Identification of TR4 Modulators for Treatment of Cushing Disease.

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ABSTRACT Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening “orphan disease” with a staggering annual health care cost that is >7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with major morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro- opiomelanocortin (POMC) gene transcription and ACTH secretion. We hypothesize that small molecule inhibitors of TR4 action would be potent inhibitors of corticotroph tumor hormone (ACTH) secretion. Such a discovery would be a transformative therapy for Cushing disease as no similar therapies exist. To identify and characterize TR4 small molecule modulators, we will perform a large scale small molecule library screen using our unique series of transactivation assays that include a TR4-directed GAL4-LBD system and POMC-promoter reporters as well as a secondary screen in human corticotroph tumor primary cultures. Our first aim will use a mammalian one-hybrid GAL4-LBD system to identify TR4 modulators from a library of 200,000 distinct compounds. Potential “hit” compounds will be confirmed by dose-response evaluation assays to calculate their EC50. Compounds identified in aim 1 will be further validated in aim 2 using a POMC transactivation assay using full-length and truncated TR4 variant plasmids together with a series of POMC promoter-driven luciferase reporters in murine corticotroph tumor AtT20 cells. A secondary screen will also be performed in human corticotroph tumor primary cultures to confirm their in vitro effects on ACTH secretion. We expect our proposal will identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD.