Figure 8. Identification of SR10067 as a highly potent synthetic REV-ERB agonist.

A, Chemical structure of the synthetic REV-ERB agonist SR10067 compared to SR9011 and SR9009. B, Results from a Gal4-REV-ERB/Gal4 UASX5 luciferase reporter cotransfection assay in HEK293 cells displaying the potent REV-ERB agonist activity of SR10067. C, Results from a cotransfection assay in HEK 293 cells with full-length REVERBα and a BMAL1 promoter driven luciferase reporter displaying the potent agonist activity of SR10067. D, Plasma and brain concentrations of SR10067 2h following i.p. injection of 30 mg kg⁻¹ of the compound. The 6h value for SR10067 in the brain is 150 ± 20 nM. E, Nuclear receptor specificity assay illustrating lack of activity of SR10067 on a wide range of nuclear receptors. The format of the assay was a cotransfection assay with Gal4 DNA binding domain – nuclear receptor fusions in HEK293 cells as previously described (see Methods). SR10067 was tested at 20 μM. Error bars indicate mean ± s.e.m. and n=3. There were no statistical differences between vehicle and drug treatment in any of the assays shown as assessed by a Student’s t test (unpaired two-tailed). F, Normalized (to Gapdh) expression of Npas2 mRNA isolated from the hypothalamic of mice injected with 30 mg kg⁻¹ of SR10067 (i.p. at ZT0) demonstrating a SR10067-dependent alteration in the circadian rhythm of expression. Expression was monitored over 24h and the results are double plotted. n=5. *P<0.05. Mean ± SEM.