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Pharmacological targeting of the mammalian clock regulates sleep architecture and emotional behaviour.

Banerjee S, Wang Y, Solt LA, Griffett K, Kazantzis M, Amador A, El-Gendy BM, Huitron-Resendiz S, Roberts AJ, Shin Y, Kamenecka TM, Burris TP - Nat Commun (2014)

Bottom Line: REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep.Interestingly, REV-ERB agonists also reduce anxiety-like behaviour.These data are consistent with increased anxiety-like behaviour of REV-ERBβ- mice, in which REV-ERB agonists have no effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458, USA.

ABSTRACT
Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and β, regulate sleep architecture and emotional behaviour in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behaviour. These data are consistent with increased anxiety-like behaviour of REV-ERBβ- mice, in which REV-ERB agonists have no effect. These results indicate that pharmacological targeting of REV-ERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety.

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SR10067 Alters Sleep Architecture and Anxiety-like BehaviorA, Actograms from wheel running cages demonstrating the effect of SR10067 injection (red bars, i.p. various concentrations) on wheel running activity in mice. B, Assessment of the dose-dependence of inhibition of wheel running activity in mice during the entire dark phase following administration of SR10067. n=6 to 8 mice per group. C, Assessment of the effect of SR10067 on wakefulness in mice. Wakefulness, SWS and REM sleep was monitored by EEG as indicated in Figs. 1 & 2. Mice were injected with SR10067 (30 mg kg−1, i.p.) or vehicle at ZT6. n=8 mice. D, Locomotor telemetry data indicating an increase in movement of mice during the period 2h after ZT6 injection of SR10067 (30 mg kg−1). n=6 mice. E, Results from the marble burying assay demonstrating that SR10067 dose-dependently reduces anxiety like behavior in the marble burying assay. n=8 mice. Values are mean ± SEM. In panel D differences between treatment groups (vehicle vs. SR) were assessed by a two tailed t test (Student’s) with significance *P < 0.05. In panels B and E, differences between groups were assessed using one-way ANOVA followed by Tukey’s post hoc test with significance *P<0.05. In panel C, potential differences between treatments were assessed by repeated measure two-way ANOVA followed by Bonferroni post hoc test with significance *P < 0.05.
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Figure 9: SR10067 Alters Sleep Architecture and Anxiety-like BehaviorA, Actograms from wheel running cages demonstrating the effect of SR10067 injection (red bars, i.p. various concentrations) on wheel running activity in mice. B, Assessment of the dose-dependence of inhibition of wheel running activity in mice during the entire dark phase following administration of SR10067. n=6 to 8 mice per group. C, Assessment of the effect of SR10067 on wakefulness in mice. Wakefulness, SWS and REM sleep was monitored by EEG as indicated in Figs. 1 & 2. Mice were injected with SR10067 (30 mg kg−1, i.p.) or vehicle at ZT6. n=8 mice. D, Locomotor telemetry data indicating an increase in movement of mice during the period 2h after ZT6 injection of SR10067 (30 mg kg−1). n=6 mice. E, Results from the marble burying assay demonstrating that SR10067 dose-dependently reduces anxiety like behavior in the marble burying assay. n=8 mice. Values are mean ± SEM. In panel D differences between treatment groups (vehicle vs. SR) were assessed by a two tailed t test (Student’s) with significance *P < 0.05. In panels B and E, differences between groups were assessed using one-way ANOVA followed by Tukey’s post hoc test with significance *P<0.05. In panel C, potential differences between treatments were assessed by repeated measure two-way ANOVA followed by Bonferroni post hoc test with significance *P < 0.05.

Mentions: SR9011 and SR9009 were the first synthetic REV-ERB agonists developed with sufficient pharmacokinetic properties to be used as in vivo chemical tools9. However, they exhibit relatively low potency for the receptor with IC50’s of approximately ~700 nM9. We hypothesized that if activation of REV-ERB with synthetic agonists did indeed lead to the wakefulness and anxiolytic effects that development of higher affinity REVERB agonists with either equivalent or improved pharmacokinetic properties should yield improvements in the in vivo potency. In order to test this hypothesis, we evaluated a synthetic REV-ERB agonist (SR10067) derived from extensive modification of the SR9009/SR9011 scaffold37,38 (Fig. 7A and Supplementary Fig. 8 & 9) that displays substantially greater potency than SR9011 and SR9009 in vitro. The potency of SR10067 is considerably better than SR9011 and SR9009 in the Gal4DBD-REV-ERB ligand binding domain (LBD) cotransfection assay (SR10067: REV-ERBα IC50=170 nM, REV-ERBβ IC50=160 nM (Fig. 7B) vs. SR9011: REV-ERBα IC50=670 nM, REV-ERBβ IC50=800 nM9). Potency of SR10067 was also considerably better in a cotransfection assay utilizing full-length REV-ERBα along with the BMAL1 promoter luciferase reporter (SR10067 IC50=140 nM (Fig. 8C) vs. SR9011 IC50=620 nM9). SR10067 displayed no significant activity at any other nuclear receptor (Fig. 7E) or a range of other receptors, ion channels and transporters assessed in the NIMH Psychoactive Drug Screening Program (Supplementary Table 1. Assessment of plasma and brain concentrations of SR10067 one and six hours after i.p. injection (30 mg kg−1) revealed that levels of the compound remain above the IC50 for the receptor six hours after administration (Fig. 7D). As a marker of in vivo efficacy of REV-ERB agonists we previously demonstrated that a single injection of SR9011 or SR9009 suppressed the circadian rhythm of Npas2 gene expression in the mouse hypothalamus9, and when we performed this experiment with SR10067 we observed similar results (Fig. 7F). Synthetic REV-ERB agonists also suppress circadian wheel running activity after a single injection9, and this was observed with SR10067. As illustrated in Figs. 8A and 8B, administration of SR10067 at various concentrations yielded a dose-dependent effect on reduction in nocturnal wheel running activity as indicated in the actograms. The ED50 for suppression of wheel running activity was 20 mg kg−1 (Fig. 8B), which is more potent than that described for SR9011 (56 mg kg−1)9 and is consistent with the improved potency of SR10067 in the REV-ERB transcriptional assays. SR10067 induced wakefulness and reduced SWS and REM sleep when injected at ZT6 similar to what was observed with SR9011 and SR9009 (Fig. 8C). The effect of SR10067 on sleep architecture was also similar to what was observed with SR9011 and SR9009 with increased number of episodes of SWS with decreased duration and decreased number of episodes and duration of REM sleep (Fig. 9). No effect on EEG power was observed (Supplementary Figure 1C). SR10067 also induced locomotion when administered into mice at ZT6 in a manner similar to SR9009 and SR9011 consistent with an increase in wakefulness (Fig. 8D). The anxiolytic activity of SR10067 was assessed in the marble burying anxiety assay and SR10067 displayed superior potency than SR9011 (ED50 of 12 mg kg−1vs. 61 mg kg−1) (Fig. 8E). This 5-fold improvement in in vivo anxiolytic potency compares favorably with the in vitro improvement in potency (4–5 fold) when comparing SR10067 to SR9011. These data clearly suggest that the anxiolytic activity is mediated by activation of REV-ERB given that two synthetic REV-ERB selective agonists, with distinct chemical structures, display anxiolytic activity that correlates to their relative activity at REV-ERB.


Pharmacological targeting of the mammalian clock regulates sleep architecture and emotional behaviour.

Banerjee S, Wang Y, Solt LA, Griffett K, Kazantzis M, Amador A, El-Gendy BM, Huitron-Resendiz S, Roberts AJ, Shin Y, Kamenecka TM, Burris TP - Nat Commun (2014)

SR10067 Alters Sleep Architecture and Anxiety-like BehaviorA, Actograms from wheel running cages demonstrating the effect of SR10067 injection (red bars, i.p. various concentrations) on wheel running activity in mice. B, Assessment of the dose-dependence of inhibition of wheel running activity in mice during the entire dark phase following administration of SR10067. n=6 to 8 mice per group. C, Assessment of the effect of SR10067 on wakefulness in mice. Wakefulness, SWS and REM sleep was monitored by EEG as indicated in Figs. 1 & 2. Mice were injected with SR10067 (30 mg kg−1, i.p.) or vehicle at ZT6. n=8 mice. D, Locomotor telemetry data indicating an increase in movement of mice during the period 2h after ZT6 injection of SR10067 (30 mg kg−1). n=6 mice. E, Results from the marble burying assay demonstrating that SR10067 dose-dependently reduces anxiety like behavior in the marble burying assay. n=8 mice. Values are mean ± SEM. In panel D differences between treatment groups (vehicle vs. SR) were assessed by a two tailed t test (Student’s) with significance *P < 0.05. In panels B and E, differences between groups were assessed using one-way ANOVA followed by Tukey’s post hoc test with significance *P<0.05. In panel C, potential differences between treatments were assessed by repeated measure two-way ANOVA followed by Bonferroni post hoc test with significance *P < 0.05.
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Figure 9: SR10067 Alters Sleep Architecture and Anxiety-like BehaviorA, Actograms from wheel running cages demonstrating the effect of SR10067 injection (red bars, i.p. various concentrations) on wheel running activity in mice. B, Assessment of the dose-dependence of inhibition of wheel running activity in mice during the entire dark phase following administration of SR10067. n=6 to 8 mice per group. C, Assessment of the effect of SR10067 on wakefulness in mice. Wakefulness, SWS and REM sleep was monitored by EEG as indicated in Figs. 1 & 2. Mice were injected with SR10067 (30 mg kg−1, i.p.) or vehicle at ZT6. n=8 mice. D, Locomotor telemetry data indicating an increase in movement of mice during the period 2h after ZT6 injection of SR10067 (30 mg kg−1). n=6 mice. E, Results from the marble burying assay demonstrating that SR10067 dose-dependently reduces anxiety like behavior in the marble burying assay. n=8 mice. Values are mean ± SEM. In panel D differences between treatment groups (vehicle vs. SR) were assessed by a two tailed t test (Student’s) with significance *P < 0.05. In panels B and E, differences between groups were assessed using one-way ANOVA followed by Tukey’s post hoc test with significance *P<0.05. In panel C, potential differences between treatments were assessed by repeated measure two-way ANOVA followed by Bonferroni post hoc test with significance *P < 0.05.
Mentions: SR9011 and SR9009 were the first synthetic REV-ERB agonists developed with sufficient pharmacokinetic properties to be used as in vivo chemical tools9. However, they exhibit relatively low potency for the receptor with IC50’s of approximately ~700 nM9. We hypothesized that if activation of REV-ERB with synthetic agonists did indeed lead to the wakefulness and anxiolytic effects that development of higher affinity REVERB agonists with either equivalent or improved pharmacokinetic properties should yield improvements in the in vivo potency. In order to test this hypothesis, we evaluated a synthetic REV-ERB agonist (SR10067) derived from extensive modification of the SR9009/SR9011 scaffold37,38 (Fig. 7A and Supplementary Fig. 8 & 9) that displays substantially greater potency than SR9011 and SR9009 in vitro. The potency of SR10067 is considerably better than SR9011 and SR9009 in the Gal4DBD-REV-ERB ligand binding domain (LBD) cotransfection assay (SR10067: REV-ERBα IC50=170 nM, REV-ERBβ IC50=160 nM (Fig. 7B) vs. SR9011: REV-ERBα IC50=670 nM, REV-ERBβ IC50=800 nM9). Potency of SR10067 was also considerably better in a cotransfection assay utilizing full-length REV-ERBα along with the BMAL1 promoter luciferase reporter (SR10067 IC50=140 nM (Fig. 8C) vs. SR9011 IC50=620 nM9). SR10067 displayed no significant activity at any other nuclear receptor (Fig. 7E) or a range of other receptors, ion channels and transporters assessed in the NIMH Psychoactive Drug Screening Program (Supplementary Table 1. Assessment of plasma and brain concentrations of SR10067 one and six hours after i.p. injection (30 mg kg−1) revealed that levels of the compound remain above the IC50 for the receptor six hours after administration (Fig. 7D). As a marker of in vivo efficacy of REV-ERB agonists we previously demonstrated that a single injection of SR9011 or SR9009 suppressed the circadian rhythm of Npas2 gene expression in the mouse hypothalamus9, and when we performed this experiment with SR10067 we observed similar results (Fig. 7F). Synthetic REV-ERB agonists also suppress circadian wheel running activity after a single injection9, and this was observed with SR10067. As illustrated in Figs. 8A and 8B, administration of SR10067 at various concentrations yielded a dose-dependent effect on reduction in nocturnal wheel running activity as indicated in the actograms. The ED50 for suppression of wheel running activity was 20 mg kg−1 (Fig. 8B), which is more potent than that described for SR9011 (56 mg kg−1)9 and is consistent with the improved potency of SR10067 in the REV-ERB transcriptional assays. SR10067 induced wakefulness and reduced SWS and REM sleep when injected at ZT6 similar to what was observed with SR9011 and SR9009 (Fig. 8C). The effect of SR10067 on sleep architecture was also similar to what was observed with SR9011 and SR9009 with increased number of episodes of SWS with decreased duration and decreased number of episodes and duration of REM sleep (Fig. 9). No effect on EEG power was observed (Supplementary Figure 1C). SR10067 also induced locomotion when administered into mice at ZT6 in a manner similar to SR9009 and SR9011 consistent with an increase in wakefulness (Fig. 8D). The anxiolytic activity of SR10067 was assessed in the marble burying anxiety assay and SR10067 displayed superior potency than SR9011 (ED50 of 12 mg kg−1vs. 61 mg kg−1) (Fig. 8E). This 5-fold improvement in in vivo anxiolytic potency compares favorably with the in vitro improvement in potency (4–5 fold) when comparing SR10067 to SR9011. These data clearly suggest that the anxiolytic activity is mediated by activation of REV-ERB given that two synthetic REV-ERB selective agonists, with distinct chemical structures, display anxiolytic activity that correlates to their relative activity at REV-ERB.

Bottom Line: REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep.Interestingly, REV-ERB agonists also reduce anxiety-like behaviour.These data are consistent with increased anxiety-like behaviour of REV-ERBβ- mice, in which REV-ERB agonists have no effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458, USA.

ABSTRACT
Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and β, regulate sleep architecture and emotional behaviour in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behaviour. These data are consistent with increased anxiety-like behaviour of REV-ERBβ- mice, in which REV-ERB agonists have no effect. These results indicate that pharmacological targeting of REV-ERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety.

Show MeSH
Related in: MedlinePlus