TGR5
TGR5 (G protein-coupled bile acid receptor). [1]
SBI-115 is a selective antagonist of TGR5 (Takeda G protein-coupled receptor 5, also known as GPCR19, G protein-coupled bile acid receptor). In HEK293 cells expressing human TGR5, SBI-115 inhibits TGR5-mediated cAMP accumulation with an IC₅₀ of approximately 120 nM . The compound functions as a competitive antagonist by occupying the receptor's binding site, thereby preventing activation by endogenous bile acids such as taurolithocholic acid (TLCA) and oleanolic acid (OA) .

SBI-115 (100-200 μM, 24 hours) prevents the proliferation of shRNA-transfected ADPKD cholangiocytes that is caused by pre-treating cystic cholangiocytes with taurolithocholic acid (TLCA)[1].

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In cystic cholangiocytes derived from ADPKD patients, SBI-115 (100 and 200 µM) dose-dependently inhibited TLCA-induced cell proliferation by 32-48%, as assessed by MTS assay and cell counting. [1]
SBI-115 (200 µM) reduced the growth of cholangiocyte spheroids formed by ADPKD cholangiocytes in 3-D hanging drop culture by approximately 30%. [1]
SBI-115 (200 µM) decreased cAMP levels in ADPKD cystic cholangiocytes that had been pre-stimulated with the TGR5 agonist TLCA by approximately 30%. This effect was absent in TGR5-depleted (via shRNA) cholangiocytes, confirming TGR5-dependent action. [1]
Concurrent treatment of ADPKD cystic cholangiocytes with SBI-115 (200 µM) and pasireotide (20 µM, an SSTR agonist) led to a greater reduction in cell proliferation (45-55% inhibition), spheroid growth, and cAMP levels compared to each drug alone. [1]
SBI-115 alone had no effect on cell proliferation, spheroid growth, or cAMP levels in the absence of TGR5 agonist stimulation. [1]
SBI-115 had no effect on forskolin-induced cAMP production in cystic cholangiocytes, indicating its action is specific to TGR5-mediated cAMP signaling. [1]

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SBI-115 inhibited taurolithocholic acid (TLCA)-induced proliferation of cystic cholangiocytes derived from ADPKD patients in a dose-dependent manner (32-48% inhibition) at concentrations of 100 and 200 μM for 24 hours, as measured by MTS assay and cell counting . At 200 μM, SBI-115 reduced the growth of cholangiocyte spheroids in 3-D hanging drop culture by approximately 30% and decreased TLCA-stimulated intracellular cAMP levels by approximately 30%, an effect that was absent in TGR5-knockdown cells, confirming on-target activity . In the absence of TGR5 agonist stimulation, SBI-115 alone had no effect on cell proliferation, spheroid growth, or cAMP levels, and it did not affect forskolin-induced cAMP production, indicating specificity for the TGR5-mediated cAMP signaling pathway . Combination treatment with SBI-115 (200 μM) and pasireotide (20 μM, a somatostatin receptor agonist) resulted in synergistic reductions in cell proliferation (45-55% inhibition), spheroid growth, and cAMP levels compared to either drug alone . SBI-115 (1-100 μM) also blocked lithocholic acid (LCA)-induced cathelicidin (CAMP) gene expression and ERK1/2 phosphorylation in bronchial epithelial BCi cells . In pancreatic cancer cell lines, SBI-115 treatment significantly reduced cell proliferation capacity and induced cell death, with accompanying disturbances in mitochondrial morphology and metabolic pathways . SBI-115 (100 μM, 24 hours) reversed the regulatory effects of bile acids on M1/M2 macrophage polarization .

In order to further investigate TGR5's role in PLD, we produced double-mutant mice with the TGR5−/−;Pkhd1del2/del2 phenotype. TGR5−/− mice demonstrated good health and reproducibility, in line with an earlier report. 29, Overall morphology (Supporting Fig. 4), liver, cholangiocyte, and primary cilium morphology (Supporting Table 4), and serum biochemistries (Supporting Table 3) were similar in WT and TGR5−/− mice. More than one hepatic cyst is present in Pkhd1del2/del2 rodents. 30, TGR5 was overexpressed in Pkhd1del2/del2 mice relative to WT, while it was not seen in TGR5−/− mice or TGR5−/−;Pkhd1del2/del2 mice, which is in line with our findings in other animal models of PLD (Supporting Fig. 5). Compared to Pkhd1del2/del2 littermates, we observed reductions in liver weight (30%), hepatic cystic areas (31%), and hepatic fibrotic areas (33%), in double mutant TGR5−/−;Pkhd1del2/del2 mice (both males and females). Supporting Table 4 and Figure 5A. The mouse serum biochemistries in each group were comparable (Supporting Table 3). Reduced quantity of PCNA-positive nuclei by three times was linked to attenuated hepatic cystogenesis in double mutant TGR5−/−;Pkhd1del2/del2 mice (Fig. 5B).

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In Pkhd1del2/del2 mice (a polycystic liver disease model), genetic elimination of TGR5 or treatment with SBI-115 reduced hepatic cystic areas by 45% and 31%, respectively, and decreased liver weight by 30% . Pre-treatment of polycystic kidney rats with the TGR5 agonist oleanolic acid (OA) increased hepatic cystogenesis by 35%, while SBI-115 treatment significantly reduced this effect . Immunohistochemistry showed a 3-fold reduction in PCNA-positive nuclei in livers of SBI-115-treated animals, indicating decreased cholangiocyte proliferation . In a collagen-induced arthritis (CIA) mouse model, oral administration of SBI-115 (80 mg/kg, daily for 28 days) was evaluated for anti-inflammatory effects . In a cecal ligation and puncture (CLP)-induced sepsis-related acute liver injury (SALI) mouse model, SBI-115 was used as a tool compound to validate TGR5-mediated protection, where it reversed the protective effects of TGR5 agonists .

The following assay protocols are described: cAMP detection assay – Cholangiocytes (10,000 cells/well) were incubated with TLCA (25 μM), SBI-115 (100 and 200 μM), and/or pasireotide (20 μM) for 15-30 minutes. Intracellular cAMP levels were measured using the Bridge-It cAMP Designer cAMP Assay Kit following the manufacturer's instructions . Cell proliferation assay (MTS) – Control and ADPKD human cystic cholangiocytes (2,500 cells/well) were plated for 24-48 hours, then treated with SBI-115 (100 or 200 μM), pasireotide (20 μM), or both for an additional 24 hours. Cell proliferation was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS), with results expressed as percent change compared to untreated control cells (set at 100%) . 3-D cholangiocyte spheroid growth assay – Spheroids were generated using the hanging drop method in 96-well plates by forming 10,000 suspended cells per drop. Spheroids formed within 24 hours and were cultured for an additional 96 hours before treatment with SBI-115 (200 μM), pasireotide (20 μM), or both. Photomicrographs were taken before and after treatment, and spheroid circumference was measured to assess growth. Treatment of ADPKD cholangiocytes with SBI-115 (200 μM) reduced spheroid growth by approximately 30% .

Cell Line: shRNA-transfected ADPKD cholangiocytes
Concentration: 100, 200 µM
Incubation Time: 24 hours
Result: Inhibited proliferation (by 32-48%) triggered by pre-treatment of cystic cholangiocytes with TLCA.

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Cell Proliferation Assay: Control and ADPKD human cystic cholangiocytes were plated at 2500 cells/well and grown for 24-48 hours. Cells were then treated with SBI-115 (100 or 200 µM), pasireotide (20 µM), or a combination, for an additional 24 hours. Cell proliferation was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) and by direct cell counting using an automated cell counter. For TGR5 antagonism studies, cells were often pre-treated with the TGR5 agonist TLCA (25 µM) to stimulate proliferation before adding SBI-115. [1]
cAMP Detection Assay: Cholangiocytes (10,000 cells/well) were incubated with SBI-115 (100 or 200 µM), pasireotide (20 µM), or a combination, for 15-30 minutes. In some experiments, cells were first stimulated with TLCA (25 µM). Intracellular cAMP levels were measured using the Bridge-It cAMP designer cAMP assay. [1]
3-D Cholangiocyte Spheroid Growth Assay: Cholangiocyte spheroids were generated using a hanging drop method. Drops containing 10,000 cholangiocytes in suspension were formed in a 96-well hanging drop plate. Spheroids formed within 24 hours and were grown for an additional 96 hours before treatment with SBI-115 (200 µM), pasireotide (20 µM), or both. Microphotographs were taken before and after treatment, and the circumference of the spheroids was measured to assess growth. [1]

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Cholangiocyte culture and treatment – Human cystic cholangiocytes from ADPKD patients were cultured and treated with SBI-115 (100 or 200 μM) for 24 hours. To stimulate TGR5-mediated proliferation, cells were pre-treated with the TGR5 agonist taurolithocholic acid (TLCA, 25 μM) before SBI-115 addition. Cell proliferation was measured by MTS assay and automated cell counting . cAMP measurement – Cholangiocytes (10,000 cells/well) were incubated with SBI-115 (100 or 200 μM) and/or pasireotide (20 μM) for 15-30 minutes. In some experiments, cells were stimulated with TLCA (25 μM) prior to treatment. cAMP levels were measured using a commercial cAMP assay kit . Pancreatic cancer cell assay – BXPC3 cells (5 μM SBI-115, 48 hours) and PANC-1 cells (10 μM SBI-115, 48 hours) were treated to assess cell proliferation, cell death, mitochondrial morphology, and metabolic pathway alterations .

Polycystic liver disease model (Pkhd1del2/del2 mice) – Double mutant TGR5⁻/⁻;Pkhd1del2/del2 mice were generated to evaluate the effect of TGR5 deficiency on hepatic cystogenesis. Compared to Pkhd1del2/del2 littermates, TGR5⁻/⁻;Pkhd1del2/del2 mice showed 30% reduction in liver weight, 31% reduction in hepatic cystic area, and 33% reduction in hepatic fibrosis area. Serum biochemistry parameters were similar across all groups . Oleanolic acid (OA)-treated polycystic kidney rats – Polycystic kidney rats were treated with the TGR5 agonist OA to increase cystogenesis by 35%. SBI-115 treatment antagonized this effect, reducing cyst formation . Collagen-induced arthritis (CIA) mouse model – DBA/1J mice were administered SBI-115 orally at 80 mg/kg daily for 28 days to evaluate anti-inflammatory effects . Sepsis-related acute liver injury (SALI) model – Mice underwent cecal ligation and puncture (CLP) to induce SALI. SBI-115 was used as a TGR5 antagonist to reverse the protective effects of TGR5 agonists, confirming the receptor's role in mitigating liver injury .

In animal studies, serum biochemistry parameters (including ALT, AST, and other markers) in SBI-115-treated animals were similar to control groups, indicating no significant hepatotoxicity at the doses tested . In the Pkhd1del2/del2 mouse model, TGR5⁻/⁻ mice (comparable to SBI-115-treated animals) were reported to be healthy and fertile, with normal gross anatomy and liver morphology, suggesting that TGR5 antagonism is well-tolerated .

[1]. TGR5 contributes to hepatic cystogenesis in rodents with polycystic liver diseases through cyclic adenosine monophosphate/Gαs signaling. Hepatology. 2017 Oct; 66(4):1197-1218.

SBI-115 (m-Tolyl-5-chloro-2-(ethanesulfonyl)pyrimidine-4-carboxylic acid ester) is a novel small molecule TGR5 antagonist. [1] It was discovered by high-throughput screening of a library of 50,000 compounds using CHO-K1 cells expressing TGR5. [1] The compound was found to be >95% pure by HPLC. [1] This study suggests that SBI-115 may be a novel potential therapy for polycystic liver disease (PLD) by inhibiting TGR5 and reducing cAMP levels in cystic bile duct cells. [1] Data suggest that in PLD, the simultaneous use of a TGR5 antagonist (SBI-115) and a somatostatin receptor agonist (paretide) to target elevated cAMP levels may be more effective than either drug alone in inhibiting bile duct cell proliferation and cyst growth. [1]

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SBI-115 (CAS: 882366-16-7) has the chemical name m-tolyl 5-chloro-2-(ethylsulfonyl)pyrimidine-4-carboxylate and molecular formula C₁₄H₁₃ClN₂O₄S . The compound was discovered through high-throughput screening of a 50,000-compound library using CHO-K1 cells expressing TGR5 and was found to be >95% pure by HPLC . SBI-115 functions as a selective TGR5 antagonist by blocking TGR5-mediated cAMP accumulation, with an IC₅₀ of approximately 120 nM . In polycystic liver disease (PLD), TGR5 is overexpressed in cystic cholangiocytes (2-3 fold increase compared to control), and SBI-115 treatment reduces cAMP levels and cholangiocyte proliferation . The combination of a TGR5 antagonist (SBI-115) and a somatostatin receptor agonist (pasireotide) may be more effective than either drug alone in inhibiting cholangiocyte proliferation and cyst growth, representing a potential novel therapeutic approach for PLD . SBI-115 has also been used as a tool compound in studies investigating TGR5's role in sepsis-related acute liver injury, where it reversed the protective effects of TGR5 agonists on hepatocyte pyroptosis via NLRP3 inflammasome inhibition . The compound has further been employed in research on macrophage polarization (M1/M2 balance) and airway inflammation . Storage: powder at -20°C for up to 3 years; in solution at -20°C for up to 6 months, protected from light and moisture .

C14H13CLN2O4S

340.782021284103

340.03

C, 49.34; H, 3.85; Cl, 10.40; N, 8.22; O, 18.78; S, 9.41

882366-16-7

18879973

White to off-white solid powder

2.9

0

6

5

22

494

0

O=C(C1C(Cl)=CN=C(S(CC)(=O)=O)N=1)OC1C=C(C)C=CC=1

IJPXOPBVXVPPEW-UHFFFAOYSA-N

InChI=1S/C14H13ClN2O4S/c1-3-22(19,20)14-16-8-11(15)12(17-14)13(18)21-10-6-4-5-9(2)7-10/h4-8H,3H2,1-2H3

(3-methylphenyl) 5-chloro-2-ethylsulfonylpyrimidine-4-carboxylate

SBI115; SBI 115; m-Tolyl 5-chloro-2-(ethylsulfonyl)pyrimidine-4-carboxylate; RefChem:925116; 882366-16-7; m-Tolyl5-chloro-2-(ethylsulfonyl)pyrimidine-4-carboxylate; SBI-115

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 68~150 mg/mL (199.5~440.2 mM)

Solubility in Formulation 1: 3 mg/mL (8.80 mM) in 2% DMSO + 40% PEG300 + 5% Tween80 + 53% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5 mg/mL (14.67 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)