A Novel Synthetic Androgen Receptor Antagonist

描述（由申请人提供）：由于雄激素非依赖性生长的发展，目前的激素消融治疗是晚期前列腺癌的主要治疗方法，是姑息性的。雄激素受体（AR）在大多数前列腺癌细胞中表达，AR的过表达对于雄激素非依赖性生长是充分和必要的，这为通过下调AR开发针对晚期前列腺癌的新疗法提供了强有力的理论依据。Dong博士的实验室已经确定了一种新的合成化合物6-氨基-2-[2-(4-叔丁基- penoxy)-乙基磺胺]- h -嘧啶-4- 1，简单命名为DL3，作为一种有效的AR拮抗剂。DL3在所有测试的前列腺癌细胞系中抑制双氢睾酮（DHT）刺激的细胞生长和基因表达，包括雄激素非依赖性细胞和抗雄激素、氟他胺（Flut）或尼鲁他胺（Nilut）的细胞。DL3的抑制作用比比卡鲁胺（biical）、Flut和Nilut更有效。它既不抑制AR核定位，也不抑制dht诱导的AR nh2端和cooh端相互作用，没有可检测到的AR激动剂活性。DL3降低AR稳定性，下调AR蛋白表达。它与二氢睾酮竞争而不是与雌二醇竞争与细胞的结合。利用AR配体结合域（LBD）的蛋白晶体结构进行对接分析，发现DL3可以与LBD结合。这些观察结果促使Dong博士假设DL3是一种新的AR拮抗剂，它与AR结合并诱导非活性AR转录机制的形成和AR降解，因此，阻断AR信号传导。我们提出了三个具体目标来验证这一假设，并在动物实验中研究DL3治疗人类前列腺癌细胞的疗效。在具体目标1中，他将表征DL3与AR的结合，并鉴定DL3结合位点的氨基酸残基。他将确定结合的生化特性，研究DL3是否与DHT竞争AR结合，并确定AR与DL3相互作用的氨基酸残基。在具体目标2中，他将研究DL3对蛋白体介导的AR降解和AR转录复合物组装的影响。他将确定DL3和biical对AR稳定性、泛素化以及与E3泛素连接酶的关联的影响。通过染色质免疫沉淀（ChIP）试验，他将研究DL3和biical治疗是否诱导在前列腺特异性抗原基因启动子区域形成转录无活性AR复合物，并鉴定该复合物中的辅助因子。在具体目标3中，董博士提出研究DL3对小鼠人前列腺癌细胞的治疗作用。他将确定并比较DL3和biical单独使用或与去势联合使用对雄激素依赖性细胞和非依赖性细胞以及Flut难治性细胞形成的肿瘤生长的影响。他将确定DL3在荷瘤小鼠中的分布，并将治疗效果与肿瘤中AR和AR靶基因的表达联系起来。这些研究将坚定地确立DL3是一种新型的AR拮抗剂，并将丰富对bic阻断AR信号传导机制的理解。我们的初步研究鉴定了一种新的合成化合物6-氨基-2-[2-（4-叔丁基-戊氧基）-乙基磺酰]- 1h -嘧啶-4- 1，为简单起见命名为DL3。DL3竞争性地抑制DHT与人前列腺癌细胞的结合，而雌二醇对人乳腺癌细胞没有抑制作用。下调雄激素受体表达，抑制雄激素诱导的前列腺癌细胞生长和基因表达。该研究将证实DL3是一种新型的AR拮抗剂，阐明DL3阻断AR信号传导的机制，并确定DL3对小鼠前列腺癌细胞的治疗效果。公共卫生相关性：我们的初步研究鉴定了一种新的合成化合物6-氨基-2-[2-（4-叔丁基-戊氧基）-乙基磺酰]- 1h -嘧啶-4- 1，为简单起见命名为DL3。DL3竞争性地抑制DHT与人前列腺癌细胞的结合，而雌二醇对人乳腺癌细胞没有抑制作用。下调雄激素受体表达，抑制雄激素诱导的前列腺癌细胞生长和基因表达。该研究将证实DL3是一种新型的AR拮抗剂，阐明DL3阻断AR信号传导的机制，并确定DL3对小鼠前列腺癌细胞的治疗效果。

DESCRIPTION (provided by applicant): Current hormonal ablation therapy, the mainstay treatment for advance prostate cancer, is palliative, due the development of androgen-independent growth. Androgen receptor (AR) is expressed in most prostate cancer cells and AR overexpression of AR is sufficient and necessary for androgen-independent growth, which provides strong rationale for developing novel therapies against advanced prostate cancer through downregulation of AR. Dr. Dong's laboratory has identified a novel synthetic compound 6-amino-2-[2-(4-tert- butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one, named DL3 for simplicity, as a potent AR antagonist. DL3 inhibits dihydrotestosterone (DHT)-stimulated cell growth and gene expression in all prostate cancer cell lines tested, including androgen-independent cells and cells resistant to antiandrogens, flutamide (Flut) or nilutamide (Nilut). The inhibitory effects of DL3 are more potent than bicalutamide (Bical), Flut, and Nilut. It inhibits neither AR nuclear localization nor DHT-induced AR NH2-terminus and COOH-terminus interaction and has no detectable AR agonist activity. DL3 reduces AR stability and downregulates of AR protein expression. It competes with DHT but not estradiol for the binding to cells. Docking analysis using protein crystal structure of AR ligand-binding domain (LBD) implies that DL3 can bind to the LBD. These observations prompted Dr. Dong to hypothesize that DL3 is a novel AR antagonist that binds to AR and induces the formation of inactive AR transcription machinery and AR degradation, and, hence, interrupts AR signaling. Three specific aims are proposed to test the hypothesis and to investigate efficacy of DL3 therapy against human prostate cancer cells in animals. In the specific aim 1, he will characterize the binding of DL3 to AR and identify amino acid residues of the DL3 binding site. He will determine the biochemical properties of the binding, investigate whether DL3 competes with DHT for AR binding, and identify amino acid residues of AR that interact with DL3. In the specific aim 2, he will investigate effects of DL3 on proteosome-mediated degradation of AR and assembly of AR transcription complex. He will determine effects of DL3 and Bical on AR stability, ubiquitination, and association with E3 ubiquitin ligase. By using the chromatin immunoprecipitation (ChIP) assay, he will investigate whether treatment with DL3 and Bical induce formation of transcription-inactive AR complex at the promoter region of prostate-specific antigen gene and identify cofactors in the complex. In the specific aim 3, Dr. Dong propose to investigate therapeutic effects of DL3 against human prostate cancer cells in mice. He will determine and compare effects of DL3 and Bical, alone or in combination with castration, on growth of tumors formed by both androgen-dependent and -independent cells and by cells refractory to Flut. He will determine DL3 distribution in tumor-bearing mice and correlate therapeutic effects with expression of AR and AR-target genes in tumors. These studies will firmly establish that DL3 is a novel AR antagonist and will enrich the understanding of mechanisms by which Bical interrupts AR signaling. Project Narrative Our preliminary studies have identified a novel synthetic compound 6-amino-2-[2-(4-tert- butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one, named DL3 for simplicity. DL3 competitively inhibits the binding of DHT to human prostate cancer cells, but not estradiol to human breast cancer cells. It downregulates androgen receptor expression and suppresses androgen-induced cell growth and gene expression in prostate cancer cells. The proposed research will establish that DL3 is a novel AR antagonist, will elucidate mechanisms by which DL3 interrupts AR signaling, and will determine therapeutic effects of DL3 against human prostate cancer cells in mice. PUBLIC HEALTH RELEVANCE: Our preliminary studies have identified a novel synthetic compound 6-amino-2-[2-(4-tertbutyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one, named DL3 for simplicity. DL3 competitively inhibits the binding of DHT to human prostate cancer cells, but not estradiol to human breast cancer cells. It downregulates androgen receptor expression and suppresses androgen-induced cell growth and gene expression in prostate cancer cells. The proposed research will establish that DL3 is a novel AR antagonist, will elucidate mechanisms by which DL3 interrupts AR signaling, and will determine therapeutic effects of DL3 against human prostate cancer cells in mice.