Mechanism of Action of Irilone as a Potentiator of Progesterone Receptor Signaling

Progesterone signaling and its proper regulation is important for reproductive function. When progesterone signaling is dysregulated, gynecological diseases can occur, for example endometriosis, uterine fibroids, and endometrial cancer. While these diseases are treated with progestin therapy, progestins can bind to multiple steroid receptors, exerting side effects of weight gain, immunosuppression, cardiovascular disease, and stroke. Discovering an alternative progestin that is selective for the progesterone receptor (PR) is ideal. One potential source of such an alternative is botanical dietary supplements, which have become increasingly popular among consumers with sales reaching $9.6 billion in 2019. Although botanical supplements are popular, the chemical structures and biological action of botanical supplements would benefit from deeper scientific investigation. Studies of Trifolium pratense L. (red clover), primarily used for the treatment of menopausal symptoms, identified phytoestrogen compounds as the chemicals that mitigate those symptoms. Interestingly, irilone, identified from red clover, potentiated progesterone signaling via a progesterone response element luciferase (PRE/Luc) assay. Potentiation is when a compound has no activity by itself but when combined with another molecule, i.e. progesterone, that compound enhances PR activity. Prior to irilone, a natural compound with the ability to potentiate progesterone signaling had not been previously reported. The purpose of this study was to determine the mechanism of action of irilone. We hypothesized that irilone was potentiating PR by blocking PR degradation and by altering PR post-translational modifications. Irilone was found to potentiate 5 nM P4 using a PRE-luciferase assay in both T47D and Ishikawa PR expressing cells. Since PR is a downstream target gene of ER, we investigated if irilone also had ER activity. Irilone increased expression of an ERE-luciferase reporter gene. Next, we investigated if irilone could stabilize PR degradation and if irilone altered PR phosphorylation via western blot. Irilone was found to increase PR protein levels, but when ER was blocked, this was mitigated. In the presence of P4, irilone did not increase phosphorylation of serine 294 on PR. Future studies will determine if irilone is altering sumoylation of PR, and if irilone can potentiate PR signaling in vivo. Determining how irilone is potentiating progesterone will help us understand PR biology and could be an effective treatment for gynecological diseases by enhancing endogenous progesterone action.