Synthetic REV-ERB Agonists Research Articles

Structural basis of synthetic agonist activation of the nuclear receptor REV-ERB

The nuclear receptor REV-ERB plays an important role in a range of physiological processes. REV-ERB behaves as a ligand-dependent transcriptional repressor and heme has been identified as a physiological agonist. Our current understanding of how ligands bind to and regulate transcriptional repression by REV-ERB is based on the structure of heme bound to REV-ERB. However, porphyrin (heme) analogues have been avoided as a source of synthetic agonists due to the wide range of heme binding proteins and potential pleotropic effects. How non-porphyrin synthetic agonists bind to and regulate REV-ERB has not yet been defined. Here, we characterize a high affinity synthetic REV-ERB agonist, STL1267, and describe its mechanism of binding to REV-ERB as well as the method by which it recruits transcriptional corepressor both of which are unique and distinct from that of heme-bound REV-ERB.

REV-ERB agonist suppresses IL-17 production in γδT cells and improves psoriatic dermatitis in a mouse model

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and cellular infiltration. Studies have shown that disease development depends on proinflammatory cytokines, such as interleukin (IL)-23 and IL-17. It has been suggested that IL-23 produced by innate immune cells, such as macrophages, stimulates a subset of helper T cells to release IL-17, promoting neutrophil recruitment and keratinocyte proliferation. However, recent studies have revealed the crucial role of γδT cells in psoriasis pathogenesis as the primary source of dermal IL-17. The nuclear receptors REV-ERBs are ligand-dependent transcription factors recognized as circadian rhythm regulators. REV-ERBs negatively regulate IL-17-producing helper T cells, whereas the involvement of REV-ERBs in regulating IL-17-producing γδT (γδT17) cells remains unclear. Here we revealed the regulatory mechanism involving γδT17 cells through REV-ERBs. γδT17 cell levels were remarkably elevated in the secondary lymphoid organs of mice that lacked an isoform of REV-ERBs. A synthetic REV-ERB agonist, SR9009, suppressed γδT17 cells in vitro and in vivo. Topical application of SR9009 to the skin reduced the inflammatory symptoms of psoriasiform dermatitis in mice. The results of this study provide a novel therapeutic approach for psoriasis targeting REV-ERBs in γδT17 cells.

REV-ERB Agonist SR9009 Regulates the Proliferation and Neurite Outgrowth/Suppression of Cultured Rat Adult Hippocampal Neural Stem/Progenitor Cells in a Concentration-Dependent Manner.

REV-ERBs are heme-binding nuclear receptors that regulate the circadian rhythm and play important roles in the regulation of proliferation and the neuronal differentiation process in neuronal stem/progenitor cells in the adult brain. However, the effects of REV-ERB activation in the adult brain remain unclear. In this study, SR9009, a synthetic REV-ERB agonist that produces anxiolytic effects in mice, was used to treat undifferentiated and neuronally differentiated cultured rat adult hippocampal neural stem/progenitor cells (AHPs). The expression of Rev-erbβ was upregulated during neurogenesis in cultured rat AHPs, and Rev-erbβ knockdown analysis indicated that REV-ERBβ regulates the proliferation and neurite outgrowth of cultured rat AHPs. The application of a low concentration (0.1µM) of the REV-ERB agonist SR9009 enhanced neurite outgrowth during neurogenesis in cultured rat AHPs, whereas the addition of a high concentration (2.5µM) of SR9009 suppressed neurite outgrowth. Further examination of the SR9009 regulatory mechanism showed that the expressions of downstream target genes of REV-ERBβ, including Ccna2 and Sez6, were modulated by SR9009. The results of this study indicated that REV-ERBβ activity in cultured rat AHPs was regulated by SR9009 in a concentration-dependent manner. Furthermore, SR9009 inhibited the growth of cultured rat AHPs through various pathways, which may provide insight into the multifunctional mechanisms of action associated with SR9009. The findings of this study may provide an improved understanding of proliferation and neuronal maturation mechanisms in cultured rat AHPs through SR9009-regulated REV-ERBβ signaling pathways.

The Putatively Specific Synthetic REV-ERB Agonist SR9009 Inhibits IgE- and IL-33-Mediated Mast Cell Activation Independently of the Circadian Clock.

The cell-autonomous circadian clock regulates IgE- and IL-33-mediated mast cell activation, both of which are key events in the development of allergic diseases. Accordingly, clock modifiers could be used to treat allergic diseases, as well as many other circadian-related diseases, such as sleep and metabolic disorders. The nuclear receptors REV-ERB-α and -β (REV-ERBs) are crucial components of the circadian clockwork. Efforts to pharmacologically target REV-ERBs using putatively specific synthetic agonists, particularly SR9009, have yielded beneficial effects on sleep and metabolism. Here, we sought to determine whether REV-ERBs are functional in the circadian clockwork in mast cells and, if so, whether SR9009 affects IgE- and IL-33-mediated mast cell activation. Bone marrow-derived mast cells (BMMCs) obtained from wild-type mice expressed REV-ERBs, and SR9009 or other synthetic REV-ERBs agonists affected the mast cell clockwork. SR9009 inhibited IgE- and IL-33-mediated mast cell activation in wild-type BMMCs in association with inhibition of Gab2/PI3K and NF-κB activation. Unexpectedly, these suppressive effects of SR9009 were observed in BMMCs following mutation of the core circadian gene Clock. These findings suggest that SR9009 inhibits IgE- and IL-33-mediated mast cell activation independently of the functional circadian clock activity. Thus, SR9009 or other synthetic REV-ERB agonists may have potential for anti-allergic agents.

The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine-induced hepatocellular carcinoma.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35mg/kg body weight ip) once a week for 8weeks. Melatonin was given at 5 or 10mgkg-1 d-1 ip beginning 4weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1ε, Rev-erbα, and Rev-erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

Rev-erb regulation of cholesterologenesis

REV-ERBα and REV-ERBβ are heme regulated nuclear receptors that are known to regulate metabolic pathways. We previously demonstrated that treatment of mice with synthetic REV-ERB agonists suppressed plasma cholesterol levels and the hepatic levels of the rate limiting enzyme in cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-CoA reductase). Here, we characterize the role of REV-ERB on the cholesterol biosynthetic pathway in greater detail. The REV-ERB agonist SR9009 reduced plasma cholesterol levels in both wild type C57Bl/6 and low density lipoprotein receptor (LDLR) null mice as well as reducing the expression of an array of genes within the cholesterol biosynthetic pathway. Consistent with these data, we observed increased expression of these genes in mice deficient in expression of Rev-erbα. Analysis of global run-on and deep sequencing (GRO-Seq) and chromatin immunoprecipitation deep sequencing (ChIP-Seq) data revealed that Rev-erb directly binds to the majority of genes involved in cholesterol biosynthesis and directly suppresses their expression. This study reveals insight into the complex mechanism by which Rev-erb directly and indirectly (via inhibition of Srebf2 expression) regulates cholesterol biosynthesis and provides information of how cholesterol levels are regulated in a circadian fashion. Additionally, these studies suggest that targeting Rev-erb may be an effective method for suppressing LDL cholesterol levels in the clinic.

The intrinsic microglial clock system regulates interleukin-6 expression.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

Anti-proliferative actions of a synthetic REV-ERBα/β agonist in breast cancer cells

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.

REV-ERBα negatively regulates the Th17 mediated immune response (IRM15P.603)

Abstract Th17 cells are a major driver of autoimmune disease such as multiple sclerosis. The differentiation of the Th17 cell lineage is dependent on RORƔt, which is part of the ROR nuclear receptor family. In addition to their involvement in immune cell lineage commitment, RORs also play important roles in circadian clock regulation. In the circadian system, the function of RORs is countered by another pair of nuclear receptors, REV-ERBα and REV-ERBβ, which are transcriptional repressors that bind to the ROR binding motif (RORE) and suppress ROR target gene expression. We tested whether REV-ERBs are also negative regulators in the context of Th17 cell differentiation. REV-ERBα is specifically induced in Th17 cells, and it directly binds to the RORE motifs at the IL17 locus and represses IL17 expression. Constant expression of REV-ERBα in T cells protects mice against Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of multiple sclerosis. Additionally, treating mice with a synthetic REV-ERB agonist can delay EAE development and reduce Th17 cell infiltration into the CNS. Together, these results suggest that REV-ERB can suppress Th17 differentiation and serve as a potential target for treatment of Th17 mediated autoimmune diseases.

Abstract 545: Suppression of Atherosclerosis by Synthetic REV-ERB Agonist

Background: Development of atherosclerosis is a complex process that involves both hyperlipidemia and inflammation. Therapies that can target both metabolic and inflammatory aspect of this disease have significant potential in the treatment of cardiovascular diseases. The nuclear receptors for heme, REV-ERBα and REV-ERBβ, play important roles in regulation of metabolism and inflammation. Recently it was demonstrated that knock-down of REV-ERBα in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model. Methods and Results: A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Eight-week-old mice with deficient LDL receptor were fed high cholesterol diet and treated with vehicle or REV-ERB agonist, SR9009 (100mg/kg, I.P.) for a period of eight weeks. Atherosclerotic plaque size was significantly reduced in mice administered SR9009 compared to control mice. In another study plasma lipid levels were monitored every two weeks for eight weeks. We demonstrate that SR9009 significantly reduced plasma lipid levels in these mice. To determine the role of REV-ERB in macrophage differentiation and polarization bone-marrow derived macrophages (BMDM) were treated with SR9009 in vitro. SR9009 treatment of BMDM reduced the polarization of BMDM to proinflammatory M1 macrophage. Gene expression of markers of M1 polarization as well as pro-inflammatory cytokine secretion was reduced in macrophages treated with SR9009. Conclusions: We demonstrate that REV-ERB targets multiple processes involved in atherosclerosis. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis.

Suppression of atherosclerosis by synthetic REV-ERB agonist

The nuclear receptors for heme, REV-ERBα and REV-ERBβ, play important roles in the regulation of metabolism and inflammation. Recently it was demonstrated that reduced REV-ERBα expression in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model. A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Atherosclerotic plaque size was significantly reduced (p < 0.05) in mice administered SR9009 (100 mg/kg) for seven weeks compared to control mice (n = 10 per group). SR9009 treatment of bone marrow-derived mouse macrophages (BMDM) reduced the polarization of BMDMs to proinflammatory M1 macrophage while increasing the polarization of BMDMs to anti-inflammatory M2 macrophages. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis.

Time in a bottle

A biological clock synchronizes animal behaviour and physiology with Earth's 24-hour rotation. Drugs targeting the clock's 'gears' show promise for treating obesity and other metabolic disorders. See Article p.62 & Letter p.123 Metabolic processes need to run like clockwork to prevent disease. Core clock proteins drive these rhythms, and the nuclear receptors REV-ERB-α and REV-ERB-β have a central role in regulating the expression of clock genes. Solt et al. report the identification of potent synthetic REV-ERB agonists, termed SR9011 and SR9009, which can alter the circadian expression of core clock genes in the hypothalami of mice. This is shown to alter the expression of metabolic genes in liver, skeletal-muscle and adipose tissue, and results in increased energy expenditure by the mice. The REV-ERB agonists reduce fat mass in diet-induced obese mice and improve dyslipidaemia and hyperglycaemia. These results suggest that synthetic REV-ERB ligands are promising candidates for the treatment of metabolic diseases. Cho et al. present genetic evidence that REV-ERB-α and REV-ERB-β are indispensible for the coordination of circadian rhythm and metabolism. Mice without REV-ERBs show disrupted expression of clock and lipid homeostatic gene networks. They have altered circadian wheel-running behaviour and deregulated lipid metabolism. These data ally REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression.

Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists

Synchronizing rhythms of behavior and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERBα and REV-ERBβ play 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 behavior 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 dyslipidemia and hyperglycemia. These results suggest that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may hold utility in the treatment of sleep disorders as well as metabolic diseases.

Abstract 1357: Inhibition of breast cancer cell growth by a synthetic ligand targeting the nuclear receptor, REV-ERB (NR1D1 and NR1D2)

Abstract Disruption of the circadian rhythm leads to increased risk for development of breast cancer in humans. The molecular mechanism underlying the link between circadian rhythm disruption and the increased risk of breast cancer in humans is not clear. The REV-ERBs are nuclear receptors that function as critical regulatory components of the circadian rhythm. We observed that REV-ERBbeta is highly overexpressed in multiple breast cancer cell lines, but not in the non-tumorigenic control breast cell line, MCF-10A. Furthermore, we show that a synthetic REV-ERB agonist that we discovered, SR9011, inhibits the proliferation of multiple breast cancer cell lines including triple negative breast cancers but has no effect on MCF10A cells. We discovered that the REV-ERBs directly regulates the expression of cyclin A2 gene suggesting a possible mechanism for inhibition of proliferation. Treatment of breast cancer cell lines with SR9011 repress the expression of cyclin A2 at both mRNA and protein levels. SR9011 treated SKBR3 cells display a reduced fraction of cells in S phase and in the G2/M phase. Administration SR9011 to synchronized SKBR3 cells results in about 8 hours delay of G2/M phase occurrence. Thus, our data suggests that REV-ERB is a valid target for development of anti-cancer agents and SR9011 holds promise for treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2011-1357