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].

在表达嵌合 Gal4 DNA 结合结构域 (DBD)-REV-ERB 配体结合结构域 (LBD) α 或 β 和 Gal4 响应性荧光素酶报告基因 (REV-) 的 HEK293 细胞中，SR9011 剂量依赖性增强 REV-ERB 依赖性抑制活性。 REV-ERBβ IC50 = 560 nM，ERBα IC50 = 790 nM。在使用全长 REV-ERBα 和 Bmal1 启动子驱动的荧光素酶报告基因的共转染测试中，SR9011 有效抑制转录（SR9011 IC50 = 620 nM）。 SR9011 以依赖于 REV-ERBα/β 的方式抑制 HepG2 细胞中 BMAL1 mRNA 的表达 [1]。此外，SR9011 可以阻止乳腺癌细胞系的生长，无论其 ER 或 HER2 状态如何。 SR9011 似乎可以在乳腺癌细胞进入 M 期之前停止其细胞周期。由于细胞周期蛋白 A (CCNA2) 被发现是 REV-ERB 的直接靶基因，因此细胞周期停滞可能是通过 SR9011 抑制该细胞周期蛋白表达来介导的。 SR9011治疗后G0/G1期细胞增加，S和G2/M期细胞减少，表明REV-ERB激活可能减少从G1期到S期和/或从S期到G2/M期的转变[2]。

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在 HEK293 细胞中使用嵌合 Gal4 DNA 结合结构域-REV-ERB 配体结合结构域报告系统进行的实验中，SR9011 剂量依赖性地增加 REV-ERB 依赖性抑制活性，对 REV-ERBα 和 REV-ERBβ 的 IC50 值分别为 790 nM 和 560 nM。
在使用全长 REV-ERBα 和由 Bmal1 启动子驱动的荧光素酶报告基因的共转染实验中，SR9011 能有效抑制转录，IC50 为 620 nM。
SR9011 以 REV-ERBα/β 依赖的方式抑制 HepG2 细胞中 BMAL1 mRNA 的表达。
在生化实验中，SR9011 增加了核受体共抑制子 NCoR 的 CoRNR 盒肽段的募集，这与直接激动剂活性一致。
SR9011 可逆地抑制了来自 Per2:luc 报告基因小鼠的 SCN 外植体的昼夜节律振荡，降低了振荡幅度但不影响周期。
在 Per2:luc 成纤维细胞中也观察到了类似的对昼夜节律振荡的影响。[1]

由于 SR9011 表现出相当大的血浆暴露量，因此在六天内给予不同剂量 SR9011 的小鼠肝脏中研究了对 REV-ERB 敏感的基因表达。响应 SR9011，REV-ERB 靶基因纤溶酶原激活剂抑制剂 1 型 (Serpine1) 显示剂量依赖性表达抑制。甾醇反应元件结合蛋白 (Srepf1) 和胆固醇 7α-羟化酶 (Cyp7a1) 的基因同样被证明对 REV-ERB 有反应，并且通过增加 SR9011 剂量而受到剂量依赖性抑制。在 D:D 环境中 12 天后，在 CT6（Rev-erbα 表达峰值）时给小鼠注射单剂量的 SR9011 或载体。车辆注射不会干扰昼夜节律运动活动。然而，SR9011 治疗导致受试者在黑暗时期丧失运动活动能力。随后的昼夜节律周期恢复正常，这与一天内药物清除一致。 SR9011 依赖性抑制 REV-ERB 反应基因 Srebf1 的体内功效 (ED50 = 56 mg/kg) (ED50 = 67 mg/kg) 与 SR9011 依赖性减少小鼠滚轮运行的效果相当连续黑暗条件下的行为[1]。

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在持续黑暗条件下，于昼夜时间 CT6 对小鼠单次腹腔注射 SR9011（100 mg/kg），导致随后主观暗期的运动活动完全丧失，正常活动在下一个昼夜周期恢复。
对跑轮行为的这种影响是剂量依赖性的，ED50 为 56 mg/kg。
在 12:12 明暗循环条件下，单次注射 SR9011 导致夜间运动活动开始时间延迟 1-3 小时，而非完全停止。
在 CT0 单次注射 SR9011（100 mg/kg，腹腔）改变了小鼠下丘脑中核心时钟基因（Per2, Cry2, Bmal1, Npas2, Clock）的昼夜表达模式。
对 Balb/c 或 C57BL/6 小鼠慢性给予 SR9011（100 mg/kg，腹腔，每日两次）10-12 天，导致由于脂肪量减少而体重下降，但不影响总食物摄入量。
在 CLAMS 实验中，SR9011 处理使耗氧量增加约 5%，表明能量消耗增加，尽管运动量减少了 15%。
SR9011 给药后，观察到夜间耗氧量峰值延迟 1-3 小时。
SR9011 改变了肝脏（例如抑制 Serpine1, Cyp7a1, Srebf1, Srebf2, Scd1, Ppargc1a/b）、骨骼肌（例如增加 Cpt1b, Fatp1, Ppargc1b, Ucp3, Hk1, Pkm2）和白色脂肪组织（例如抑制 Dgat1, Dgat2, Mgat, Plin1, Hsl）中代谢基因的昼夜表达。
在瘦型 C57BL/6 小鼠中，SR9011 处理（100 mg/kg，腹腔，每日两次，持续 10 天）降低了空腹血浆甘油三酯和总胆固醇水平。[1]

在这项研究中，研究人员开发了两种REV-ERBα/β激动剂，它们具有足够的血浆/脑暴露，可以在体内评估其效果。SR9009和SR9009（图1a，补充图1）均剂量依赖性地增加了在表达嵌合Gal4 DNA结合结构域（DBD）-REV-ERB配体结合结构域α或β和Gal4反应性萤光素酶报告子的HEK293细胞中评估的REV-ERB依赖性阻遏物活性（图1b）（SR9009:REV-ERBαIC50=670 nM，REV-ERBβIC50=800 nM；SR9011:REV-ERB a IC50=790 nM，REV-ERBβIC50=560 nM）。REV-ERB配体GSK4112（补充图2）没有暴露于血浆中，其活性有限（图1b）。在使用全长REV-ERBα以及由Bmal1启动子驱动的萤光素酶报告子的共转染试验中，SR9011和SR9009均有效地抑制了转录（图1c）（SR9009 IC50=710 nM；SR9011 IC50=620 nM）。SR9011和SR9009以REV-ERBα/β依赖的方式抑制HepG2细胞中BMAL1 mRNA的表达（补充图3）。与这两种化合物作为REV-ERB的直接激动剂的作用一致，我们注意到，使用生化分析，这些化合物增加了NCoR的CoRNR盒肽片段的募集（补充图4）。SR9009与REV-ERBα的直接结合也通过圆二色谱分析得到证实（补充图5）（Kd=800 nM）。这两种化合物都没有在其他核受体上表现出活性12,13（补充图6）[1]。

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使用生化实验测量在 SR9011 存在下，核受体共抑制子 NCoR 的 CoRNR 盒肽段向 REV-ERB 的募集情况，结果显示募集增加，这与激动剂活性一致。[1]

HEK293 细胞在 96 孔板（1×106/孔）中生长，并使用 Lipofectamine 瞬时转染。每孔用总共 200 ng DNA 转染细胞，其中包含 pGL4 mIL-17 萤火虫荧光素酶报告基因构建体、pGL4 mIL-17 + CNS-5 萤火虫荧光素酶报告基因构建体或 pGL4 mIL-17 2kB RORE 突变体（100 ng/孔）、肌动蛋白启动子海肾荧光素酶报告基因（50 ng/孔）以及单独的对照载体或测试 DNA（50 ng/孔的全长 RORα 或全长 RORγ）。所有 48 种人类核受体均出现在特异性测定中，SR9009 的测试浓度为 20 μM。该测定的形式是在 HEK293 细胞中使用 Gal4 DNA 结合域-核受体融合物进行共转染测定[1]。

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将编码嵌合 Gal4 DNA 结合结构域与 REV-ERBα 或 REV-ERBβ 配体结合结构域融合蛋白的载体，以及一个 Gal4 响应性荧光素酶报告基因共转染到 HEK293 细胞中。用 SR9011 处理细胞，测量荧光素酶活性以评估 REV-ERB 依赖性抑制活性。
对于全长受体实验，将全长 REV-ERBα 的表达载体和由 Bmal1 启动子驱动的荧光素酶报告质粒共转染到 HEK293 细胞中。用 SR9011 处理后，测量荧光素酶活性以评估转录抑制。
用 SR9011 处理 HepG2 细胞，通过 QPCR 定量 BMAL1 mRNA 水平以评估 REV-ERB 依赖性基因抑制。
培养来自 Per2:luc 报告基因小鼠的 SCN 外植体或成纤维细胞，并用 SR9011 处理。随时间监测生物发光，以评估该化合物对昼夜节律振荡幅度和周期的影响。[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 displayed reasonable plasma exposure in mice, enabling the evaluation of its effects in vivo. [1]

Mice treated with SR9011 did not display a decrease in strength and continued to move in an open field assay.
Complete blood counts in treated mice showed no signs of overt toxicity.
Despite significant increases in body weight over a short period in monkeys (mentioned in context of REV-ERB agonists' general profile), no elevation of liver enzyme transaminase levels greater than 2-fold over baseline was observed at any dose of the REV-ERB agonists 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. SR-9011 has been demonstrated that it is specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and has no effect on the viability of normal cells or tissues.

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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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SR9011 is a synthetic agonist of the nuclear receptors REV-ERBα and REV-ERBβ, which are key components of the circadian clock regulatory machinery.
By pharmacologically targeting REV-ERB, SR9011 modulates circadian behavior, core clock gene expression, and the circadian pattern of numerous metabolic genes in peripheral tissues.
Its effects include increasing energy expenditure, promoting fatty acid and glucose oxidation in muscle, suppressing lipogenesis and cholesterol synthesis in the liver, and reducing triglyceride synthesis and storage in adipose tissue.
These actions lead to decreased adiposity and improved plasma lipid profiles in mouse models.
The study suggests that synthetic REV-ERB ligands like SR9011 may have potential utility in treating sleep disorders (by phase-shifting circadian rhythms) 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

110

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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