Rev-erbalpha/REV-ERBalpha (mouse/human reverse erythroblastosis virus alpha). GSK2945 hydrochloride is a highly specific antagonist (inverse agonist) of the nuclear receptor Rev-erbalpha/REV-ERBalpha. Rev-erbalpha is a transcription factor and key component of the circadian clock, regulating the expression of clock genes (e.g., Bmal1) and metabolic genes (e.g., CYP7A1, SREBP1c, FAS). As a repressor, Rev-erbalpha binds to RORE (Rev-erb Response Element) sequences and recruits co-repressors (NCoR, HDAC3) to suppress gene transcription. GSK2945 antagonizes Rev-erbalpha activity by blocking its repressive function, thereby de-repressing target gene expression. By inhibiting Rev-erbalpha, GSK2945 increases the expression of CYP7A1, the rate-limiting enzyme in the bile acid synthesis pathway from cholesterol, leading to enhanced cholesterol metabolism and reduced plasma cholesterol levels. GSK2945 also upregulates LRH-1 (liver receptor homolog-1, NR5A2), another nuclear receptor involved in bile acid synthesis.

GSK2945 hydrochloride increases, in a dose-dependent manner (EC50 = 2.05 μM), the transcriptional activity of the Rev-erbα and Bmal1 (the target gene of REV-ERB) luciferase reporter genes. [1]. Cyp7a1/CYP7A1 levels in mouse and human primary hepatocytes are increased by treatment with GSK2945 hydrochloride (20 μM; 12 hours and 24 hours). Treatment with GSK2945 hydrochloride (20 μM) also raises the levels of Lrh-1/LRH-1 mRNA and protein, which is a recognized hepatic activator of Cyp7a1/CYP7A1 [1].

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In vitro, GSK2945 hydrochloride increases the expression of CYP7A1 and LRH-1 in hepatic cell lines. Treatment with GSK2945 hydrochloride (20 uM) raises the levels of Lrh-1/LRH-1 mRNA and protein in cultured hepatocytes. The compound also elevates CYP7A1 mRNA and protein levels, indicating enhanced bile acid synthetic capacity. In cell-based reporter assays using a Rev-erbalpha-responsive luciferase reporter plasmid, GSK2945 hydrochloride antagonizes Rev-erbalpha-mediated transcriptional repression with EC50 values of 21.5 uM (mouse Rev-erbalpha) and 20.8 uM (human REV-ERBalpha). These EC50 values reflect the concentration required to achieve 50% of the maximal antagonism (de-repression) effect. The compound is highly specific for Rev-erbalpha/REV-ERBalpha, with minimal off-target activity against other nuclear receptors (e.g., LXRalpha, PPARgamma, FXR, RORalpha) at relevant concentrations. GSK2945 hydrochloride is used as a tool to dissect the roles of Rev-erbalpha in metabolism, especially in hepatic lipid and cholesterol homeostasis.

GSK2945 hydrochloride (0–10 mg/kg; intraperitoneal; twice daily for 7 days; male C57BL/6 mice) treatment lowered wild-type plasma cholesterol in rats and elevated mouse liver cholesterol 7α-hydroxylase (Cyp7a1) levels [1].

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In vivo, GSK2945 hydrochloride (0-10 mg/kg; intraperitoneal; twice daily for 7 days; male C57BL/6 mice) lowers plasma cholesterol levels in wild-type mice. The compound (3-10 mg/kg) significantly reduces total plasma cholesterol compared to vehicle control. The cholesterol-lowering effect is mediated by the induction of CYP7A1, the rate-limiting enzyme for the conversion of cholesterol to bile acids. GSK2945 hydrochloride elevates mouse liver cholesterol 7alpha-hydroxylase (Cyp7a1) levels and increases Cyp7a1 enzymatic activity in hepatic microsomes. The compound also upregulates liver Lrh-1 expression. In hypercholesterolemic mouse models (e.g., high-fat diet-fed mice or ApoE-/- mice), GSK2945 hydrochloride treatment further reduces plasma cholesterol and triglyceride levels. The compound is generally well-tolerated at doses up to 10 mg/kg, with no significant weight loss or hepatotoxicity (based on ALT/AST levels). GSK2945 hydrochloride is also active in rat models, lowering plasma cholesterol. These data support the potential of Rev-erbalpha antagonism as a strategy for treating dyslipidemia and metabolic syndrome.

For a non-cellular receptor binding assay, surface plasmon resonance (SPR) or a fluorescence polarization (FP)-based competitive binding assay can be performed. For SPR: Recombinant human REV-ERBalpha ligand-binding domain (LBD) protein is immobilized onto a CM5 sensor chip via amine coupling (EDC/NHS chemistry). Varying concentrations of GSK2945 hydrochloride (0.1-1000 uM) are flowed over the immobilized receptor in running buffer (HBS-EP+, 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) at 25degC. Association (2-3 min) and dissociation (5-10 min) phases are recorded. The KD is calculated using a 1:1 Langmuir binding model. For FP competitive binding assay: A fluorescently labeled Rev-erbalpha ligand (e.g., a tracer) is incubated with REV-ERBalpha-LBD protein in black 384-well plates in binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 0.01% NP-40). Increasing concentrations of GSK2945 hydrochloride (0.1-1000 uM) are added, and after equilibration (60-120 min at room temperature), fluorescence polarization (excitation 485 nm, emission 520 nm) is measured. The IC50 is determined, and Ki is calculated. For a radioligand binding assay: 3H- or 125I-labeled Rev-erbalpha agonist is used as the tracer. REV-ERBalpha protein is immobilized on scintillation proximity assay (SPA) beads, and competition with unlabeled GSK2945 hydrochloride is measured.

RT-PCR[1]
Cell Types: Mouse(male, CD1) and human(male, Caucasian) primary hepatocytes
Tested Concentrations: 20 μM
Incubation Duration: 12 hrs (hours) and 24 hrs (hours)
Experimental Results: Led to significant increases in mRNA and protein(at 24- h) expression of Cyp7a1. mRNA and protein(at 24-h) levels of CYP7A1 were increased in human primary hepatocyte. Lrh-1/LRH-1 was upregulated.

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For cell-based assays, HepG2 human hepatoma cells or primary mouse hepatocytes are used. Cells are seeded in 12- or 24-well plates at 2-4 × 10^5 cells/well in DMEM with 10% FBS, 100 U/mL penicillin, 100 microg/mL streptomycin, and cultured for 24-48 hours at 37degC, 5% CO2. For CYP7A1 and LRH-1 expression studies, cells are treated with GSK2945 hydrochloride (0.1-100 uM) in serum-free medium for 24-48 hours. For qPCR analysis: Total RNA is extracted using TRIzol reagent, reverse-transcribed to cDNA using a reverse transcriptase kit, and qPCR is performed with SYBR Green or TaqMan probes (CYP7A1, LRH-1, beta-actin or GAPDH as internal control). For protein analysis: Cells are lysed in RIPA buffer with protease and phosphatase inhibitors. Lysates (20-50 ug protein) are separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-CYP7A1, anti-LRH-1, or anti-beta-actin antibodies. Bands are detected by chemiluminescence and quantified by densitometry. For Rev-erbalpha reporter gene assays: HEK293T cells are co-transfected with a Rev-erbalpha expression plasmid, a (RORE)-luciferase reporter plasmid (containing RORE elements), and a Renilla luciferase control plasmid for normalization. After 24 hours of transfection, cells are treated with GSK2945 hydrochloride (0.1-1000 uM) for 24-48 hours. Luciferase activity is measured using dual-luciferase assay kit (Promega). GSK2945 is expected to increase luciferase signal (de-repression) in a concentration-dependent manner. The EC50 is calculated from the dose-response curve. Each condition should be tested in triplicate wells, and at least three independent experiments performed. For cell viability assays, cells are treated with GSK2945 hydrochloride (1-1000 uM) for 48-72 hours, and MTT or CellTiter-Glo is used to assess cytotoxicity. GSK2945 is generally not toxic at concentrations up to 100 uM.

Animal/Disease Models: Male C57BL/6 mice (8-10 weeks of age)[1]
Doses: 0 mg/kg or 10 mg/kg
Route of Administration: intraperitoneal (ip)injection; twice every day; for 7 days
Experimental Results: Increased hepatic mouse cholesterol 7α- hydroxylase (Cyp7a1) level and lowered plasma cholesterol in wild-type mice.

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For in vivo studies, male C57BL/6J mice (8-10 weeks old, 20-25 g) are used. GSK2945 hydrochloride is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG400, 5% Tween 80 in saline; or 10% DMSO, 90% corn oil). The compound is administered intraperitoneally (i.p.) at doses of 1, 3, 6, and 10 mg/kg (volume 5-10 mL/kg), twice daily (every 12 hours) for 7 days. Vehicle control group receives the same volume of vehicle. For hypercholesterolemic studies, mice may be fed a high-fat diet (HFD) containing 60% kcal from fat for 4-8 weeks before and during treatment, or ApoE-/- mice on normal chow are used. Body weights are measured daily. On day 7, 12 hours after the last dose, mice are fasted for 4-6 hours and then anesthetized with isoflurane. Blood is collected by cardiac puncture into EDTA-coated tubes. Plasma is separated by centrifugation (2,000 × g, 10 min, 4degC) and stored at -80degC for lipid analysis. Total cholesterol (TC), triglycerides (TG), HDL-cholesterol, and LDL-cholesterol are measured using enzymatic colorimetric kits (e.g., Wako, Stanbio). Livers are harvested, weighed, and flash-frozen in liquid nitrogen. For CYP7A1 activity assay, liver microsomes are prepared by differential centrifugation. Microsomal protein (100 ug) is incubated with [14C]-cholesterol or a colorimetric substrate, and the production of 7alpha-hydroxycholesterol (for CYP7A1) is measured by HPLC, TLC, or colorimetric plate reader. For gene expression analysis, liver tissue is homogenized, and RNA and protein are extracted for qPCR (Cyp7a1, Lrh-1, Srebp1c, Fas, Bmal1) and Western blot. Plasma cholesterol-lowering efficacy is typically observed at 3-10 mg/kg, with maximum effect at 10 mg/kg. The compound is well-tolerated with no significant changes in behavior, weight, or gross organ pathology. For long-term studies (14-28 days), monitoring of liver function tests (ALT, AST) and kidney function (creatinine, BUN) is recommended. All animal procedures must be approved by the IACUC.

No detailed pharmacokinetic (PK) data are available for GSK2945 hydrochloride in the literature. As a small-molecule antagonist of Rev-erbalpha, GSK2945 is expected to have moderate oral bioavailability and be metabolized by liver enzymes. In rodent studies, the compound is typically administered intraperitoneally at 1-10 mg/kg, with efficacy observed after 7 days of twice-daily dosing. The plasma half-life (t1/2) is not reported, but the need for twice-daily dosing suggests that the half-life may be relatively short (2-6 hours) in mice. The compound likely undergoes hepatic metabolism, possibly by cytochrome P450 enzymes (CYP3A4, CYP2C9). GSK2945 hydrochloride is soluble in DMSO and can be formulated in aqueous vehicles for injection. For PK studies, a standard protocol would involve administration of GSK2945 (1-10 mg/kg, i.p. or i.v.) to male C57BL/6 mice (n=3-5 per time point). Blood samples (50-100 uL) are collected via tail vein at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dose. Plasma is separated, and GSK2945 concentrations are quantified by LC-MS/MS using an internal standard (e.g., deuterated GSK2945). PK parameters (Cmax, Tmax, AUC, t1/2, CL, Vd, bioavailability) are calculated using non-compartmental analysis. The hydrochloride salt form (HCl) enhances water solubility compared to the free base, improving bioavailability. GSK2945 is not an approved drug; detailed PK are not typically published for research compounds.

No specific toxicity data are available for GSK2945 hydrochloride from standardized toxicology studies. In published in vivo studies, GSK2945 hydrochloride administered intraperitoneally at doses up to 10 mg/kg twice daily for 7 days in mice is generally well-tolerated, with no reported mortality, significant body weight loss, or adverse behavioral changes. No specific organ toxicity (liver, kidney, spleen) has been reported based on histological examination. However, long-term toxicity studies (≥28 days) have not been conducted. The compound's mechanism of action as a Rev-erbalpha antagonist is not expected to cause acute toxicity, but chronic disruption of the circadian clock and metabolic pathways could theoretically lead to metabolic disturbances, though this is not documented for GSK2945. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been performed. GSK2945 hydrochloride is a research chemical and is not intended for human use. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The hydrochloride salt is not associated with additional toxicity beyond the parent compound.

[1]. REV-ERBα Regulates CYP7A1 Through Repression of Liver Receptor Homolog-1. Drug Metab Dispos. 2018 Mar;46(3):248-258.

REV-ERBalpha (also known as NR1D1) is a nuclear receptor and transcription factor that functions as a key repressor of the circadian clock. It is a receptor for heme, which acts as a ligand, and is involved in regulating the expression of clock genes (such as Bmal1), as well as metabolic genes controlling lipid and bile acid metabolism, adipogenesis, and inflammation. Rev-erbalpha is a therapeutic target for metabolic disorders, including dyslipidemia, atherosclerosis, type 2 diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). GSK2945 (developed by GlaxoSmithKline) is a tertiary amine Rev-erbalpha antagonist (sometimes referred to as an inverse agonist). It is important to note that there is potential confusion in the literature: some sources describe GSK2945 as a Rev-erbalpha agonist, while others describe it as an antagonist/inverse agonist. GSK2945 hydrochloride is a chemical probe used to investigate the function of Rev-erbalpha in circadian rhythm and metabolism. The hydrochloride salt is used to improve solubility and stability. As of 2026, no Rev-erbalpha antagonist has been approved for clinical use, though several are in preclinical development. GSK2945 hydrochloride is for research use only and is not approved for human therapy.

C20H19CL3N2O2S

457.80

GSK2945;1438071-12-5

Light brown to brown solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO :~20.83 mg/mL (~45.50 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.54 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.54 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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