REV-ERBα(IC50 = 790 nM); REV-ERBβ(IC50 = 560 nM)
In HEK293 cells expressing chimeric Gal4 DNA-binding domain (DBD)-REV-ERB ligand-binding domain (LBD) α or β and a Gal4-responsive luciferase reporter gene (REV-), SR9011 dose-dependently boosts REV-ERB-dependent repressor activity. REV-ERBβ IC50 = 560 nM, ERBα IC50 = 790 nM. SR9011 potently suppressed transcription (SR9011 IC50=620 nM) in a co-transfection test using full-length REV-ERBα and a luciferase reporter driven by the Bmal1 promoter. SR9011 suppresses the expression of BMAL1 mRNA in HepG2 cells in a manner that is dependent on REV-ERBα/β [1]. In addition, SR9011 prevents the growth of breast cancer cell lines irrespective of their ER or HER2 status. It seems that SR9011 stops breast cancer cells' cell cycle before they reach the M phase. Since cyclin A (CCNA2) was found to be a direct target gene of REV-ERB, cell cycle arrest may be mediated by SR9011's suppression of this cyclin's expression. G0/G1 phase cells increased and S and G2/M phase cells decreased after treatment with SR9011, indicating that REV-ERB activation may reduce the transition from G1 to S phase and/or from S phase to G2/M phase [2].

In HEK293 cells expressing chimeric Gal4 DNA-binding domain (DBD)-REV-ERB ligand-binding domain (LBD) α or β and a Gal4-responsive luciferase reporter gene (REV-), SR9011 dose-dependently boosts REV-ERB-dependent repressor activity. REV-ERBβ IC50 = 560 nM, ERBα IC50 = 790 nM. SR9011 potently suppressed transcription (SR9011 IC50=620 nM) in a co-transfection test using full-length REV-ERBα and a luciferase reporter driven by the Bmal1 promoter. SR9011 suppresses the expression of BMAL1 mRNA in HepG2 cells in a manner that is dependent on REV-ERBα/β [1]. In addition, SR9011 prevents the growth of breast cancer cell lines irrespective of their ER or HER2 status. It seems that SR9011 stops breast cancer cells' cell cycle before they reach the M phase. Since cyclin A (CCNA2) was found to be a direct target gene of REV-ERB, cell cycle arrest may be mediated by SR9011's suppression of this cyclin's expression. G0/G1 phase cells increased and S and G2/M phase cells decreased after treatment with SR9011, indicating that REV-ERB activation may reduce the transition from G1 to S phase and/or from S phase to G2/M phase [2].

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In a chimeric Gal4 DNA Binding Domain-REV-ERB ligand binding domain reporter assay in HEK293 cells, SR9011 dose-dependently increased REV-ERB-dependent repressor activity with IC50 values of 790 nM for REV-ERBα and 560 nM for REV-ERBβ.
In a cotransfection assay using full-length REV-ERBα and a luciferase reporter driven by the Bmal1 promoter, SR9011 potently suppressed transcription with an IC50 of 620 nM.
SR9011 suppressed BMAL1 mRNA expression in HepG2 cells in a REV-ERBα/β-dependent manner.
SR9011 increased the recruitment of the CoRNR box peptide fragment of NCoR in a biochemical assay, consistent with direct agonist activity.
SR9011 inhibited circadian oscillations in SCN explants cultured from Per2:luc reporter mice, reversibly suppressing the amplitude of oscillations without affecting the period.
Similar effects on circadian oscillations were observed in Per2:luc fibroblasts. [1]

Since SR9011 demonstrated a respectable plasma exposure, the expression of genes sensitive to REV-ERB was investigated in the livers of mice given varying dosages of SR9011 for a period of six days. In response to SR9011, the REV-ERB target gene plasminogen activator inhibitor type 1 (Serpine1) shows dose-dependent expression inhibition. The genes for sterol response element binding protein (Srepf1) and cholesterol 7α-hydroxylase (Cyp7a1) were similarly demonstrated to be responsive to REV-ERB and to be dose-dependently inhibited by increasing dosages of SR9011. After 12 days in D:D circumstances, mice were injected at CT6 (peak expression of Rev-erbα) with a single dosage of SR9011 or vehicle. There is no disruption to circadian locomotor activity by vehicle injection. SR9011 treatment, however, resulted in the loss of locomotor activity in the subjects during the dark period. The following circadian cycle returns to normal, which is consistent with the drug clearing in a day. The efficacy (ED50 = 56 mg/kg) of the SR9011-dependent suppression of the REV-ERB responsive gene, Srebf1, in vivo (ED50 = 67 mg/kg) was comparable to that of the SR9011-dependent reduction in mouse wheel running behavior under continuous dark conditions[1].

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A single intraperitoneal injection of SR9011 (100 mg/kg) at circadian time (CT) 6 in mice under constant darkness (D:D) resulted in a complete loss of locomotor activity during the subsequent subjective dark phase, with normal activity resuming the next circadian cycle.
This effect on wheel-running behavior was dose-dependent with an ED50 of 56 mg/kg.
Under 12:12 light:dark (L:D) conditions, a single injection of SR9011 caused a 1-3 hour delay in the onset of nocturnal locomotor activity, rather than complete cessation.
A single injection of SR9011 (100 mg/kg, i.p.) at CT0 altered the circadian expression pattern of core clock genes (Per2, Cry2, Bmal1, Npas2, Clock) in the mouse hypothalamus.
Chronic administration of SR9011 (100 mg/kg, i.p., twice daily) to Balb/c or C57BL/6 mice for 10-12 days resulted in weight loss due to decreased fat mass, without affecting total food intake.
In CLAMS (comprehensive laboratory animal monitoring system) studies, SR9011 treatment increased oxygen consumption (VO2) by approximately 5%, indicating increased energy expenditure, despite a 15% decrease in movement.
A delay of 1-3 hours in the nocturnal peak of VO2 was observed after SR9011 administration.
SR9011 altered the circadian expression of metabolic genes in liver (e.g., suppressed Serpine1, Cyp7a1, Srebf1, Srebf2, Scd1, Ppargc1a/b), skeletal muscle (e.g., increased Cpt1b, Fatp1, Ppargc1b, Ucp3, Hk1, Pkm2), and white adipose tissue (e.g., suppressed Dgat1, Dgat2, Mgat, Plin1, Hsl).
In lean C57BL/6 mice, SR9011 treatment (100 mg/kg, i.p., twice daily for 10 days) decreased fasting plasma triglyceride and total cholesterol levels. [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].

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A biochemical assay was used to measure the recruitment of the CoRNR box peptide fragment of the nuclear receptor corepressor (NCoR) to REV-ERB in the presence of SR9011, demonstrating increased recruitment consistent with agonist activity. [1]

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].

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HEK293 cells were transfected with vectors encoding a chimeric Gal4 DNA Binding Domain fused to the REV-ERBα or REV-ERBβ ligand binding domain, along with a Gal4-responsive luciferase reporter gene. Cells were treated with SR9011, and luciferase activity was measured to assess REV-ERB-dependent repressor activity.
For the full-length receptor assay, HEK293 cells were cotransfected with an expression vector for full-length REV-ERBα and a luciferase reporter plasmid driven by the Bmal1 promoter. Following treatment with SR9011, luciferase activity was measured to evaluate transcriptional suppression.
HepG2 cells were treated with SR9011, and BMAL1 mRNA levels were quantified by QPCR to assess REV-ERB-dependent gene suppression.
SCN explants or fibroblasts from Per2:luc reporter mice were cultured and treated with SR9011. Bioluminescence was monitored over time to assess the compound's effect on circadian oscillation amplitude and period. [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]

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For circadian behavior and gene expression studies under constant darkness, C57BL/6 mice were housed in wheel cages, entrained to a standard light:dark cycle, and then released into constant dark (D:D) conditions. After 12 days in D:D, a single intraperitoneal injection of SR9011 (100 mg/kg) or vehicle was administered at circadian time (CT) 6 or CT0. Locomotor activity was monitored, and hypothalamic tissues were collected at various CT time points for gene expression analysis by QPCR.
For studies under a 12:12 light:dark cycle, mice were maintained on this cycle and received a single intraperitoneal injection of SR9011 (100 mg/kg). Locomotor activity and hypothalamic gene expression were assessed.
For chronic metabolic studies, Balb/c or C57BL/6 mice were administered SR9011 intraperitoneally at a dose of 100 mg/kg, twice daily (at CT0 and CT12), for 10 to 12 days. Body weight, body composition (via DEXA), and metabolic parameters (via CLAMS) were monitored.
For gene expression profiling in metabolic tissues, C57BL/6 mice received a single intraperitoneal injection of SR9011 (100 mg/kg) at CT0. Groups of mice were sacrificed at CT0, CT6, CT12, and CT18, and liver, skeletal muscle, and white adipose tissue were collected for QPCR analysis.
For lipid-lowering studies in lean mice, C57BL/6 mice were administered SR9011 intraperitoneally at 100 mg/kg, twice daily for 10 days, after which fasting plasma triglycerides and cholesterol were measured. [1]

The compound showed reasonable plasma exposure levels in mice, allowing for assessment of its in vivo effects. [1]

Mice treated with SR9011 did not show a decline in strength and continued to be active in the open field test. Blood tests of the treated mice showed no obvious signs of toxicity. Although the monkeys experienced a significant increase in body weight over a short period (mentioned in the discussion of the general characteristics of REV-ERB agonists), no increase in liver transaminase levels more than twice the baseline level was observed in any dose of REV-ERB agonist tested. [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.

SR-9011 is a REV-ERB agonist. Studies have shown that SR-9011 has a specific killing effect on cancer cells and oncogene-induced senescent cells (including melanocytic nevi) without affecting the viability of normal cells or tissues. Synchronizing behavioral and metabolic processes is crucial for cardiovascular health and the prevention of metabolic diseases. Nuclear receptors REV-ERB-α and REV-ERB-β play an indispensable role in regulating the expression of core clock proteins that drive activity and metabolic rhythms. This article describes the identification of a potent synthetic REV-ERB agonist with in vivo activity. Administration of synthetic REV-ERB ligands altered the diurnal rhythmic behavior and diurnal rhythmic patterns of core clock gene expression in mice. The diurnal rhythmic expression patterns of a range of metabolic genes in the liver, skeletal muscle, and adipose tissue were also altered, leading to increased energy expenditure. Treatment of diet-induced obese mice with REV-ERB agonists reduced obesity by decreasing fat mass and significantly improving dyslipidemia and hyperglycemia. These results suggest that synthetic REV-ERB ligands targeting circadian rhythms may be beneficial for treating sleep disorders and metabolic diseases. [1]

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SR9011 is a synthetic agonist of the nuclear receptors REV-ERBα and REV-ERBβ, which are key components of the circadian clock regulation mechanism.
By targeting REV-ERB, SR9011 can regulate circadian rhythm behavior, core clock gene expression, and the circadian rhythm patterns of numerous metabolic genes in peripheral tissues.
Its effects include increasing energy expenditure, promoting the oxidation of fatty acids and glucose in muscles, inhibiting lipogenesis and cholesterol synthesis in the liver, and reducing the synthesis and storage of triglycerides in adipose tissue.
These effects lead to reduced fat and improved plasma lipid profiles in mouse models.
This study suggests that synthetic REV-ERB ligands like SR9011 may be beneficial for treating sleep disorders and metabolic diseases. SR9011 may have potential uses in treating sleep disorders (by regulating circadian rhythm phases) and metabolic diseases (by increasing energy expenditure and reducing obesity). [1]

C23H31CLN4O3S

479.04

478.18

C, 57.67; H, 6.52; Cl, 7.40; N, 11.70; O, 10.02; S, 6.69

1379686-29-9

SR9011 hydrochloride;2070014-94-5

57394021

Light yellow to yellow solid powder

1.3±0.1 g/cm3

642.8±50.0 °C at 760 mmHg

342.6±30.1 °C

0.0±1.9 mmHg at 25°C

1.595

5.74

1

5

10

32

600

0

PPUYOYQTTWJTIU-UHFFFAOYSA-N

InChI=1S/C23H31ClN4O3S/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)

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.22 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 (5.22 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.)