Withanolide Derivative 2,3-Dihydro-3β-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa)

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3 beta-methoxy withaferin-A (3 beta mWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3 beta mWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3 beta mWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 mu M and that 3 beta mWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [K-d], 5.9 mu M). We propose that 3 beta mWi-A is a novel modulator of circadian rhythms.

Automated summary

Withanolide derivatives, particularly 3 beta mWi-A, have been shown to lengthen the circadian clock, upregulate the expression of key genes involved in circadian rhythms, and act as an inverse agonist for RORa. These findings suggest that 3 beta mWi-A may serve as a novel modulator of circadian rhythms.