Journal: Cell reports

Article Title: Blockade of CCR5 suppresses paclitaxel-induced peripheral neuropathic pain caused by increased deoxycholic acid.

doi: 10.1016/j.celrep.2023.113386

Figure Lengend Snippet: Figure 6. DCA upregulates CCR5 expression through a TGR5/CREB-dependent mechanism (A) Reduced mechanical hyperalgesia in CCR5/ rats after DCA administration (n = 8). *p < 0.05 and **p < 0.01 by two-way ANOVA followed by Sida´ k’s multiple- comparison test. (B) TGR5 and FXR expressions in rat DRG by western blot (n = 4). (C and D) CCR5 mRNA levels in DRG neurons after siRNA knockdown of TGR5 and FXR in vitro (C) and with different DCA and TGR5 antagonist SBI-115 concentrations in vitro (D) (n = 3 cells). (E and F) Expression of TGR5 in sections of PIPN rat DRG. Double immunofluorescence images (E) and quantification analysis (F) detected the colocalization of TGR5 and markers of different population of DRG neurons. Scale bar, 50 mm. (G and H) Colocalization of TGR5 and CCR5 in PIPN rat DRG. Scale bar, 50 mm. (I–K) TGR5 knockdown in L4–L5 DRG neurons via shTGR5 AAV injections. (I) Experimental timeline. Knockdown efficacy confirmed by western blot (J) and immunofluorescence (K). n = 6 rats. Scale bar, 50 mm. (L and M) Protein expressions in rat DRG (n = 5). (N–R) Protein expressions in DRG neurons in vitro with DCA (N–O) and PKA- and PKC-selective inhibitors H89 and Go6983 (P) and transfected with siTGR5 (Q and R) in vitro (n = 3 or 4 cells).

Article Snippet: Following antibodies were used: CCR5 (1:100, Abcam#ab11466), IB4 (1:200, Invitrogen#I21411), CGRP (1:200, CST#14959; 1:200, Abcam#ab81887), NF200 (1:200, CST#2836), and TGR5 (1:200, Novus#NBP2-23669).

Techniques: Expressing, Comparison, Western Blot, Knockdown, In Vitro, Transfection

Journal: Cell reports

Article Title: Blockade of CCR5 suppresses paclitaxel-induced peripheral neuropathic pain caused by increased deoxycholic acid.

doi: 10.1016/j.celrep.2023.113386

Figure Lengend Snippet: Figure 7. Blockade of CCR5 by maraviroc or CCL5 neutralizing antibody improves recovery from PIPN (A) Acute treatment with maraviroc or CCL5 neuAb reverses established PIPN at 1 and 3 h after administration (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001, PIPN group vs. PIPN + maraviroc group; #p < 0.05, ##p < 0.01, and ###p < 0.001, PIPN group vs. PIPN + CCL5 neuAb group; p values by two-way ANOVA followed by Sida´ k’s multiple-comparison test. (B) Survival of 24-month-old rats during 14-day follow-up (n = 8). (C and D) Expression of CGRP in rat DRG after maraviroc and CCL5 neuAb treatment. Immunofluorescence images (C) and quantification analysis of the number of CGRP-IR neurons (D). Scale bar, 100 mm. *p < 0.05 and **p < 0.01 by one-way ANOVA with Tukey’s multiple-comparison post hoc test. (E) Expression of CCR5 and CCL5 in pathological classes of breast cancer patients (N = 1,097) and glioblastoma patients (N = 156) by UALCAN database. (F) Correlation of CCR5 and CCL5, TGR5, and FXR in tumor microenvironment by TIMER database. All data are presented as mean ± SEM.

Article Snippet: Following antibodies were used: CCR5 (1:100, Abcam#ab11466), IB4 (1:200, Invitrogen#I21411), CGRP (1:200, CST#14959; 1:200, Abcam#ab81887), NF200 (1:200, CST#2836), and TGR5 (1:200, Novus#NBP2-23669).

Techniques: Neutralizing Assay, Comparison, Expressing

Journal: Nutrients

Article Title: Glycochenodeoxycholate Affects Iron Homeostasis via Up-Regulating Hepcidin Expression

doi: 10.3390/nu14153176

Figure Lengend Snippet: Effects of GCDCA on the changes of BMP6/ALK3 signaling related receptors in HepG2 cells. ( A ) Relative gene expression of TGR5 and FXR after GCDCA treatment. ( B ) Representative western blotting bands of TGR5 and FXR under GCDCA treatment, as well as their fold changes in optical density relative to controls. ( C ) Relative gene expression of hepcidin and FXR after treatment of a FXR activator (GW4064, 10 µM for 2 h). ( D ) The effects of GW4064 on the phosphorylation of SMAD1/5/8 (normalized by total SMAD1) and protein level of FXR. ( E – H ) The effect of guggulsterone (GS, a FXR inhibitor, pretreated HepG2 cells for 18 h at 20 µM) on GCDCA-induced activation of FXR-BMP6/ALK3 signaling proteins and hepcidin expression. Values were shown as means ± SD. *, p < 0.05, GCDCA treatment group vs. control group. **, p < 0.01. ***, p < 0.001. †, p < 0.05, other experimental groups vs. control group. ††, p < 0.01. ##, p < 0.01, GCDCA treatment group vs. other experimental groups. ###, p < 0.001. Each experiment was performed at least three times.

Article Snippet: Primary antibodies rabbit anti-TGR5 (1:1000, NBP2-23669) and rabbit anti-ferroportin (1:1000, NBP1-21502) were purchased from Novus Biologicals (Littleton, Colorado, USA).

Techniques: Gene Expression, Western Blot, Phospho-proteomics, Activation Assay, Expressing, Control

Journal: European journal of medicinal chemistry

Article Title: Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

doi: 10.1016/j.ejmech.2024.116616

Figure Lengend Snippet: Fig. 1. General overview of virtual screenings and targeted docking calculations performed at the TGR5. Compounds in bold cyan text refer to PAM activity; compounds in bold black text refer to agonistic activity. A) Initial hit compounds (K3, K5, K7, K16, Table S1) with weak PAM effect that arose from a virtual screening against the intrahelical binding site (IBS) in a homology model of the TGR5 using the ZINC15 lead-like library. B) Allosteric pocket exploration performed on PDB ID 7cfm; W75-pocket emerged as the highest-ranked pocket and KS5 as an additional common pocket in the top three. C) Virtual screening against PDB IDs 7cfm (IBS and W75-pocket) and 7cfn (IBS) using ZINC20 lead-like and drug-like libraries, which led to one PAM (K76). D) PAM K76 was used as a query for a similarity search in ZINC through SmallWorld. Retrieved compounds were then docked into PDB ID 7cfm in the W75-pocket. This docking analysis yielded three agonists, K85, K86, K91, and one PAM, K94. E) MD simulations to explore the conformations of the receptor and the stability of initial hit K7 with wildtype receptor and W75A mutant. The control systems (systems 1 and 2, Table S2) were clustered with cpptraj. Three representative frames of each control system, plus PDB ID 7xtq, were used for another screening using DOCK3.7 and a ZINC20 custom library which led to one agonist (K96) and two PAMs (K97, K100).

Article Snippet: GPBAR1 transcript variant 3 (NM_170699) Human Untagged cDNA Clone (SC123312) was obtained from OriGene Technologies GmbH (Herford, Germany) and subcloned into the pcDNA3.1/Zeo(+) mammalian expression vector (Thermo Fisher Scientific) using the restriction sites NotI/NotI (New England Biolabs).

Techniques: Activity Assay, Binding Assay, Mutagenesis, Control

Journal: European journal of medicinal chemistry

Article Title: Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

doi: 10.1016/j.ejmech.2024.116616

Figure Lengend Snippet: Fig. 2. Pocket exploration of the TGR5 (PDB ID 7cfm). A) Predicted pockets obtained with the “Site Finder” module of Molecular Operating Environment (MOE version 2019.01). Green spheres indicate the predicted sites from the highest ranking one in dark green to the lowest ranking one in light green. B) Docking of initial hits (K3, K5, K7, and K16 in teal; Table S1) with SwissDock, i.e. to the whole receptor surface. The highest-ranked pocket in both analyses is in proximity to W75ECL1

Article Snippet: GPBAR1 transcript variant 3 (NM_170699) Human Untagged cDNA Clone (SC123312) was obtained from OriGene Technologies GmbH (Herford, Germany) and subcloned into the pcDNA3.1/Zeo(+) mammalian expression vector (Thermo Fisher Scientific) using the restriction sites NotI/NotI (New England Biolabs).

Techniques:

Journal: European journal of medicinal chemistry

Article Title: Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

doi: 10.1016/j.ejmech.2024.116616

Figure Lengend Snippet: Fig. 3. Bioactivity of the in silico predicted compounds at the TGR5 in cellular in vitro assays. Green bars represent reference values (obtained with LCA, P395, or Forskolin). A) Concentration-response curve of the orthosteric agonist LCA at the TGR5 with (dark gray) and without (light gray) co-treatment with 30 μM PAM K76, determined in the CRE-Luciferase assay. LCA was tested at increasing concentrations (0.01 μM–10 μM) in stable TGR5-expressing HEK EPAC cells. B) Agonistic activities of tested compounds at the TGR5 led to intracellular cAMP accumulation in a TGR5-dependent manner. TGR5 HEK EPAC cells were treated with 30 μM compound (full bars). To confirm TGR5 specificity, non-TGR5- expressing HEK EPAC cells were treated in the same way (striped bars). Note that for K76 no specificity control was performed due to its inactivity. The TGR5 agonist LCA (10 μM) and the direct stimulator of adenylyl cyclase For skolin (10 μM) were included as positive controls (green bars). C) Agonistic activities of tested compounds at the TGR5 determined by CRE-Luciferase as says. Compounds were tested at 30 μM concentration in stable TGR5-expressing HEK EPAC cells (gray bars). To determine potential TGR5-independent un specific effects, non-TGR5-expressing HEK EPAC cells were treated in the same way (striped bars). D) PAM activities of tested compounds at TGR5 determined by CRE-Luciferase assays. Compounds were tested at 30 μM final concentration in co-treatment with the orthosteric agonists LCA (3 μM, left) or P395 (3 nM, right) in stable TGR5-expressing HEK EPAC cells (dotted bars; activation of orthosteric agonists alone shown by dashed line). Results are expressed as percent increase in fold activation (A, C, D) compared or fluorescence emission ratio (B) to vehicle control (0.1 or 0.2 % DMSO). All bars and points represent mean ± SD of at least three independent biological replicates (n ≥3) measured in technical quadruplicates (A, C, D) or triplicates (B). One-way ANOVA fol lowed by Dunnett’s post hoc test (****p ≤0.0001, ***p ≤0.001, **p ≤0.01, *p ≤0.05, n. s. p > 0.05) compared to vehicle control in TGR5 HEK EPAC cells, HEK EPAC (control) cells (B, C) or to orthosteric agonist alone (LCA 3 μM or P395 3 nM; D), respectively.

Article Snippet: GPBAR1 transcript variant 3 (NM_170699) Human Untagged cDNA Clone (SC123312) was obtained from OriGene Technologies GmbH (Herford, Germany) and subcloned into the pcDNA3.1/Zeo(+) mammalian expression vector (Thermo Fisher Scientific) using the restriction sites NotI/NotI (New England Biolabs).

Techniques: In Silico, In Vitro, Concentration Assay, Luciferase, Expressing, Control, Activation Assay, Fluorescence

Journal: European journal of medicinal chemistry

Article Title: Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

doi: 10.1016/j.ejmech.2024.116616

Figure Lengend Snippet: Fig. 5. Ligands show concentration-dependent activities as TGR5 agonists in vitro and Concentration-response curves for agonist K91. A) Concentration-dependent agonistic activities of tested compounds at the TGR5 determined by CRE-Luciferase assays. Compounds were tested in decreasing concentrations (30 μM–0.3 μM) in stable TGR5-expressing HEK EPAC cells in a CRE-Luciferase assay to determine their agonistic activities at the TGR5 (gray bars). The TGR5 agonist LCA (10 μM) was included as a positive control (green bar). Apparent EC50 value (EC50 app) of K91 was 19 μM. B) Concentration-dependent agonistic activities of tested compounds at the TGR5 determined in a cAMP accumulations assay. TGR5 HEK EPAC cells were treated with the indicated compounds in decreasing concentrations (30 μM–0.3 μM) in stable TGR5-expressing HEK EPAC cells in a cAMP accumulation assay. The TGR5 agonist LCA (10 μM) and the direct stimulator of adenylyl cyclase Forskolin (10 μM) were included as positive controls (green bars). Apparent EC50 value (EC50 app) of K91 was 30 μM. C) Concentration-response curve for the agonist K91 at the TGR5, determined in CRE-Luciferase assay. The compound was tested at increasing concentrations (0.3 μM–30 μM) in stable TGR5-expressing HEK EPAC cells to determine its agonistic activity at the TGR5. D) Concentration-response curve for the agonist K91 at the TGR5, determined in cAMP accumulation assay. The compound was tested at increasing concentrations in stable TGR5-expressing HEK EPAC cells. Results are expressed as fold activation (A, C) or percent increase in fluorescence emission ratio (B, D) compared to vehicle control (0.1 % DMSO). All bar charts and points represent the mean ± SD of at least three independent biological replicates (n ≥3) measured in technical quadruplicates (A, C) or triplicates (B, D). One-way ANOVA followed by Dunnett’s post hoc test (****p ≤0.0001, ***p ≤0.001, **p ≤0.01, *p ≤0.05, n. s. p > 0.05 compared to vehicle control.

Article Snippet: GPBAR1 transcript variant 3 (NM_170699) Human Untagged cDNA Clone (SC123312) was obtained from OriGene Technologies GmbH (Herford, Germany) and subcloned into the pcDNA3.1/Zeo(+) mammalian expression vector (Thermo Fisher Scientific) using the restriction sites NotI/NotI (New England Biolabs).

Techniques: Concentration Assay, In Vitro, Luciferase, Expressing, Positive Control, Activity Assay, Activation Assay, Fluorescence, Control

Journal: Molecular metabolism

Article Title: Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

doi: 10.1016/j.molmet.2018.03.007

Figure Lengend Snippet: Figure 4: Bile acids stimulate GLP-1, NT, and PYY secretion by activation of basolateral, but not luminal, TGR5 receptors. Data are shown as means 1 SEM. All X-Y plot data with exception of C, D, F, I and J are from isolated perfused rat small intestine. A1,2: Effects of luminal and vascular administration of a poorly absorbable TGR5 agonist on GLP-1, NT and PYY secretion, n¼6. B1,2: Effects of luminal and vascular administration of GW4064 (a FXR agonist) on GLP-1, NT and PYY secretion, n¼6. C,D: Activation of TGR5 in response to different bile-acids in cells transfected with either the human TGR5 receptor (C) or rat TGR5 receptor (D). E: Co-localization of TGR5 and GLP-1/PYY in rat small intestine. F: Expression of TGR5 in isolated pancreatic a-, b- or d-cells or intestinal L-cells from the mouse, n¼3. G: Plasma GLP-1 (total) concentrations in response to a complex BA-mix (n¼6), match total conc. of UDCA (n¼8) or 0.9%NaCl (neg. control, n ¼ 3). H1,2: Effects of vascular TUDCA, TCDCA or TDCA on GLP-1, NT and PYY secretion, n¼6. I and J: Effects of a complex BA-mix on the secretion of GLP-1 and PYY from isolated perfused mouse small intestine. I: WT mice, J: TGR5 KO mice, n ¼ 6. In all perfusion experiments, bombesin (BBS) was administered in the end of the experiment and used as positive control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: 35,000 COS-7 cells per well were seeded in 96-well plates coated with poly-Dlysine and modified for either human or rat TGR5 expression using a transient calcium phosphate precipitation transfection procedure [21], using a pCMV6-XL5 or pCMV6-Entry vector, respectively (Cat. no. SC123312 and RN210451, OriGene, Technologies Inc., Rockville, MD).

Techniques: Activation Assay, Isolation, Transfection, Expressing, Clinical Proteomics, Control, Positive Control

Journal: Molecular metabolism

Article Title: Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

doi: 10.1016/j.molmet.2018.03.007

Figure Lengend Snippet: Figure 5: TGR5 activation or bile acids has no direct effects on glucagon or insulin secretion. Data are shown as means ? 1 SEM. All data are from isolated perfused rat pancreas. A. Glucagon (1,2) and insulin (3,4) secretion at low glucose (3.5 mmol/L) in response to the TGR5 agonist RO9272296 or a complex BA-mix. B: Glucagon (1,2) and insulin (3,4) secretion at high glucose (10 mmol/L) in response to the TGR5 agonist RO9272296 or a complex BA-mix. L-arginine (Arg) was included at the end of all experiment and used a positive control. n ¼ 6 for all experiments. *P < 0.05, **/##P < 0.01, ***/###P < 0.001, ****P < 0.0001. Stars indicate significance compared to baseline and hashes indicate significance between treatments.

Article Snippet: 35,000 COS-7 cells per well were seeded in 96-well plates coated with poly-Dlysine and modified for either human or rat TGR5 expression using a transient calcium phosphate precipitation transfection procedure [21], using a pCMV6-XL5 or pCMV6-Entry vector, respectively (Cat. no. SC123312 and RN210451, OriGene, Technologies Inc., Rockville, MD).

Techniques: Activation Assay, Isolation, Positive Control

Journal: Molecular metabolism

Article Title: Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

doi: 10.1016/j.molmet.2018.03.007

Figure Lengend Snippet: Figure 6: Proposed model of bile acid stimulated secretion of appetite and metabolism regulating hormones. (1) Food intake, and in particular fat consumption, stimulates the secretion of bile acids into the upper small intestine by CCK-mediated contraction of the gallbladder. (2) In addition to their well-known role in facilitating fat absorption (by micelle formation) bile acids activate TGR5 receptors which are located at the basolateral membranes of the enterocytes and therefore are activated secondary to bile acid (BA) absorption. Upon activation, the secretion of GIP, GLP-1, NT, and PYY is stimulated. Conjugated bile acids are absorbed through the secondary active transporter ileal-bile acid transporter (IBAT) which is predominantly expressed in the lower part of the small intestine, whereas unconjugated bile acids (which are more lipophilic) spontaneously cross the intestinal mucosal layer. (3) Eliminating bile acid absorption by BA-sequestrants (which cross bind both conjugated and unconjugated BAs into large unabsorbable complexes) or by direct IBAT inhibition (which attenuates the absorption of conjugated BAs) therefore eliminates BA-stimulated gut hormone secretion. (4) Collectively, the absorption mechanisms results in a very efficient BA absorption so about 95% of the secreted bile acids are returned to the liver through the enterohepatic circulation. (5) The majority of the returned bile acids are extracted by the liver (where they are reconjugated and rehydroxylated, allowing the same pool of bile acids to be secreted several times during the day), (6) Only 3e10% pass the liver and eventually ends up in the systemic circulation, (7) The pool is further diluted with a factor of about three since the hepatic return constitutes about 1/3 of the total venous return. (8) Therefore, only a small fraction of the secreted bile acid makes it to the systemic circulation.

Article Snippet: 35,000 COS-7 cells per well were seeded in 96-well plates coated with poly-Dlysine and modified for either human or rat TGR5 expression using a transient calcium phosphate precipitation transfection procedure [21], using a pCMV6-XL5 or pCMV6-Entry vector, respectively (Cat. no. SC123312 and RN210451, OriGene, Technologies Inc., Rockville, MD).

Techniques: Activation Assay, Inhibition

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: TRC210258, a novel TGR5 agonist, reduces glycemic and dyslipidemic cardiovascular risk in animal models of diabesity

doi: 10.2147/DMSO.S50209

Figure Lengend Snippet: Activation of TGR5 by TRC210258. ( A ) Effect of TRC210258 on TGR5-mediated cAMP formation. EC 50 value expressed in nM. ( B ) Effect of TRC210258 on CRE luciferase reporter. EC 50 value expressed in nM. ( C ) Effect of TRC210258 on GLP1 secretion from NCI-H716, a human enteroendocrine cell line. GLP1 levels expressed in pM. Each value is the average of two experiments, each performed in duplicate. * P <0.05, TRC210258 versus vehicle control. Abbreviations: DMSO, dimethyl sulfoxide; TRC210258, N-(4-chlorophenyl)-2-(4-fluorophenoxy)-N-methylimidazo [1, 2-a] pyrimidine-3-carboxamide; GLP, glucagon like peptide 1; CRE, cyclic AMP responsive element; pM, picomole; conc, concentration.

Article Snippet: Chinese Overy Hamster-K1 (CHO-K1) cells (American Type Culture Collection, Manassas, VA, USA, catalog number CCL-61) were transiently transfected with human TGR5 expression vector (Origene, Rockville, MD, USA).

Techniques: Activation Assay, Luciferase, Concentration Assay

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: TRC210258, a novel TGR5 agonist, reduces glycemic and dyslipidemic cardiovascular risk in animal models of diabesity

doi: 10.2147/DMSO.S50209

Figure Lengend Snippet: Schematic representation summarizing TRC210258-mediated TGR5 activation and improvement of glycemic and dyslipidemic cardiovascular risk. Abbreviations: GLP1, glucagon like peptide-1; D2, deiodinase 2; wt, weight; TG, triglyceride; CV, cardiovascular; TRC210258, N-(4-chlorophenyl)-2-(4-fluorophenoxy)-N-methylimidazo [1, 2-a] pyrimidine-3-carboxamide.

Article Snippet: Chinese Overy Hamster-K1 (CHO-K1) cells (American Type Culture Collection, Manassas, VA, USA, catalog number CCL-61) were transiently transfected with human TGR5 expression vector (Origene, Rockville, MD, USA).

Techniques: Activation Assay