acid (CDCA, Cat. No. 700198P), 1α,25-dihydroxyvitamin D₃ (D1530), fenofibrate, rosiglitazone, GW501516 (SML1491), GW3964 (G6295), thyroxin (T1775), GW4064 (Cat. No. G5172), (Z)-guggulsterone (Z-GUG; Cat. No. 78251) were purchased form Sigma-Aldrich, now Merck, Darmstadt, Germany). Obeticholic acid (Synonyms: OCA; INT-747; 6-ECDCA; 6-ethylchenodeoxycholic acid; Cat. No HY-12222) was purchased form MCE MedChem Express (NJ, United States). CITCO were obtained from Tocris (3683/10, Minneapolis, MN, United States). Tauro-beta-muricholic acid (Tβ-MCA) has been obtained from Cayman (Cay20289-5; Ann Arbor, MI, United States).

Human hepatocellular carcinoma HepG2 cells (purchased from the European Collection of Cell Cultures, ECACC, Salisbury, United Kingdom) were cultured in antibiotic-free Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, now Merck, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% sodium pyruvate. The HepaRG™ cells (Biopredic, Rennes, France) were seeded at the density of 26,600 cells/cm² and kept in William’s medium, supplemented with 5 µg/ml insulin, 50 µM hydrocortisone, 10% Hyclone fetal serum (GE Healthcare Life Sciences) and 1% L-glutamine. 14 days after seeding, the HepaRG cells were differentiated to hepatocyte-like cells using 1.5% DMSO in culture media for another 14 days. Human enteroendocrine colon cancer NCI-H716 cells (ATCC-CCL-251, ECACC) were cultured in antibiotic-free RPMI-1640 medium, supplemented with 10% FBS. For each experiment, the NCI-H716 cells were seeded onto 96-well Matrigel (Corning®) coated plates (1 × 10⁵ cells/well) and differentiated for 48 h. GLUTag cells were kindly provided by Dr. Colette Roche (Centre de Recherche en Cancérologie de Lyon, INSERM U1052, Lyon, France) with the permission of Dr. Daniel J. Drucker (Lunenfeld Tanenbaum Research Institute Mt. Sinai Hospital, Toronto). GLUTag cells were cultured in DMEM supplemented with 10% FBS. Primary human hepatocytes were purchased from Biopredic (Rennes, France; Lot N. HEP2201023, Human Long-term hepatocytes in monolayer, Caucasian male, 71 years old).

For transient transfection, HepG2 cells were seeded at the density of 40,000 cells/cm². In the case of the determination of GPBAR1 activation, cells were transfected using Lipofectamine 2000® (ThermoFisher Scientific, Waltham, MA, United States) with 200 ng CRE luciferase reporter vector (pGL4.29[luc2P/CRE/Hygro], Promega, Hercules, CA, United States), together with 150 ng GPBAR1 (GPBAR1-pcDNA3.1+/C-(K)-DYK) (Genscript, Piscataway, NJ, United States) or empty vector pcDNA3.1 and 50 ng pRL-TK Renilla luciferase vector (Promega). The next day, cells were challenged with tested ligands in indicated concentrations for 5 h. Forskolin (FSK) was used as a positive control for the generation of cAMP. All the other transient transfection reporter gene assays were performed with Lipofectamine® 3000 (ThermoFisher Scientific). Briefly, cells were transfected with 150 ng/well luciferase reporter gene constructs (p(DR3)₃-luc, SHP-luc, p3A4-luc, pTAT-(GRE)2-TK-luc, p2B6-luc, pGL4.35[luc2P⁄9XGAL4UAS⁄Hygro]), FXRE-luc together with 100 ng/well of the corresponding full-length fragment nuclear receptor expression vectors (pSG5-hVDR, pSG5-hFXR, pSG5-hRXRα, pSG5-hPXR, CAR3 variant) or the ligand binding domains of human, pCMX-GAL4-hFXR, pCMX-GA4L-LXRα, pCMX-GA4L-LXRα, pCMX-GA4L-TRα, pCMX-GA4L-PPARα, pCMX-GAL4-PPARγ, pCMX-GAL4-PPARδ) and 30 ng/well of pRL-TK Renilla luciferase vector 24 h prior to the treatment. Construct are described in our previous papers (Dvorak et al., 2008; Carazo et al., 2018; Stefela et al., 2020). The firefly luciferase activity was measured using the Dual Luciferase® Reporter Assay System (Promega) and normalized to Renilla luciferase activity. Experiments in an agonistic mode were performed with OCA, CDCA or tested compounds (all compounds at 10 μM). Experiments in an antagonistic mode were performed by co-treating cells with 1 µM OCA and tested compounds at 10 and 40 µM concentration or at increasing concentrations (ranging from 0.001 to 200 µM) in the case of IC₅₀ determination. The EC₅₀ and IC₅₀ are theoretical concentrations of a tested compound that provide half-maximal activation and inhibition, respectively, in reporter gene assays. EC₅₀ and IC₅₀ were calculated using GraphPad PRISM ver. 9.1.0. software (San Diego, CA, United States) employing a non-linear regression module. All the experiments have been repeated at least three times and each experiment was performed in biological triplicates (n = 3). Results are presented as fold change to control nontreated (NT) samples. Vehicle (0.1% DMSO) was used as a solvent in all samples including control samples.

The substitutions of amino acids Ser270, Tyr89 and Glu169 for glycines were inserted in the GPBAR1 gene cloned into the pcDNA3.1 and shuttle expression vector using a Quick Change XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, United States). The mutagenic primers were created using Quick Change Primer Design software from Agilent. (Ser270: 5′-CCT​CTC​CCT​AGG​AGG​CGC​CAG​TGC​AGC-3′ (F) 5′- GCT​GCA​CTG​GCA​CCG​CCT​AGG​GAG​AGG - 3′; Tyr89: 5′- GTC​CTG​CCT​CCT​CGT​CGG​CTT​GGC​TCC​CAA​CTT​C – 3′ (F) 5′- GAA​GTT​GGG​AGC​CAA​GCC​GAC​GAG​GAG​GCA​GGA​C – 3′ (R); Glu169: 5′- CAG​GAG​CCC​ATA​GAC​GCC​GAG​GTA​CAG​GTA​GGG – 3′ (F), 5′- CCC​TAC​CTG​TAC​CTC​GGC​GTC​TAT​GGG​CTC​CTG – 3′ (R). The plasmid constructs were transformed into E. coli XL10-Gold ultracompetent cells according to the manufacturer’s guideline. The final gene mutations were confirmed by sequencing.

GLUTag cells were transfected with non-targeting scrambled siRNA (siRNA control) or with the combination of siRNAs specific for Gpbar1 (Silencer® Select Pre-designed siRNA, LOT# ASO2HKN6 and ASO2HKN7, Life Technologies, Carlsbad, CA, United States) twice at 24 and 72 h after seeding using Lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, CA, United States). GLUTag cells were then treated with tested molecules at the day 4 post-seeding.

Total RNA from HepaRG cells or primary human hepatocyte samples was isolated by the phenol/chloroform method with TRI-reagent (Merck) according to the manufacturer's protocol. The cDNA was synthesized using a Tetro cDNA Synthesis Kit (Bioline, now Meridian Bioscience, Memphis, United States), and RT-qPCR was run in the Quant Studio 6 instrument using the Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, United States) according to the MIQE protocols. All the probes were obtained from ThermoFisher Scientific: ABCB11 (BSEP, Hs00184824_m1), NR0B2 (SHP, Hs00222677_m1). Data were normalized to beta-2 microglobulin (B2M (Mm00437762_m1) as the reference gene and were evaluated by the ΔΔCq method. All the experiments have been repeated three times and each experiment was performed in biological triplicates (n = 3). Results are presented as fold change of mRNA expression to control nontreated (NT) samples.

Protein determination was performed from whole-cell lysates of terminally differentiated HepaRG cells treated for 48 h. Protein levels were quantified using a PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, United States) according to the manufacturer's protocol. The western blotting analysis was performed as described in our previous paper (Stefela et al., 2020) with mouse monoclonal anti-SHP antibody (clone OTI5F10, Cat. No. TA806319, Origene, Rockville, MD, United States) and antibody against GAPDH (Cell Signaling Technology, Leiden, the Netherlands) as a loading control. Protein concentration was measured using the BCA protein assay (Sigma-Aldrich/Merck, Prague, Czech Republic). Protein expression quantification was done using densitometric software (LabImage, Kapelan Bio-Imaging, Germany).

The LanthaScreen® TR-FRET FXR Coactivator Assay (goat, PV4833, ThermoFisher Scientific) was performed to assess the affinity of tested compounds to FXR ligand-binding domain (LBD) in agonistic and antagonistic models. In the antagonistic assays, the FXR LBD was co-incubated with OCA or GW4064, together with tested ligands in increasing concentrations. The assay was performed according to the manufacturer’s protocols. After an 1 h incubation period at room temperature, the TR-FRET ratio of 520/495 nm was measured using the Biotek plate reader and used to calculate the IC₅₀ values from the concentration-response curves of each compound using GraphPad Prism version 9.1.0. software. Data have been obtained from three independent experiments performed in 4 replicates.

After serum starvation, differentiated NCI-H716 and NCI-H716 cells were washed in PBS and incubated with the tested compound in Hanks' Balanced Salt Solution (HBSS) supplemented with 0.2% (w/v) BSA and 50 µM Diprotin A (Sigma-Aldrich/Merck) for 1 or 2 h, respectively. Supernatants were collected and centrifuged. The quantity of GLP-1 was determined using the Glucagon Like Peptide-1 (Active) ELISA kit (EZGLP1T-36K, Merck Millipore, Burlington, MA, United States) according to the manufacturer's instructions. Data were normalized to protein concentration, and they are presented as a fold protein increase to control nontreated (NT) samples. Vehicle (0.1% DMSO) was present in the control as well as in other samples. All the experiments have been repeated three times and each experiment was performed in biological triplicates (n = 3).

Differentiated NCI-H716 cells were serum-starved, washed in PBS, and the cell culture medium was changed to Hanks' Balanced Salt solution (HBSS) supplemented with 0.2% (w/v) BSA and 500 µM 3-isobutyl-1-methylxanthine and incubated for 45 min at 37°C. Cells were stimulated with tested compounds or forskolin for 30 min, and the amount of cAMP generated was measured using the cAMP-Glo™ Assay (V1501, Promega, Hercules, CA, United States). The changes in cAMP levels (ΔcAMP) are presented as cAMP levels in treated samples after subtraction of the cAMP levels in the nontreated (NT) samples. All the experiments have been repeated three times and each experiment was performed in biological triplicates (n = 3).

Statistical analyses were performed using GraphPad Prism 9.1.0. software (GraphPad Software, Inc., San Diego, CA, United States), with a p-value of <0.05 considered statistically significant. All data are presented as the mean ± standard deviations (SDs) based on at least three independent experiments (n = 3). A one-way analysis of variance (ANOVA) with a Dunnett’s or Bonferroni’s post-hoc test was applied to the data if more than two groups were being analyzed. The half maximal inhibitory concentration (IC₅₀) and the half-maximal response (EC₅₀) values were calculated using nonlinear fitting of concentration-response curves (log(inhibitor) vs. normalized response) or (log(agonist) vs. response (three parameters)), respectively.

For the procedure of FXR LBD docking experiments and molecular dynamics, see Supplementary Material .

The 3D structures of ligand molecules were designed in PerkinElmer Chem3D (version 19.0.1.28). The energy minimization was done utilizing an inbuilt MM2 force field. Ligands were then exported to PDB files. All ligands were prepared for docking by the AutoDockTools 1.5.6 (Morris et al., 2009) python script “prepare_ligand4.py”. This procedure consists of assigning Gasteiger charges, merging non-polar hydrogens, building the torsion tree, and then exporting the data to PDBQT.

The preparation of the receptor (PDB 7CFN) was performed in the AutoDockTools using a standard protocol. In particular, all chains but R together with water molecules were deleted, non-polar hydrogens were merged, and Kollman charges were calculated. The grid box, which securely covered the whole LBD and the pocket entrance on the extracellular part of the receptor, was defined as a cube with a side length of 30 Grid points (1 Å spacing) and with its center at X: 98, Y: 124, Z: 119, roughly at the level of the INT-777 D-ring.

Molecular docking was performed with AutoDock Vina 1.1.2 (Trott and Olson, 2010). The exhaustiveness was set to 16, the rest of the parameters were kept at default values. Five independent runs were conducted, and the average affinity for the corresponding poses was taken as the final affinity value. Visualization of docking results was generated in Chimera 1.14 (Pettersen et al., 2004). Only residues within a 5 Å distance from the INT-777 pose are displayed. All other residues and all non-polar hydrogens are omitted for clarity. LigPlot+ v.2.2 (Laskowski and Swindells, 2011) was employed to generate the 2D ligand-protein interaction diagram.

All commercial reagents and solvents were used without purification. Melting points were determined with a Hund/Wetzlar micromelting point apparatus (Germany), and are uncorrected. ROESY NMR spectra were obtained using a Bruker Avance III™ HD 500 MHz and/or a JEOL ECZ500 spectrometer, both operating at 125.7 MHz for ¹³C and 500 MHz for ¹H. The assignment of hydrogen and carbon signals was based on a combination of 1D and 2D NMR experiments (³H, ¹³C-APT, ¹H,¹H COSY, ¹H,¹³C HSQC and ¹H,¹³C HMBC). Proton and carbon NMR spectra were measured in a Bruker AVANCE III™ 400 or 500 MHz with chemical shifts given in parts per million (ppm) (δ relative to residual solvent peak for ¹H and ¹³C). Coupling constants (J) are given in Hz. The HR-MS spectra were performed with LCQ Advantage (ThermoFisher Scientific, Waltham, MA, United States) using ESI mode. Thin-layer chromatography (TLC) was performed on silica gel (Merck, 60 µm). For column chromatography, neutral silica gel 60 µm (Fluka, Buchs, Switzerland) was used. Analytical samples were dried over phosphorus pentoxide at 50°C/0.25 kPa. The purity of final compounds was assessed by HPLC analysis with ELS detection (evaporative light scattering), and all corresponding chromatographs are enclosed in Supplementary Material.

Analysis was carried out on a HPLC Gilson system (United States) equipped with ELS detector. Solvent A was DCM/AcOH (1000:1), and solvent B was MeOH/AcOH (1000:1). Analysis was performed in isocratic setup as 95/5 A/B with flow rate 1 ml/min, column: Supelco, bare-silica LC-SI 5 μm, 150 × 4.6 mm. The sample was prepared by dissolving the sample (1 mg) in DCM (1 ml) and by then being sonicated for 5 min 20 µl was injected into the LC system.

Analysis was carried out on LC-MS system LCQ Advantage (Thermo Fisher Scientific). Ions were detected in negative ESI ion mode, with m/z range from 250 to 1500 Da. Solvent A was water/acetonitrile (98:2), and solvent B was acetonitrile/isopropanol/water (95:3:2), with 5 mM ammonium formate in both. Gradient setup: 0-25-30-30.1-45 min, 50-100-100-50-50% of solvent B and flow rate 150 µL/min, column: Phenomenex, C4, Jupiter® 5 μm, 250 × 4.6 mm. The sample was prepared by dissolving the sample (1 mg) in solution A/B (1:3, 1 ml) and by then being sonicated for 5 min. 10 µl was injected into the LCMS system.

General Procedure for Grignard Reaction. A solution of 3α-hydroxy-7-oxo-5β-cholan-24-oic acid (1, 1.28 mmol, 500 mg) was added dropwise at room temperature to a solution of Grignard reagent (6.4 mmol) in dry THF (10 ml) under an inert atmosphere. Upon addition, a cloud-like precipitate formed. The solution was then vigorously stirred and heated to reflux. The progress of the reaction was monitored by TLC. After 2 h, the reaction mixture was acidified with aqueous 1M HCl to pH 2 and extracted with EtOAc (3 × 75 ml). The combined extracts were washed with water, brine, dried over Na₂SO₄, and the solvents were evaporated. The crude product was purified by column chromatography on silica gel (MeOH/DCM, 2:98 to 5:95 v/v), followed by purification on semi-preparative HPLC (Column, Luna® 5 µm bare-silica 250 × 21.2 mm, Isocratic MeOH/DCM, 3:97 v/v, 15 ml/min, injected in either DCM or THF - if insoluble in DCM).

3α-Hydroxy-7-oxo-5β-cholan-24-oic acid (1). 7-Oxolithocholic acid methyl ester (Stefela et al., 2020) (8.2 g, 20.27 mmol) was dissolved in 300 ml of 5% NaOH in MeOH/H₂O (1:1) and heated to 50°C. After 2 h, aqueous solution of HCl (5%) was added to pH 3. The product was extracted with EtOAc (3 × 200 ml), combined organic extracts were washed with brine (1 × 300 ml) and dried over anhydrous Na₂SO₄. After solvent evaporation, the oily residue (8.2 g) was purified by flash chromatography (EtOAc/hexane/AcOH, 30/70/1, v/v/v), and further crystallized from boiling EtOAc to afford compound 1 (7.6 g, 96% yield). R_f (TLC) = 0.43 (acetone/hexane/AcOH, 40/60/1), mp 202–203 °C (EtOAc), lit. (Fieser and Rajagopalan, 1950), 202–203°C. [α]_D ²⁵ −29.6 (c 0.28, MeOH). ¹H NMR (401 MHz, MeOD): δ 3.53 (tt, J ₁ = 10.5 Hz, J ₂ = 4.7 Hz, ¹H, H-3), 2.99 (ddd, J ₁ = 12.5, J ₂ = 6.0 Hz, J ₃ = 1.1 Hz, 1H, H-6a), 2.54 (t, J = 11.3 Hz, ₁H, H-8), 1.23 (s, 3H, H-19), 0.96 (d, J = 6.5 Hz, 3H, H-21), 0.71 (s, 3H, H-18). ¹³C NMR (101 MHz, MeOD): δ 215.1 (C-7), 178.1 (C-24), 71.5 (C-3), 56.3, 50.7, 50.4, 47.5, 46.4, 44.4, 43.8, 40.3, 38.2, 36.6, 36.3, 35.2, 32.3, 32.0, 30.6, 29.3, 25.8, 23.5, 22.8, 18.8, 12.5. MS (ESI neg): m/z 389.3 (100%, M−H), 435.3 (5%, M+FA−H), 779.5 (3%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₄H₃₇O₃ [M−H], 389.26938; found, 389.26973. For C₂₄H₃₈O₄ calcd C 73.81, H 9.81. Found: C 73.72, H 9.57.

(E)-3α-Hydroxy-7-ethylidene-5β-cholan-24-oic acid (7-ELCA, 2a). Sodium hydride (60% in mineral oil, 59 mg, 1.48 mmol) was added to a solution of ethyltriphenylphosphonium bromide (550 mg, 0.51 mmol) in dry THF (15 ml) under an inert atmosphere. The reaction mixture was refluxed until a deep orange color formed. Then, the solution was cooled to 50°C, and a solution of 7-keto-LCA (1, 200 mg, 1.48 mmol) in dry THF (10 ml) was slowly added dropwise. After overnight reflux, the reaction mixture was poured into a beaker with crushed ice and extracted with EtOAc (3 × 50 ml). The combined extracts were washed with water, brine, dried over Na₂SO₄, and solvents evaporated. The crude product was purified by column chromatography on silica gel (MeOH/DCM, 2:98 to 5:95 v/v), followed by purification on semi-preparative HPLC (Column, Luna® 5 µm bare-silica 250 × 21.2 mm, Isocratic MeOH/DCM, 3:97 v/v, 15 ml/min, injected in DCM) affording compound 2a as a slightly yellowish powder (6 mg, 3%): R_f (TLC) = 0.69 (EtOAc/hexane/AcOH, 50/50/1), mp 67–72°C. ¹H NMR (500 MHz, MeOD): δ 5.30 (q, J = 6.7 Hz, ₁H, H-1′), 3.58–3.50 (m, 1H, H-3), 1.59–1.56 (m, 3H, H-2′), 1.08 (s, 3H, H-19), 0.96 (d, J = 6.5 Hz, 3H, H-21), 0.71 (s, 3H, H-18). ¹³C NMR (126 MHz, MeOD): δ 176.5 (C-24), 140.9 (C-6), 115.6 (C-1′), 72.0 (C-3), 56.4, 51.4, 46.5, 44.3, 44.12, 44.09, 40.5, 37.1, 37.1, 36.6, 36.1, 32.6, 32.3, 31.8, 31.0, 29.1, 26.5, 24.3, 22.1, 18.9, 13.3, 12.7. MS (ESI neg): m/z 401.3 (76%, M−H), 447.3 (100%, M+FA−H), 803.6 (10%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₆H₄₁O₃ [M-H], 401.3061; found, 401.3062. LCMS method B (ESI neg, t_R = 17.45 min). Purity 97.5% (HPLC method A, t_R = 5.26 min).

3α,7α-Dihydroxy-7β-methyl-5β-cholan-24-oic acid (7β-methyl-CDCA, 2b). Compound 2b was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2b (153 mg, 29%) was obtained as white solids: R_f (TLC) = 0.28 (DCM/MeOH, 5/95), mp 85–88°C, lit. (Une et al., 1989) 96–99°C, [α]_D ²⁵ + 29.9 (c 0.15, MeOH). ¹H NMR (401 MHz, CDCl₃): δ 3.49 (tt, J = 11.0, 4.5 Hz, 1H, H-3), 1.22 (s, 3H, H-1′), 0.95 (d, J = 6.4 Hz, 3H, H-21), 0.87 (s, 3H, H-19), 0.68 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.0 (C-24), 73.2 (C-7), 72.1 (C-3), 54.9, 51.5, 44.4, 44.2, 43.3, 42.1, 40.2, 38.5, 36.2, 35.7, 35.5, 34.7, 33.7, 31.0, 30.9, 30.5, 28.6, 28.2, 23.0, 21.4, 18.6, 12.4. MS (ESI neg): m/z 405.3 (100%, M−H), 451.3 (11%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₅H₄₁O₄ [M−H], 405.30103; found, 405.30043. LCMS method B (ESI neg., t_R = 12.66 min). Purity 95.6% (HPLC method A, t_R = 6.53 min).

3α,7α-Dihydroxy-7β-ethyl-5β-cholan-24-oic acid (7β-ethyl-CDCA, 2c). Compound 2c was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2c (254 mg, 47%) was obtained as white solids: R_f (TLC) = 0.21 (EtOAc/hexane/AcOH, 50/50/1), mp 112–114 °C (EtOAc), lit. (Une et al., 1989) 102–103°C, [α]_D ²⁵ + 32.8 (c 0.27, MeOH). ¹H NMR (401 MHz, MeOD): δ 3.41 (tt, J = 11.2, 4.5 Hz, 1H, H-3), 0.97 (d, J = 6.5 Hz, 3H, H-21), 0.91–0.83 (m, 6H, H-19 and H-2′), 0.74 (s, 3H, H-18). ¹³C NMR (101 MHz, MeOD): δ 178.3 (C-24), 76.1 (C-7), 72.8 (C-3), 56.4, 52.6, 45.3, 43.3, 41.6, 40.7, 39.8, 39.4, 37.8, 37.3, 36.9, 36.7, 35.6, 32.3, 32.1, 31.2, 29.3, 27.8, 23.4, 22.6, 19.0, 12.6, 9.9. MS (ESI neg): m/z 419.3 (100%, M-H), 465.3 (60%, M+FA-H), 479.3 (44%, M+AcOH−H), 839.6 (75%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₆H₄₃O₄ [M−H], 419.31668; found, 419.31647. LCMS method B (ESI−, t_R = 14.36 min). Purity 95.6% (HPLC method A, t_R = 6.44 min).

3α,7α-Dihydroxy-7β-vinyl-5β-cholan-24-oic acid (7β-vinyl-CDCA, 2d). Compound 2d was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2d (273 mg, 51%) was obtained as white solids: R_f (TLC) = 0.24 (EtOAc/hexane/AcOH, 50/50/1), mp 90–95°C, [α]_D ²⁵ + 9.3 (c 0.10, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 5.92 (dd, J ₁ = 17.3 Hz, J ₂ = 10.7 Hz, 1H, H-1′), 5.15 (dd, J ₁ = 17.3 Hz, J ₂ = 1.1 Hz, 1H, (Z)-H-2′), 4.91 (dd, J ₁ = 10.8 Hz, J ₂ = 1.0 Hz, 1H, (E)-H-2′), 3.55–3.45 (m, 1H, H-3), 0.96–0.87 (m, 6H, H-19 and H-21), 0.67 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.3 (C-24), 150.3 (C-1′), 110.2 (C-2′), 75.8 (C-7), 72.1 (C-3), 55.1, 51.2, 43.8, 43.7, 41.8, 41.3, 40.0, 38.7, 35.6, 35.5, 35.2, 34.6, 31.0, 30.9, 30.5, 28.5, 27.9, 22.9, 21.1, 18.5, 12.3. MS (ESI neg): m/z 417.3 (80%, M−H), 463.3 (100%, M+FA−H), 477.3 (50%, M+AcOH−H), 835.6 (35%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₆H₄₁O₄ [M−H], 417.30103; found 417.30066. LCMS method B (ESI neg., t_R = 13.22 min). Purity 96.5% (HPLC method A, t_R = 6.00 min).

3α,7α-Dihydroxy-7β-ethynyl-5β-cholan-24-oic acid (7β-ethynyl-CDCA, 2e). Compound 2e was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2e (370 mg) was obtained as white solids that were re-dissolved in DCM (7 ml). After gentle evaporation with a steam of air, precipitate formed. Filtration afforded solid material that was washed with HPLC grade pentane (3 × 5 ml), dried by high vacuum to obtain 2e as a fine white powder (337 mg, 63%). R_f (TLC) = 0.35 (DCM/MeOH, 5/95), mp 122–125°C, [α]_D ²⁵ + 48.6 (c 0.15, MeOH). ¹H NMR (401 MHz, CDCl₃): δ 3.50–3.37 (m, 1H, H-3), 2.40 (s, 1H, H-2′), 0.92 (d, J = 6.6 Hz, 3H, H-21), 0.91 (s, 3H, H-19), 0.69 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 177.4 (C-24), 90.7 (C-1′), 71.7 (C-2′), 71.7 (C-7), 69.2 (C-3), 55.2, 50.9, 43.7, 43.5, 42.8, 41.6, 39.8, 38.2, 35.4, 35.3, 34.9, 34.4, 30.9, 30.9, 30.3, 28.4, 26.2, 22.8, 20.9, 18.4, 12.1. MS (ESI neg): m/z 415.3 (100%, M−H), 461.3 (10%, M+FA−H). HR-MS (ESI neg): m/z calcd for C₂₆H₄₁O₄ [M−H], 417.30103; found 417.30066. LCMS method B (ESI neg., t_R = 12.27 min). Purity 95.4% (HPLC method A, t_R = 5.78 min).

3α,7α-Dihydroxy-7β-propyl-5β-cholan-24-oic acid (7β-propyl-CDCA, 2f). Compound 2f was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2f (203 mg, 36%) was obtained as white solids. R_f (TLC) = 0.26 (DCM/MeOH, 5/95), mp 100–105 °C, lit. (Une et al., 1989) 102–103°C, [α]_D ²⁵ + 30.1 (c 0.13, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 3.49 (tt, J ₁ = 11.0 Hz, J ₂ = 4.5 Hz, ¹H, H-3), 0.94 (d, J = 6.4 Hz, 3H, H-21), 0.88 (t, J = 6.9 Hz, 3H, H-3′), 0.84 (s, 3H, H-19), 0.70 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.2 (C-24), 75.4 (C-7), 72.1 (C-3), 54.9, 51.6, 47.6, 44.4, 41.8, 40.5, 40.3, 39.7, 38.8, 36.3, 35.7, 35.5, 34.5, 31.1, 30.9, 30.5, 28.5, 27.1, 22.9, 21.6, 18.6, 18.5, 14.7, 12.4. MS (ESI neg): m/z 433.3 (100%, M−H), 479.3 (6%, M+FA−H). HR-MS (ESI neg): m/z calcd for C₂₇H₄₅O₄ [M−H], 433.33233; found 433.33180. LCMS method B (ESI neg., t_R = 16.63 min). Purity 97.8% (HPLC method A, t_R = 6.27 min).

3α,7α-Dihydroxy-7β-allyl-5β-cholan-24-oic acid (7β-allyl-CDCA, 2g). Compound 2g was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2g (220 mg, 40%) was obtained as white solids. R_f (TLC) = 0.29 (EtOAc/hexane/AcOH, 50/50/1), mp 90–93°C, [α]_D ²⁵ + 42.9 (c 0.11, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 5.82 (ddt, J ₁ = 17.3, J ₂ = 10.1, J ₃ = 7.4 Hz, 1H, H-2′), 5.10 (dd, J ₁ = 10.2, J ₂ = 2.2 Hz, 1H, (E)-H-3′), 5.04 (dd, J ₁ = 17.1, J ₂ = 2.1 Hz, 1H, (Z)-3′), 3.50 (tt, J ₁ = 11.1, J ₂ = 4.5 Hz, 1H, H-3), 0.95 (d, J = 6.4 Hz, 3H, H-21), 0.82 (s, 3H, H-19), 0.70 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.0 (C-24), 134.8 (C-2′), 118.6 (C-3′), 74.8 (C-7), 72.1 (C-3), 54.9, 51.7, 48.9, 44.4, 41.7, 41.2, 40.4, 39.8, 38.6, 36.6, 35.7, 35.5, 34.5, 31.0, 30.9, 30.5, 28.5, 27.7, 22.9, 21.6, 18.6, 12.4. MS (ESI neg): m/z 431.3 (100%, M−H), 477.3 (50%, M+FA−H), 491.3 (35%, M+AcOH−H), 863.6 (45%, 2M−H). HR-MS (ESI neg): m/z calcd for C₂₇H₄₃O₄ [M−H], 431.31668; found 431.31629. LCMS method B (ESI neg., t_R = 14.92 min). Purity 96.0% (HPLC method A, t_R = 8.45 min).

3α,7α-Dihydroxy-7β-isopropyl-5β-cholan-24-oic acid (7β-isopropyl-CDCA, 2h). Compound 2h was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2h (200 mg, 36%) was obtained as white solids. Crystallization from DCM/MeOH (2 ml/1 drop) afforded 40 mg of crystals (40 mg). R_f (TLC) = 0.56 (DCM/MeOH/AcOH, 5/95/1), mp 95–100 °C (DCM:MeOH, 2 ml:1 drop), [α]_D ²⁵ + 34.0 (c 0.19, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 3.50 (tt, J ₁ = 11.1, J ₂ = 4.6 Hz, 1H, H-3), 0.95 (d, J = 6.4 Hz, 3H, H-21), 0.89 (d, J = 5.5 Hz, 3H, H-2′), 0.87 (d, J = 5.5 Hz, 3H, H-2′), 0.83 (s, 3H, H-19), 0.72 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.3 (C-24), 77.48 (C-7, CDCl₃ overlap), 72.1 (C-3), 54.8, 51.6, 44.6, 41.2, 40.4, 39.1, 39.1, 36.9, 36.6, 35.8, 35.5, 34.4, 31.8, 31.1, 30.9, 30.5, 28.4, 27.3, 22.9, 21.7, 18.8, 18.6, 16.7, 12.4. MS (ESI neg): m/z 433.3 (100%, M−H), 479.3 (4%, M+FA−H). HR-MS (ESI neg): m/z calcd for C₂₇H₄₅O₄ [M−H], 433.33233; found 433.33195. LCMS method B (ESI neg., t_R = 15.70 min). Purity 99.1% (HPLC method A, t_R = 7.97 min).

3α,7α-Dihydroxy-7β-cyclopropyl-5β-cholan-24-oic acid (7β-cyclopropyl-CDCA, 2i). Compound 2i was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2i (185 mg, 33%) was obtained as white solids. R_f (TLC) = 0.38 (DCM/MeOH, 10/90), mp 78–82°C, [α]_D ²⁵ + 28.3 (c 0.37, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 3.54–3.42 (m, 1H, H-3), 0.95 (d, J = 6.4 Hz, 3H, H-21), 0.88 (s, 3H, H-19), 0.69 (s, 3H, H-18), 0.58–0.12 (m, 4H, H-2′). ¹³C NMR (101 MHz, CDCl₃): δ 179.4 (C-24), 72.6 (C-7), 72.1 (C-3), 54.9, 50.8, 45.5, 44.4, 41.8, 40.0, 39.1, 38.8, 35.9, 35.7, 35.4, 34.7, 31.2, 30.9, 30.4, 28.6, 27.5, 24.8, 23.0, 21.3, 18.6, 12.3, 4.5, 2.7. MS (ESI neg): m/z 431.3 (65%, M−H), 477.3 (100%, M+FA−H), 491.3 (56%, M+AcOH−H), 863.6 (37%, 2M-H). HR-MS (ESI neg): m/z calcd for C₂₇H₄₃O₄ [M−H], 431.31668; found 431.31619. LCMS method B (ESI neg., t_R = 15.15 min). Purity 96.2% (HPLC method A, t_R = 5.74 min).

3α,7α-Dihydroxy-7β-(pent-4-en)-5β-cholan-24-oic acid (7β-pentenyl-CDCA, 2j). Compound 2j was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2j (198 mg, 34%) was obtained as white solids. R_f (TLC) = 0.26 (DCM/MeOH, 5/95), 87–90°C, [α]_D ²⁵ + 34.9 (c 0.34, CHCl₃). ¹H NMR (401 MHz, CDCl₃): δ 5.78 (ddt, J ₁ = 16.9, J ₂ = 10.1, J ₃ = 6.7 Hz, ¹H, H-4′), 5.00 (dq, J ₁ = 17.2, J ₂ = 1.7 Hz, ¹H, (Z)-5′), 4.97–4.93 (m, ¹H, (E)-5′), 3.55–3.43 (m, ¹H, H-3), 0.94 (d, J = 6.4 Hz, 3H, H-21), 0.84 (s, 3H, H-19), 0.70 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.5 (C-24), 138.8 (C-4′), 114.9 (C-5′), 75.3 (C-7), 72.1 (C-3), 54.9, 51.6, 44.5, 44.4, 41.7, 40.7, 40.4, 39.6, 38.8, 36.4, 35.7, 35.5, 34.5, 34.4, 31.2, 30.9, 30.5, 28.5, 27.3, 24.6, 23.0, 21.6, 18.6, 12.5. MS (ESI neg): m/z 459.3 (60%, M−H), 505.4 (100%, M+FA−H), 519.4 (47%, M+AcOH−H), 919.7 (45%, 2 M−H). HR-MS (ESI neg): m/z calcd for C₂₉H₄₇O₄ [M-H], 459.34798; found 459.34770. LCMS method B (ESI neg., t_R = 17.73 min). Purity 99.4% (HPLC method A, t_R = 4.31 min).

3α,7α-Dihydroxy-7β-nonyl-5β-cholan-24-oic acid (7β-nonyl-CDCA, 2k). Compound 2k was prepared according to General Procedure for Grignard Reaction. Starting from compound 7-keto-LCA (1, 500 mg, 1.28 mmol), compound 2k (235 mg, 35%) was obtained as white solids. R_f (TLC) = 0.27 (DCM/MeOH, 5/95), mp 78–80°C, [α]_D ²⁵ + 30.6 (c 0.36, CHCl³). ¹H NMR (401 MHz, CDCl₃): δ 3.49 (tt, J ₁ = 11.0, J ₂ = 6.2 Hz, 1H, H-3), 0.94 (d, J = 6.4 Hz, 3H, H-21), 0.87 (t, J = 6.5 Hz, 3H, H-9′), 0.84 (s, 3H, H-19), 0.70 (s, 3H, H-18). ¹³C NMR (101 MHz, CDCl₃): δ 179.2 (C-24), 75.4 (C-7), 72.1 (C-3), 54.9, 51.6, 45.1, 44.4, 41.8, 40.5, 40.4, 39.7, 38.8, 36.4, 35.7, 35.5, 34.5, 32.1, 31.1, 31.0, 30.5, 30.3, 29.7, 29.4, 28.5, 27.2, 25.2, 23.0, 22.8, 21.6, 18.6, 14.2, 12.4. MS (ESI neg): m/z 517.4 (58%, M−H), 563.4 (100%, M+FA−H), 577.4 (60%, M+AcOH−H), 1035.9 (69%, 2M−H). HR-MS (ESI neg): m/z calcd for C₃₃H₅₇O₄ [M−H], 517.4262; found 517.4258. LCMS method B (ESI neg., t_R = 25.75 min). Purity 97.9% (HPLC method A, t_R = 4.36 min).

7-Ketolithocholic acid (1) was prepared by a three-step synthesis (Figure 1) from commercially available chenodeoxycholic acid (CDCA). First, the carboxylic moiety was protected as methyl ester, followed by the selective oxidation of a 7-hydroxy substituent (Stefela et al., 2020). The regioselectivity of the oxidation towards the C-7 substituent is given by the different reactivity of C-3 equatorial and C-7 axial hydroxy groups, which has been described in the literature (Haslewood, 1942; Fieser and Rajagopalan, 1949; Fieser and Rajagopalan, 1950). The protection of carboxylic moiety as ester facilitates the separation of products after the oxidation step on the column chromatography. Finally, the ester moiety was hydrolyzed under basic conditions.

(E)-7-Ethylidene derivative (7-ELCA, 7-ethylidene-LCA, 2a) was prepared by the Wittig reaction using ethyltriphenylphosphonium bromide as an alkylating reagent (Posa et al., 2014). Similar to the published data, only the E-isomer was obtained. Its structure was confirmed by ROESY NMR (Supplementary Figure S9), exhibiting contacts of the double-bond hydrogen with hydrogen atoms in positions C-14 and C-15. Next, compounds 2b-2k were prepared by the addition of Grignard reagent on the C-7 carbonyl group. The addition proceeded exclusively from the β-side of the steroid skeleton to form a new equatorial C-C bond, as reported by other groups (Une et al., 1989; Bjedov et al., 2017). The stereochemistry at C-7 was assigned and confirmed by several ROESY NMR experiments. For example, the olefinic CH protons of 2d and 2g had clear contacts to hydrogen atoms in position C-6β and C-8, which confirms that the allyl substituent is in position C-7β and the hydroxyl group in position C-7α (Supplementary Figures S10, S11). The structure of 7-ethyl derivative 2c does not exhibit such clear contacts of 7-substituent with the steroid skeleton. The structure was confirmed by the catalytic hydrogenation on palladium in ethanol of 7β-vinyl derivative 2d that afforded compound with an identical ¹H NMR spectrum with that of compound 2c. Finally, the crystal data (Figure 2) of compound 2h (7β-isopropyl-CDCA) showed an alkyl substituent in position C-7β and a hydroxyl group in position C-7α.

To determine the activity of novel derivatives on FXR, we performed luciferase gene reporter assays using a human FXR expression construct in human hepatocyte-derived HepG2 cells. We found that the alkyl substitution to the 7β position led to the complete abrogation of the capacity to activate FXR for all derivatives at 10 µM concentration (Figure 3A). Moreover, the introduction of cyclopropyl (2i) and nonyl (2k) moieties resulted in significant inhibition of the FXR basal activation.

Subsequently, we evaluated whether the introduction of alkyl substituents to the C-7β position would result in an antagonistic capacity toward FXR. For this purpose, we co-treated HepG2 cells with tested compounds together with different known FXR agonists including the highly potent semisynthetic bile acid OCA (1 μM, Figure 3B), non-steroidal ligand GW4064 (1 µM) or endogenous bile acid CDCA (20 μM, Supplementary Table S1). Our results show that the FXR antagonizing behavior appears to be dependent on the length and level of unsaturation of the alkyl substituent at the 7β position. The FXR antagonizing capacity increased with the longer alkyl chain: methyl (2b) < ethyl (2c) < propyl (2f) derivative (Figure 3B). Furthermore, the FXR antagonizing capacity was improved for the branched isopropyl (2h) and cyclopropyl (2i) analogs as compared with the propyl (2f) derivative (Figure 3B). Longer substituents (≥ C₅, 2j, 2k) may, however, affect viability at higher concentrations, as the IC₅₀ of 7β-nonyl derivative (2k) was determined about 15 µM in various cell lines using the MTS viability assay (Supplementary Table S2). This effect might relate to the increased lipophilicity of compounds 2j and 2k. Contrarily, compounds 2a-2h exhibited no effects on cellular viability with IC₅₀ value > 100 µM (Supplementary Figure S1, Supplementary Table S2).

Interestingly, the ability of vinyl (2d), allyl (2g), and pent-4-enyl (2j) derivatives to antagonize the OCA-stimulated FXR activation was maintained with the presence of a double bond in the alkyl moiety. However, this antagonizing capacity might be dependent on the double bond position as well as the spatial orientation of the lipophilic moiety, as 7-ELCA (2a) with (E)-7-ethylidene substituent was established as the most potent FXR antagonist. On the other hand, the compound 2e bearing ethynyl substituent failed to maintain any strong antagonistic effect and exhibited only minor antagonist activity.

In summary, 7-ELCA (2a), 7β-propyl-CDCA (2f), and 7β-isopropyl-CDCA (2h) were demonstrated as the most potent FXR antagonists. These compounds inhibited FXR activation in a concentration-dependent manner by about 95%, 85%, and 90% at 100 µM concentration, respectively, with IC₅₀ values of 15.1 ± 0.7 µM (7-ELCA, 2a, Figure 3C), 20.5 ± 1.0 µM (7β-propyl-CDCA, 2f, Figure 3D) and 17.5 ± 1.7 µM (7β-isopropyl-CDCA, 2h, Figure 3E), respectively. Known FXR antagonists tauro-β-muricholic acid (Tβ-MCA) and Z-GUG were used as controls in these experiments. However, their FXR antagonistic capacity to inhibit FXR activation by the highly potent agonist OCA was limited, and Tβ-MCA decreased the FXR activity by 15% and Z-GUG by 70% at the concentration of 100 μM (IC₅₀ for Z-GUG = 46.0 ± 7.1 μM, Figure 3F).

The FXR LBD structure with the co-crystallized CDCA and OCA, as well as docked poses for 7-ELCA and 7β-isopropyl-CDCA, underwent short molecular dynamics simulations to understand how they could potentially interact within the ligand binding pocket (LBP). Information from different FXR crystal structures shows that the positions of most α-helices are conserved for steroid-based ligands, with the exception of helices α11 and α12, which suggests a single flexible LBP. Due to the high chemical similarity between our series and co-crystallized ligands, we postulated that all ligands could occupy a similar binding site (respective side-chains are depicted as sticks in Figure 4A). However, crystal structures and docking poses can only represent a static snapshot of this interaction, which drove us to use simulations as a model to represent the dynamic equilibrium. CDCA (Figure 4B) and OCA (Figure 4C) have conserved electrostatic interactions between the carboxylate moieties His294, Arg331 and Arg264, the latter leading to the reorganization of the loop between helices α1 and α2. Additionally, both OCA and CDCA presented recurrent hydrogen interactions between the 7α-hydroxyl’s group and Ser332 and Tyr369, which were less prominent in our antagonists. The work from Merk et al. describes the lipophilic contact between the Trp454 and the hydrophobic β-face of CDCA’s A-ring as crucial for FXR full activation (Merk et al., 2019). Similar hydrophobic contact was conserved for most of our proposed antagonists (Supplementary Figure S8B), with exception of guggulsterone (Supplementary Figure S8C), which was unstable in our simulations. Trp454 interaction can also bring the 3α-hydroxyl group into a suitable position to interact with Tyr361 and His447. However, interactions between the 3α-hydroxyl group and Tyr361/His447, although represented in the crystal structure, were not conserved in simulations (Supplementary Figure S8A and Zenodo open data repository under the doi:10.5281/zenodo.3898392).

Simulations of the antagonist muricholic acid’s (MCA) docking pose (Figure 4D) generated a similar interaction profile with a more stable interaction with His447 (Supplementary Figure S9A). We hypothesize that the free His447 could influence the conformation of the α11 and so the heterodimerization interface. However, the extent of this conformational change would need to be addressed by longer monomeric simulations and simulations with the heterodimer. Contrastingly, our proposed antagonists 7-ELCA (2a) and 7β-isopropyl-CDCA (2h) shared similar features with OCA and CDCA, such as a stable interaction with the loop L:α1–α2’s residues (Figure 4E), which suggests a competitive mechanism of action against the natural ligand. Specifically, 7-ELCA had no interactions with Ser332 and Tyr361. Due to the lack of 7α-hydroxyl’s group (Figure 4F), the counterpart ethylidene moiety established hydrophobic contacts with both Phe366 and Phe372.

The time-resolved fluorescence energy transfer (TR-FRET) FXR co-activator association assay with the recombinant FXR LBD was used to assess the FXR antagonism of the most potent FXR antagonists 7-ELCA, 7β-propyl-CDCA and 7β-isopropyl-CDCA in the cell-free system. We found that all three compounds inhibited OCA-induced recruitment of the co-activator peptide SRC-2 to FXR LBD in a concentration-dependent manner with IC₅₀ values 18.0 ± 2.7 µM (7-ELCA), 26.5 ± 2.0 µM, (7β-propyl-CDCA), 25.0 ± 3.7 µM (7β-isopropyl-CDCA, Figures 5A–C), respectively. Moreover, the derivatives antagonized the interaction of SRC-2 and FXR promoted by the nonsteroidal FXR activator GW4064 with IC₅₀ values 6.6 ± 2.9 µM (7-ELCA), 13.8 ± 2.5 µM, (7β-propyl-CDCA), 35.2 ± 5.4 µM (7β-isopropyl-CDCA, Figures 5D–F), respectively.

To further corroborate the FXR antagonistic properties of 7-ELCA and 7β-propyl-CDCA, we examined their effects on the expression of FXR downstream genes in the presence and absence of the FXR agonist OCA in terminally differentiated HepaRG cells (Figures 6A,B) and primary human hepatocytes (Figures 6C,D). 7-ELCA and 7β-propyl-CDCA (40 µM) reduced BSEP (Figures 6A,C) and SHP (Figures 6B,D) mRNA levels upregulated by OCA (1 µM). The treatment with 7-ELCA and 7β-propyl-CDCA, per se, did not have an impact on the expression of FXR target genes BSEP and SHP mRNA. In addition, we confirmed the FXR antagonistic effect of 7-ELCA and 7β-propyl-CDCA on SHP protein expression in HepaRG cells treated with the FXR agonist OCA (Figure 6E). The effects of 7-ELCA and 7β-propyl-CDCA were much stronger on SHP protein downregulation than on SHP mRNA expression after treatment with OCA in HepaRG cells (Figures 6B,E). We suppose that the phenomenon is due to the longer treatment intervals in protein expression experiments. Altogether, these data suggest that 7-ELCA and 7β-propyl-CDCA act as FXR antagonists in hepatic cells.

To examine the specificity of the most potent FXR antagonist in this study, 7-ELCA, we assessed its interaction with a wide range of nuclear receptors known to interact with BAs or to regulate metabolic processes, including vitamin D receptor (VDR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors α, γ, β/δ (PPAR α, γ, β/δ), glucocorticoid receptor (GR), liver X receptor α, β (LXR α, β) and thyroid receptor (TRα). As shown in Figure 7A, 7-ELCA did not activate any of the examined nuclear receptors. In addition, 7-ELCA did not antagonize the activation of the nuclear receptors stimulated by their model ligands at the 10 µM concentration (Figure 7B). Taken together, 7-ELCA is a selective FXR antagonist when considering interactions with the tested nuclear receptors.

Upon the activation of the membrane GPBAR1, BAs bind to the ligand-binding pocket of GPBRA1 and trigger downstream signaling via cAMP generation followed by the activation of downstream kinases and cAMP response element (CRE) in the nucleus (Kawamata et al., 2003). Tested compounds (10 µM) were analyzed for their ability to activate a CRE-luc construct when compared to LCA (10 µM) as a known GPBAR1 agonist (Figure 8A). Only 7-ELCA (2a) was able to significantly increase CRE-luc activation more than LCA (10 µM). 7β-propyl-CDCA (2f), 7β-allyl-CDCA (2g) and 7β-cyclopropyl-CDCA (2i) displayed comparable activities to LCA. Other compounds significantly activated GPBAR1 with activity lower than LCA at the concentration of 10 μM and compounds 7β-ethynyl-CDCA (2e), 7β-pentenyl-CDCA (2j) and 7β-nonyl-CDCA (2k) exhibited weak or no ability to act as agonists of GPBAR1. As demonstrated in Figure 7B, the activation of CRE-luc by LCA or other compounds was dependent on the co-transfection of GPBAR1, because the mock co-transfection of an empty vector did not lead to augmented luciferase activity of CRE-luc. Consistently with the data, the treatment with 10 µM 7-ELCA led to more significant production of cAMP compared to the treatment with 10 µM LCA in differentiated enteroendocrine NCI-H716 cells (Figure 8C).

The concentration-response study (Figure 8D) underlined the superiority of 7-ELCA in GPBAR1 activation with the EC₅₀ value being lower by about 2 orders of magnitude when compared to LCA activity (0.026 ± 0.006 µM vs. 1.54 ± 0.4 µM, respectively).

The activation of GPBAR1 by BAs in colonic L-cells is known to result in the secretion of the incretin GLP-1, which in turn stimulates insulin secretion from pancreatic cells. To further evaluate the activity of 7-ELCA on GPBAR1, we exposed differentiated colonic human NCI-H716 L-cells to 7-ELCA. We observed a significant increase of GLP-1 secretion into the culture media after the treatment with 7-ELCA (Figure 8E). In addition, the GLP-1 secretion induced by 7-ELCA (10 µM) was significantly stronger compared to LCA (10 µM). Interestingly, 7-ELCA can also increase GPBAR1 mRNA expression in NCI-H716 cells (Figure 8F). We determined GLP-1 secretion in control and Gpbar1 siRNA transfected GLUTag cells. The treatment of 7-ELCA (10 µM) led to a significant increase of the GLP-1 secretion in the control cells. However, when the Gpbar1 expression was silenced by Gpbar1 siRNA, 7-ELCA treatment did not lead to the significantly increased GLP-1 production (Figure 8G). To summarize, our results show that 7-ELCA is a potent steroidal GPBAR1 agonist with an EC₅₀ value at nanomolar concentration and its effect on GLP-1 production is dependent on the GPBAR1 expression.

For a long time, only homology models of GPBAR1 have been available. Recently, the cryo-electron microscopy structure of GPBAR-G_s unveiled an oval LBP with hydrophilic residues accumulated at the bottom part of the cavity with the rest of the LBD surface formed predominantly by hydrophobic amino acids (Yang et al., 2020). For this study, the PDB 7CFN model was used due to the structural similarity of our compounds and the bound ligand (6-ethyl-23(S)-methyl-cholic acid, INT-777). The molecular docking study revealed that LCA binds to GPBAR1 perpendicularly to the cytoplasmatic membrane (Figure 9A). The A-ring of LCA faces the hydrophilic bottom of the cavity where it forms a hydrogen bond between 3-hydroxyl group and amino acid residues Tyr240 and Ser270. The rest of the LBD is fairly hydrophobic and further stabilizes the LCA preferred pose. The flexible sidechain connected to the D-ring and ended by a carboxyl group freely floats in the outward direction from the pocket. However, it is not long enough to exhibit any interaction with polar groups that form the outer surface around the pocket entrance.

The docking study showed that the docking score of LCA to GPBAR1 (−9.2 kcal/mol, Figure 9B) is worse than in the case of 7-ELCA (−9.9 kcal/mol, Figure 9C). In addition, 7-ELCA had the best docking score towards GPBAR1 among all alkylated derivatives (Supplementary Figure S2). All tested ligands adopted a similar position as LCA in the LBD. If present, the C-7 hydroxyl always forms a hydrogen bond with Ser247. The C-7 alkyls face towards a strongly hydrophobic pocket cleft formed by Phe161, Leu166, Val170, Leu244, Ser247, and Val248, as presented with the 7-ELCA in Figure 9D. The alkylation at the position C-7 might help ligands to pose in the pocket tightly. Furthermore, the alkylation on C-7 influences the hydrogen bond formation between a ligand and Ser270. Ligands with two-carbons substituents on C-7 form only one hydrogen bond interaction between the C-3 hydroxyl and Tyr240. The Ser270 hydroxyl is spatially too far away because the whole ligand’s C-7 two-carbons substituent drags the ligand towards the hydrophobic pocket cleft to exploit hydrophobic interactions. On the other hand, compounds with three-carbons substituents are wide enough to reach both the hydrophobic pocket cleft with its C-7 substituent and polar Tyr240 and Ser270 groups with its C-3 hydroxyl. LCA has no C-7 alkyl substituent and therefore is not attracted so strongly towards the hydrophobic pocket cleft and prefers a position where hydrogen bonds are formed with both Tyr240 and Ser270.

Finally, deeper insight into the interactions between GPBAR1 and 7-ELCA was obtained in luciferase reporter gene experiments with mutated GPBAR1 variants (Figure 9E). Amino acids Ser270, Glu169, and Tyr89, identified previously as potentially important for the interaction of ligands with GPBAR1 in various receptor models (D'Amore et al., 2014; Gertzen et al., 2015; Macchiarulo et al., 2013), were replaced with glycine, a neutral amino acid incapable to form hydrophobic or hydrophilic interactions. The mutation of Ser270 blocked the capacity of LCA to activate CRE-luc reporter construct. However, the activity of 7-ELCA was partially preserved in the same experiment. This might be explained by the hydrogen bond between Ser270 and 3-hydroxyl group of LCA, which is not present in the case of 7-ELCA. These data suggest that Ser270 may be important for the activation of GPBAR1 (Figure 9E). On the other hand, Glu169 is not crucial for GPBAR1 activation by LCA, but rather it helps to stabilize ligands in the LBD. The mutation of the residue Tyr89 did not affect GPBAR1 activation, which is consistent with our previous data (Stefela et al., 2020).