Efficacy and Mechanism of Novel Androgen Receptor Inhibitors

描述(申请人提供)：雄激素受体(AR)是前列腺癌(PCa)的最佳分子靶点。新的抗雄激素可能在治疗原发和复发的前列腺癌方面都有用。这样的抗雄激素不一定是竞争性拮抗剂，可以想象的是可以阻断AR信号中的下游事件。我们已经使用细胞筛选来鉴定两种新的、有效的抗雄激素：一种是FDA批准的药物，另一种是天然产品。这些化合物作为非竞争性拮抗剂发挥作用，并与经典的抗雄激素如羟基氟他胺或比卡鲁胺协同作用。初步证据表明，一种化合物在体内是有效的，特别是与Bic联合使用。这笔赠款的长期目标是开发一种更好的前列腺癌治疗方法。 目的1：小鼠的药效研究。我们将通过IP注射治疗动物，测试候选化合物与比卡鲁胺单独或联合使用，并以去势为阳性对照。我们将测量对前列腺重量和形态的影响，并将使用RT-PCR来确定它可以在多大程度上下调前列腺癌中AR依赖基因的表达。如果成功，我们将确定比卡鲁胺与该化合物的最佳比例，以实现最大的反应。这些研究将为将这种化合物转化为临床治疗人类激素难治性前列腺癌奠定基础。 目的2：确定先导化合物的作用机制。这两种已鉴定的化合物在细胞系统中都具有显著的效力。确定它们的分子作用机制将有助于阐明控制AR活性的生物学机制，并可能导致新的治疗靶点。我们将利用类固醇受体生物学的广泛知识来表征化合物抑制AR活性的步骤。我们将使用基于FRET的分析来确定对分子内和分子间构象变化的影响。我们将通过细胞分离和蛋白质印迹来测试对AR核定位的影响。由于这些化合物特异性地抑制AR，而不是密切相关的糖皮质激素受体，我们将使用结构域转座来测试AR的哪些区域介导其对抑制的敏感性。这将有助于识别与这些区域相互作用的因素，以及可能受化合物调节的因素。我们将使用定量RT-PCR和染色质免疫沉淀(CHIP)来测量测试化合物对基因调控和启动子占有率的影响，以确定它们是整体影响AR转录，还是仅影响特定启动子亚群。这些方法将明确AR抑制的精确机制，有助于识别细胞内靶点，并将扩大我们对AR生物学的知识，促进新的前列腺癌治疗策略。

DESCRIPTION (provided by applicant): The androgen receptor (AR) is the single best molecular target for prostate cancer (PCa). Novel anti-androgens will likely be useful in treating both primary and recurrent PCa. Such anti-androgens need not be competitive antagonists, and could conceivably block downstream events in AR signaling. We have used cellular screens to identify two novel, potent anti-androgens: one is an FDA-approved drug, and the other a natural product. These compounds function as non-competitive antagonists, and synergize with classical anti-androgens such as hydroxy-flutamide or bicalutamide. Preliminary evidence suggests one compound is effective in vivo, especially in combination with BiC. The long term goals of this grant are to develop a better therapy for PCa. Aim 1: Efficacy studies in mice. We will treat animals for via IP injection, testing the candidate compound with bicalutamide alone or in combination, with castration as a positive control. We will measure effects on prostate weight and morphology, and will use RT-PCR to determine the degree to which it can down-regulate AR-dependent gene expression in the prostate gland. If successful, we will then establish an optimal ratio of the compound with bicalutamide to achieve maximal responses. These studies will underlie translation of this compound to the clinic for the treatment of hormone-refractory PCa in humans. Aim 2: Determine the mechanism of action of lead compounds. Both compounds identified have marked potency in cellular systems. Determination of their molecular mechanism of action will help elucidate biological mechanisms that control AR activity, and may lead to new therapeutic targets. We will use the extensive knowledge of steroid receptor biology to characterize at which step compounds inhibit AR activity. We will use FRET-based assays to determine effects on intramolecular and intermolecular conformational change. We will test for effects on AR nuclear localization via cell fractionation and western blot. Since the compounds specifically inhibit AR, but not the closely related glucocorticoid receptor, we will use domain transposition to test which regions of AR mediate its sensitivity to inhibition. This will facilitate identification of factors that interact with these regions, and which may be modulated by the compounds. We will measure effects of test compounds on gene regulation and promoter occupancy using quantitative RT-PCR and chromatin immunoprecipitation (ChIP) to determine whether they affect AR transcription globally, or only at specific subsets of promoters. These approaches will pinpoint the precise mechanisms of AR inhibition, will facilitate identification of intracellular targets, and will expand our knowledge of AR biology, facilitating new treatment strategies for PCa.