REV-ERBα(IC50 = 790 nM); REV-ERBβ(IC50 = 560 nM)

In HEK293 cells expressing a chimeric Gal4 DNA Binding Domain (DBD) - REV-ERB ligand binding domain (LBD) α or β and a Gal4-responsive luciferase reporter (REV-ERBα IC50=790 nM, REV-ERBβ IC50=560 nM), SR9011 dose-dependently boosts the REV-ERB-dependent repressor activity. In a cotransfection test with full-length REV-ERBα and a luciferase reporter powered by the Bmal1 promoter, SR9011 (IC50=620 nM) effectively and potently inhibits transcription. SR9011 inhibits the expression of BMAL1 mRNA in HepG2 cells in a manner that is dependent on REV-ERBα/β[1]. In addition, SR9011 inhibits the growth of breast cancer cell lines irrespective of their ER or HER2 status. It seems that SR9011 stops the breast cancer cells' cell cycle before they enter the M phase. Since cyclin A (CCNA2) is a direct target gene of REV-ERB, the cell cycle arrest may be mediated by SR9011's reduction of this cyclin's expression. Increases in G0/G1 phase cells and decreases in S and G2/M phase cells following SR9011 treatment imply that REV-ERB activation may be causing a reduced transition from G1 to S phase and/or from S to G2/M phase[2].

Since SR9011 exhibits a respectable plasma exposure, the expression of genes sensitive to REV-ERB is investigated in the livers of mice given varying dosages of SR9011 for a period of six days. As a REV-ERB target gene, the plasminogen activator inhibitor type 1 gene (Serpine1) exhibits dose-dependent expression reduction in response to SR9011. It has also been demonstrated that the REV-ERB-responsive genes cholesterol 7α-hydroxylase (Cyp7a1) and sterol response element binding protein (Srepf1) are inhibited dose-dependently with increasing concentrations of SR9011. Following 12 days in D:D circumstances, mice get a single injection of either SR9011 or a vehicle at CT6, which represents the maximal expression of Rev-erbα. There is no change in circadian locomotor activity after vehicle injection. Nevertheless, during the subject dark phase, a single dose of SR9011 causes a decrease of locomotor activity. The resumption of normal activity to the following circadian cycle is consistent with the medications clearing in less than twenty-four hours. The efficacy (ED50=56 mg/kg) of the SR9011-dependent reduction in wheel running behavior in mice kept in permanent darkness is comparable to that of the SR9011-mediated in vivo inhibition of the REV-ERB responsive gene Srebf1 (ED50=67 mg/kg)[1].

In this study, researchers developed two REV-ERBα/β agonists with sufficient plasma/brain exposure to allow evaluation of their effects in vivo. Both SR9009 and SR9009 (Fig. 1a, Supplementary Fig. 1) dose-dependently increased the REV-ERB-dependent repressor activity assessed in HEK293 cells expressing a chimeric Gal4 DNA Binding Domain (DBD) - REV-ERB ligand binding domain (LBD) α or β and a Gal4-responsive luciferase reporter (Fig. 1b) (SR9009: REV-ERBα IC50=670 nM, REV-ERBβ IC50=800 nM; SR9011: REV-ERBα IC50=790 nM, REV-ERBβ IC50=560 nM). The REV-ERB ligand GSK4112 (Supplementary Fig. 2), which exhibits no plasma exposure displays limited activity (Fig. 1b). Both SR9011 and SR9009 potently and efficaciously suppressed transcription in a cotransfection assay using full-length REV-ERBα along with a luciferase reporter driven by the Bmal1 promoter (Fig. 1c) (SR9009 IC50=710 nM; SR9011 IC50=620 nM). SR9011 and SR9009 suppressed the expression of BMAL1 mRNA in HepG2 cells in a REV-ERBα/β-dependent manner (Supplementary Fig. 3). Consistent with both compounds functioning as direct agonists of REV-ERB, we noted that the compounds increased the recruitment of the CoRNR box peptide fragment of NCoR using a biochemical assay (Supplementary Fig. 4). Direct binding of the SR9009 to REV-ERBα was also confirmed using circular dichrosim analysis (Supplementary Fig. 5) (Kd=800 nM). Neither compound exhibited activity at other nuclear receptors12,13 (Supplementary Fig. 6)[1].

Luciferase assay[2]
HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. Cells were plated in 96-well plates at a density of 15 X 103 cells/well 24 h before transfection. Eight hour post-transfection, the cells were treated with SR9011 or DMSO. Twenty-four hours post-treatment, the luciferase activity was measured using the Dual-GloTM luciferase assay system. The values indicated represent the means ± S.E. from three independently transfected wells. The experiments were repeated three times, and representative experiments are shown.

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Cell culture, compound treatment, overexpression and knockdown[2]
MCF10A, MDA-MB-231, MCF-7, MDA-MB-361, SKBR3, BT474 are from ATCC. Cells were plated in 6-well plates one day before treatment. The cells were treated with SR9011 or DMSO for 24 hr and harvested for RNA isolation or western blot. For over expression, the cells were infected with adenovirus for 24 hours and then switched to regular growth media. Twenty-four hours later, the cells were harvested to isolate total RNA. For knockdown assay, the control siRNA, human REV-ERBβ siRNA were transfected with LipofectamineTM RNAiMAX by using reverse transfection following the manufacture’s instruction. After 24 hours, cells were harvested to perform quantitative PCR assay.

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MTT assay[2]
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays were performed according to the manufacturer’s manual. Briefly, 3 × 10~3 to 5 × 10~3 cells per well were plated in 96-well plates. Twenty-four hours later, cells were treated with SR9011 or DMSO. Seventy-two hours after treatment, the cells were labeled with 1.2 mM MTT and incubated for 4 hours. DMSO was then added and readings were taken on a plate reader at 540 nm.

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HEK293 cells are grown in 96-well plates (1×106/well) and are transiently transfected using Lipofectamine. Cells are transfected with a total of 200 ng of DNA per well consisting of the pGL4 mIL-17 firefly luciferase reporter construct, the pGL4 mIL-17 + CNS-5 firefly luciferase reporter construct, or the pGL4 mIL-17 2kB RORE mutant (100 ng/well) , an actin promoter Renilla reniformis luciferase reporter (50 ng/well), and either control vector alone or the test DNA (full-length RORα or full-length RORγ at 50 ng/well). All 48 human nuclear receptors are represented in the specificity assay and SR9009 is tested at a concentration of 20 μM. The format of the assay is a cotransfection assay with Gal4 DNA binding domain-nuclear receptor fusions in HEK293 cells[1].

For circadian gene expression experiments male C57BL6 mice (8–10 weeks of age) were either maintained on a L:D (12h:12h) cycle or on constant darkness. At circadian time (CT) 0 animals were administered a single dose of 100 mg/kg SR9009 or SR9011 (i.p.) and groups of animals (n=6) were sacrificed at CT0, CT6, CT12 and CT18. Gene expression was determined by real time QPCR.[1]

[1]. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature. 2012 Mar 29;485(7396):62-8.

[2]. Anti-proliferative actions of a synthetic REV-ERBα/β agonist in breast cancer cells. Biochem Pharmacol. 2015 Aug 15;96(4):315-22.

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-β have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.[1]

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REV-ERBα and REV-ERBβ are nuclear receptors that are ligand-dependent transcriptional repressors. Heme is the natural ligand for these receptors, but several synthetic agonists and antagonists have been designed recently. The gene that encodes REV-ERBα, NR1D1, is closely associated with ERBB2, the gene that encodes the HER2 oncogene, which is amplified in HER2(+) breast cancers. We examined the effect of a synthetic REV-ERB agonist, SR9011, on a range of estrogen receptor positive (ER(+)), ER(-), HER2(+), HER2(-) and triple negative breast cancer cell lines. We found that SR9011 suppressed proliferation of the breast cancer cell lines regardless of their ER or HER2 status. SR9011 had no effect on MCF10A cell proliferation. SR9011 appears to pause the cell cycle of the breast cancer cells prior to M phase. Cyclin A (CCNA2) was identified as a direct target gene of REV-ERB suggesting that suppression of expression of this cyclin by SR9011 may mediate the cell cycle arrest. These data indicate that synthetic REV-ERB ligands may hold utility in treatment of diseases associated with uncontrolled cellular proliferation such as cancer.[2]

C23H32CL2N4O3S

515.50

514.157

2070014-94-5

SR9011;1379686-29-9

119081411

Light yellow to brown solid powder

2

5

10

33

600

0

ADTZBBQVEUEODS-UHFFFAOYSA-N

InChI=1S/C23H31ClN4O3S.ClH/c1-2-3-4-12-25-23(29)27-13-11-19(16-27)15-26(14-18-5-7-20(24)8-6-18)17-21-9-10-22(32-21)28(30)31;/h5-10,19H,2-4,11-17H2,1H3,(H,25,29);1H

3-[[(4-chlorophenyl)methyl-[(5-nitrothiophen-2-yl)methyl]amino]methyl]-N-pentylpyrrolidine-1-carboxamide;hydrochloride

R9011 hydrochloride; SR9011 (hydrochloride); 2070014-94-5; 3-[[(4-chlorophenyl)methyl-[(5-nitrothiophen-2-yl)methyl]amino]methyl]-N-pentylpyrrolidine-1-carboxamide;hydrochloride;

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, 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: ≥ 32 mg/mL (62.08 mM)
H2O: < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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