Identification of TR4 Modulators for Treatment of Cushing Disease.

摘要 库欣病(CD)是由一种分泌促肾上腺皮质激素(ACTH)的垂体瘤引起的 这会导致过量的肾上腺皮质醇。这是一种危及生命的“孤儿疾病”，每年都有令人震惊的 医疗保健费用是普通患者的7倍。手术切除是目前的一线治疗方法，但 这种病经常复发。重复手术、放射治疗和双侧肾上腺切除术并不总是 成功，并与重大发病率相关。目前可用的药物或正在进行临床试验的CD药物不能 靶向垂体促肾上腺皮质细胞肿瘤本身并逃避控制在长期使用中是常见的。一个明显的未满足 存在对提供生化和肿瘤控制的有效和安全的治疗方法的需求。我们假设 直接靶向促肾上腺皮质激素肿瘤来调节ACTH，反过来，糖皮质激素的分泌是最好的方式 治疗CD。我们最近证实了孤儿睾丸受体4(TR4，也称为NR2C2)是一种 下丘脑-垂体-肾上腺(HPA)轴功能的有力调节者，并直接调节前体 阿片黑素皮质素(POMC)基因转录和ACTH分泌。我们假设小分子抑制剂 TR4的作用可能是促肾上腺皮质激素(ACTH)分泌的有效抑制物。这样的发现 将是库欣病的一种变革性疗法，因为目前还没有类似的疗法。识别和刻画 TR4小分子调制器，我们将用我们独特的小分子文库进行大规模筛选 包括TR4-GAL4-LBD系统和POMC-启动子报告在内的一系列反式激活分析 以及在人促肾上腺皮质细胞肿瘤原代培养中的二次筛选。我们的第一个目标是用一种哺乳动物 单杂交GAL4-LBD系统，用于从200,000种不同化合物库中识别TR4调节剂。潜力 “Hit”化合物将通过剂量-反应评估分析来确认，以计算它们的EC50。化合物 在AIM 1中确定的将在AIM 2中使用POMC反式激活实验进一步验证，该实验使用全长和 截短的TR4变异质粒与一系列POMC启动子驱动的荧光素酶报告基因在小鼠体内的表达 促肾上腺皮质激素肿瘤AtT20细胞。对原发人促肾上腺皮质细胞肿瘤也将进行二次筛查 以确定它们在体外对ACTH分泌的影响。我们希望我们的提案将确定并严格执行 验证有效且特定地消除TR4作用的化合物，以抑制ACTH分泌并导致 安全有效的铅TR4抑制化合物的发现可进一步发展为潜力 CD的药物疗法。

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.