Cell experiment:

MCF10A, MDA-MB-231, MCF-7, MDA-MB-361, SKBR3, BT474 cells are plated in 6-well plates one day before treatment. The MTT cell proliferation assays are performed. Briefly, 3×103 to 5 × 103 cells per well are plated in 96-well plates. Twenty-four hours later, cells are treated with SR9011 (0, 2, 4, 6, 8 and 10 μM) or DMSO. Seventy-two hours after treatment, the cells are labeled with 1.2 mM MTT and incubated for 4 hours. DMSO is then added and readings are taken on a plate reader at 540 nm[2].

Animal experiment:

Mice[1] For circadian gene expression experiments male C57BL6 mice (8-10 weeks of age) are either maintained on a L:D (12h:12h) cycle or on constant darkness. At circadian time (CT) 0 animals are administered a single dose of 100 mg/kg SR9011 (i.p.) and groups of animals (n=6) are sacrificed at CT0, CT6, CT12 and CT18. Gene expression is determined by real time QPCR.

IC50: 790 nM for REV-ERB-α; 560 nM for REV-ERB-β

SR9011 is a REV-ERB-α/β agonist.

REV-ERB nuclear receptors have a critical role in feedback regulation of the circadian oscillator. Both Bmal1 and Clock are direct REV-ERB target genes and loss of REV-ERB-α alters circadian behaviour.

In vitro: SR9011 dose-dependently increased the REV-ERB-dependent repressor activity assessed in HEK293 cells. In addition, SR9011 potently and efficaciously suppressed transcription in a cotransfection assay using full-length REV-ERB-a along with a luciferase reporter driven by the Bmal1 promoter. SR9011 also suppressed the expression of BMAL1 messenger RNA in HepG2 cells in a REV-ERB-a/b-dependent manner. Moreover, SR9011 inhibited the activity of the SCNclock, with reversible inhibition of circadian oscillations [1].

In vivo: In the mice under constant darkness conditions, it was observed that the SR9011-dependent decrease in wheel running behaviour was dose-dependent and that the potency (half-maximum effective dose 56 mg/kg) was similar to the potency of SR9011-mediated suppression of a REV-ERB responsive gene, Srebf1, in vivo. Moreover, Tau was not affected by treatment with SR9011, and the recovery after the drug to resume the normal rhythm was similar to the effect observed after removal of the drug from the SCN explants [1].

Clinical trial: Up to now, SR9011 is still in the preclinical development stage.

Reference:
[1] Solt LA,Wang Y,Banerjee S,Hughes T,Kojetin DJ,Lundasen T,Shin Y,Liu J,Cameron MD,Noel R,Yoo SH,Takahashi JS,Butler AA,Kamenecka TM,Burris TP.  Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature.2012 Mar 29;485(7396):62-8.