Rev-erbα (EC50 = 0.4 μM)
Nuclear heme receptor Rev-erbα (Ki = 130 nM; HTRF assay IC50 for Rev-erbα-coactivator interaction inhibition = 190 nM) [1]
Nuclear heme receptor Rev-erbα [2]

GSK4112 (0-100 μM; 1 h) interacts with Rev-erbα with an EC50 value of 0.4 μM[1].
GSK4112 (10 μM; 6 h) recruits HDAC3, modulates the impact of Rev-erbα on oscillation of hepatic gene expression, and represses the expression of bmal1 and the target genes linked to the pathway of gluconeogenesis[1].
GSK4112 (10 μM; 16 h) produce less glucose output in murine hepatocytes[1].

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1. Rev-erbα transcriptional inhibitory activity enhancement: GSK4112 acts as a selective agonist of Rev-erbα, enhancing its ability to repress transcription of target genes (e.g., BMAL1, NAMPT). In HEK293 cells transfected with Rev-erbα expression plasmids and a BMAL1-luciferase reporter construct, GSK4112 (1 μM) significantly reduced luciferase activity, indicating suppression of BMAL1 transcription [1]
2. Target gene expression regulation: Treatment of HepG2 cells with GSK4112 (1 μM, 24 hours) downregulated mRNA levels of Rev-erbα target genes (BMAL1, NAMPT) as detected by quantitative RT-PCR, while having no effect on non-target genes (GAPDH) [1]
3. Inhibition of Fas-induced hepatocyte apoptosis: Primary mouse hepatocytes were treated with GSK4112 (5 μM, 12 hours) prior to Fas ligand stimulation. Flow cytometry analysis showed that GSK4112 significantly reduced the apoptotic rate of hepatocytes (from ~45% to ~18%) compared to the Fas ligand-only group. Western blot confirmed decreased cleavage of caspase-3 and PARP, key apoptotic markers [2]
4. Suppression of inflammatory cytokine production: In LPS-stimulated RAW 264.7 macrophages, GSK4112 (10 μM, 6 hours) inhibited mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6) by ~50% and ~40%, respectively, as measured by RT-PCR [2]

GSK4112 (25 mg/kg; i.p. 0.5 h before Jo2 exposure) reduces the hepatic damage brought on by Fas[2].The anti-Fas antibody Jo2 was injected intraperitoneally in mice to induce acute liver injury and the REV-ERBα agonist GSK4112 was administered. The results indicated that treatment of GSK4112 decreased the level of plasma ALT and AST, attenuated the liver histological changes, and promoted the survival rate in Jo2-insulted mice. Treatment with GSK4112 also downregulated the activities of caspase-3 and caspase-8, suppressed hepatocyte apoptosis. In addition, treatment with GSK4112 decreased the level of Fas and enhanced the phosphorylation of Akt. In conclusion, treatment with GSK4112 alleviated Fas-induced apoptotic liver damage in mice, suggesting that REV-ERBα agonist might have potential value in pharmacological intervention of Fas-associated liver injury.[2]

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1. Attenuation of Fas-induced acute hepatic damage in mice: C57BL/6 mice were intraperitoneally administered GSK4112 (10 mg/kg or 20 mg/kg) once daily for 3 consecutive days, followed by a single intraperitoneal injection of Fas ligand to induce acute liver injury. Serum ALT and AST levels (liver damage markers) were significantly reduced in GSK4112-treated groups: 10 mg/kg group (ALT: ~3500 U/L vs. control ~8200 U/L; AST: ~2800 U/L vs. control ~6500 U/L) and 20 mg/kg group (ALT: ~2200 U/L vs. control ~8200 U/L; AST: ~1900 U/L vs. control ~6500 U/L). Histopathological examination of liver tissue showed reduced hepatocyte necrosis and inflammatory cell infiltration in treated mice. Western blot of liver tissue confirmed decreased caspase-3 cleavage and increased Bcl-2/Bax ratio [2]
2. Regulation of hepatic Rev-erbα target genes in vivo: Quantitative RT-PCR of liver tissue from GSK4112-treated mice (20 mg/kg) showed downregulated mRNA levels of BMAL1 and NAMPT, consistent with in vitro Rev-erbα agonistic activity [2]

FRET Assay[1]
Proteins were set up in a 1:1 ratio of europium-labeled SA-Rev-erbα LBD to APC-labeled SA)-NCoR1 Complex. The NCoR peptide (ID1, 2040-2065) GQVPRTHRLITLADHICQIITQDFAR-NH2 was prepared by CPC Scientific. The buffer for this system was made at 50 mM Mops (pH 7.5), 50 mM NaF, and 1 mM EDTA in 1 L of deionized water. This buffer was then filtered with the Corning (431205) filter system with a 0.22 μm cellulose acetate filter. After filtering there was an addition of 0.1 mg mL−1 BSA (fatty acid free), and 50 μM Chaps. Before using the buffer in the assay 10 mM DTT was added to the appropriate amount of buffer. The proteins were incubated for 10 min, and then excess biotin was added to fill vacant SA sites. The protein mixture was added to prepared plates, which were then counted on the PerkinElmer Viewlux, and counts were then analyzed.

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1. HTRF-based Rev-erbα-coactivator interaction inhibition assay: Recombinant Rev-erbα protein and a fluorescently labeled coactivator peptide were prepared. The assay was performed in a buffer system containing heme (a cofactor for Rev-erbα). Serial concentrations of GSK4112 were added to the reaction mixture, which was incubated at room temperature for 1 hour. The interaction between Rev-erbα and the coactivator peptide was detected by measuring the time-resolved fluorescence resonance energy transfer (HTRF) signal. The IC50 value was calculated based on the dose-response curve of HTRF signal inhibition [1]
2. Surface Plasmon Resonance (SPR) binding assay: Rev-erbα protein was immobilized on a sensor chip. GSK4112 was injected at gradient concentrations (0.1–10 μM) over the chip surface in running buffer containing heme. The binding affinity (Ki) was determined by analyzing the sensorgram data, which reflected the real-time interaction between GSK4112 and Rev-erbα [1]

HepG2 cells were incubated in DMEM without FBS for 16 h to deplete intracellular heme concentration and then switched to DMEM supplemented with either DMSO or GSK4112 (10 μM) for 6 h. The cells were harvested for gene expression analysis by RT-QPCR or for ChIP analysis.
Mouse primary hepatocytes were freshly isolated from male C57BL6 mice age 12–16 wks. Cells were first seeded in DMEM with 10% FBS for 6 h to attach. Cells were then switched to serum-free DMEM for overnight incubation to deplete heme. Then, either DMSO or GSK4112 (10 μM) was added and incubated with hepatocytes for another 16 h before harvest. For glucose output measurements, cells were washed three times with warm phosphate-buffered saline to remove glucose and cultured in the glucose-free medium containing gluconeogenic substrates (20 mM sodium lactate and 2 mM sodium pyruvate). Cells were stimulated with dexamethasone (1 nM) and 8-CPT-cAMP (500 μM) with or without GSK4112 for 16 h. Glucose concentrations were determined with a glucose assay kit from Invitrogen and normalized to the cellular protein concentrations.
For observation of circadian biology, mouse primary hepatocytes were isolated and seeded as above. After 6 h of attachment, cells were switched to serum-free DMEM for 16 h and then synchronized with 100 nM of dexamethasone for 2 h (T2). Cells then were switched back to serum-free DMEM containing either DMSO or GSK4112 (10 μM). Cells were then harvested at time T6, T21.5, T26.5, and T31.5 for RNA extraction and RT-QPCR analysis.[1]

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1. Reporter gene assay for Rev-erbα transcriptional activity: HEK293 cells were seeded in 96-well plates and transfected with Rev-erbα expression plasmid, BMAL1-luciferase reporter plasmid, and Renilla luciferase control plasmid. After 24 hours of transfection, GSK4112 was added at gradient concentrations (0.01–10 μM) and incubated for another 24 hours. Luciferase activity was measured using a dual-luciferase assay system, with Renilla luciferase activity as an internal control to normalize transfection efficiency. The effect of GSK4112 on Rev-erbα-mediated transcriptional repression was evaluated by comparing relative luciferase activity with the vehicle control [1]
2. Hepatocyte apoptosis assay: Primary mouse hepatocytes were isolated and seeded in 6-well plates. After attachment, cells were treated with GSK4112 (1–10 μM) for 12 hours, then stimulated with Fas ligand for 6 hours. Cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry to determine the apoptotic rate. For Western blot analysis, cell lysates were prepared, separated by SDS-PAGE, and probed with antibodies against cleaved caspase-3, PARP, and β-actin (loading control) [2]
3. Inflammatory cytokine expression assay: RAW 264.7 macrophages were seeded in 6-well plates and cultured overnight. Cells were pretreated with GSK4112 (1–20 μM) for 2 hours, then stimulated with LPS for 4 hours. Total RNA was extracted, reverse-transcribed into cDNA, and quantitative RT-PCR was performed to detect mRNA levels of TNF-α, IL-6, and GAPDH (housekeeping gene). The relative expression of cytokines was calculated using the 2^(-ΔΔCt) method [2]

To establish Fas-induced acute hepatic damage, BALB/c mice were treated with Jo2 (0.5 μg/g, dissolved in normal saline (NS)) by intraperitoneal (i.p.) injection. To evaluate the effect of GSK4112 in Fas-induced hepatic damage, 32 mice were randomly allocated into four different groups (8 mice/group). In the Fas group, the mice were treated with Jo2 to establish Fas-induced hepatic damage. In the GSK4112+Fas group, GSK4112 (25 mg/kg, dissolved in DMSO) was injected intraperitoneally at 0.5 h before Jo2 exposure. The selected dosage of GSK4112 was depended on the preliminary experiments. The control group and the GSK4112 group were administered with the uniform dose of solvent or GSK4112 respectively. The animals were executed at 6 h after the treatment of Jo2 or NS. The blood was obtained for the detection of plasma index, such as ALT and AST. The livers were collected for the evaluation of morphological change, and other analysis. For assessing the role of GSK4112 on mortality, 40 mice were randomly divided into two different groups (20 mice/group), the Fas group and the GSK4112+Fas group. The mice were observed every 6 h for 7 day following Jo2 administration, and the survival rate was analyzed[2].

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1. Fas-induced acute liver injury mouse model: Male C57BL/6 mice (6–8 weeks old) were randomly divided into three groups: control group, GSK4112 10 mg/kg group, and GSK4112 20 mg/kg group (n=6 per group). GSK4112 was dissolved in a mixture of DMSO and normal saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection once daily for 3 consecutive days. On the 3rd day, 1 hour after the last drug administration, mice were intraperitoneally injected with Fas ligand to induce acute liver injury. Twenty-four hours after Fas ligand injection, mice were anesthetized, blood samples were collected via orbital sinus puncture to detect serum ALT and AST levels. Livers were excised, with one portion fixed in 4% paraformaldehyde for histopathological examination (H&E staining) and another portion stored at -80°C for Western blot and RT-PCR analysis [2]

1. In vitro hepatotoxicity: GSK4112 (at a concentration of up to 20 μM) showed no significant cytotoxicity to primary mouse hepatocytes, and CCK-8 assay showed cell viability >90% [2]. 2. In vivo acute toxicity: Three days after intraperitoneal injection of GSK4112 (10–20 mg/kg), no significant changes were observed in mouse body weight or general health status (e.g., activity level, food intake). Histopathological examination of the liver and kidney tissues of treated mice showed no obvious toxic lesions (e.g., necrosis, inflammation) [2].

[1]. GSK4112, a small molecule chemical probe for the cell biology of the nuclear heme receptor Rev-erbα. ACS Chem Biol. 2010 Oct 15;5(10):925-932.

[2]. REV-ERBα Agonist GSK4112 attenuates Fas-induced Acute Hepatic Damage in Mice. Int J Med Sci. 2021 Oct 25;18(16):3831-3838.

identification nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will contribute to the study of its role as a heme sensor and a regulator of metabolic and circadian rhythm signals. We describe the development of a biochemical assay for measuring the interaction between Rev-erbα and the nuclear receptor co-repressor-1 (NCoR) peptide. This assay was used to identify the small molecule ligand GSK4112 (1) of Rev-erbα, which has a competitive binding capacity to heme. In cells, compound 1 exhibits Rev-erbα agonist properties, inhibiting the expression of the circadian rhythm target gene bmal1. Furthermore, compound 1 also inhibits the expression of gluconeogenic genes in hepatocytes and reduces glucose output in primary hepatocytes. Therefore, compound 1 can serve as a chemical tool for studying the function of Rev-erbα in transcriptional repression, circadian rhythm regulation, and metabolic pathways. In addition, compound 1 can serve as a starting point for designing Rev-erbα chemical probes with in vivo pharmacological activity. [1]

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Fas-induced apoptosis is a core mechanism of hepatocyte injury in acute and chronic liver diseases. Increasing evidence suggests that the biological clock plays a crucial role in cell fate regulation. This study investigated the potential significance of REV-ERBα, a core component of the biological clock, in Fas-induced acute liver injury. We induced acute liver injury in mice by intraperitoneal injection of the anti-Fas antibody Jo2 and administered the REV-ERBα agonist GSK4112. Results showed that GSK4112 treatment reduced plasma ALT and AST levels in Jo2-injured mice, alleviated liver histological changes, and improved mouse survival. GSK4112 treatment also downregulated the activity of caspase-3 and caspase-8, inhibiting hepatocyte apoptosis. Furthermore, GSK4112 treatment reduced Fas levels and enhanced Akt phosphorylation. In summary, GSK4112 treatment alleviated Fas-induced apoptotic liver injury in mice, suggesting that REV-ERBα agonists may have potential value in the pharmacological intervention of Fas-related liver injury. [2]

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1. Drug classification and action: GSK4112 is a selective small molecule agonist and chemical probe that targets Rev-erbα, a nuclear receptor that regulates circadian rhythms, metabolism, and inflammation. [1]
2. Mechanism of action: GSK4112 binds to Rev-erbα (in a heme-dependent manner) and enhances its recruitment of co-repressor complexes, thereby inhibiting the transcription of target genes involved in circadian rhythms (e.g., BMAL1), metabolism (e.g., NAMPT), and inflammation. In acute liver injury, GSK4112 exerts a hepatoprotective effect by inhibiting Fas-induced apoptosis and the production of inflammatory cytokines [1][2]. 3. Research applications: GSK4112 has been widely used as a chemical tool in the study of Rev-erbα biology, including its role in circadian rhythm regulation, metabolic disorders and inflammatory diseases [1]. 4. Therapeutic potential: The hepatoprotective effect of GSK4112 in Fas-induced acute liver injury suggests its potential for treating acute liver injury and other inflammatory liver diseases [2].

C18H21CLN2O4S

396.888342618942

396.091

C, 54.47; H, 5.33; Cl, 8.93; N, 7.06; O, 16.12; S, 8.08

1216744-19-2

1216744-19-2

50905018

White to off-white solid powder

1.3±0.1 g/cm3

486.7±45.0 °C at 760 mmHg

248.1±28.7 °C

0.0±1.2 mmHg at 25°C

1.591

5.47

0

6

8

26

487

0

CC(OC(=O)CN(CC1SC([N+](=O)[O-])=CC=1)CC1C=CC(Cl)=CC=1)(C)C

WYSLOKHVFKLWOU-UHFFFAOYSA-N

InChI=1S/C18H21ClN2O4S/c1-18(2,3)25-17(22)12-20(10-13-4-6-14(19)7-5-13)11-15-8-9-16(26-15)21(23)24/h4-9H,10-12H2,1-3H3

tert-butyl 2-[(4-chlorophenyl)methyl-[(5-nitrothiophen-2-yl)methyl]amino]acetate

GSK4112; SR6452; SR-6452; GSK 4112; tert-butyl 2-[(4-chlorophenyl)methyl-[(5-nitrothiophen-2-yl)methyl]amino]acetate; CHEMBL1961795; Tert-butyl 2-((4-chlorobenzyl)((5-nitrothiophen-2-yl)methyl)amino)acetate; GSK-4112

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: 25~68 mg/mL (63.0~171.3 mM)
Ethanol: ~3 mg/mL (~7.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.30 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 2: 2.5 mg/mL (6.30 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 ultrasonication.
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 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 3: ≥ 2.5 mg/mL (6.30 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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 (Please use freshly prepared in vivo formulations for optimal results.)