Rev-erbα (EC50 = 0.4 μM)

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].

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]

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.

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]

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].

[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.

The identification of nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will enable studies of its role as a heme sensor and regulator of metabolic and circadian signaling. We describe the development of a biochemical assay measuring the interaction between Rev-erbα and a peptide from the nuclear receptor corepressor-1 (NCoR). The assay was utilized to identify a small molecule ligand for Rev-erbα, GSK4112 (1), that was competitive with heme. In cells, 1 profiled as a Rev-erbα agonist in cells to inhibit expression of the circadian target gene bmal1. In addition, 1 repressed the expression of gluconeogenic genes in liver cells and reduced glucose output in primary hepatocytes. Therefore, 1 is useful as a chemical tool to probe the function of Rev-erbα in transcriptional repression, regulation of circadian biology, and metabolic pathways. Additionally, 1 may serve as a starting point for design of Rev-erbα chemical probes with in vivo pharmacological activity.[1]

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Fas-induced apoptosis is a central mechanism of hepatocyte damage during acute and chronic hepatic disorders. Increasing evidence suggests that circadian clock plays critical roles in the regulation of cell fates. In the present study, the potential significance of REV-ERBα, a core ingredient of circadian clock, in Fas-induced acute liver injury has been investigated. 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]

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.)