A Novel Synthetic Androgen Receptor Antagonist

描述(申请人提供)：目前的激素消融治疗是晚期前列腺癌的主要治疗方法，由于雄激素非依赖性生长的发展，它是姑息性的。雄激素受体(AR)在大多数前列腺癌细胞中表达，AR的过度表达是雄激素非依赖性生长所必需的，这为通过下调AR来开发治疗晚期前列腺癌的新疗法提供了强有力的理论基础。董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-叔基-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one，)，为简单起见，命名为DL3，是一种有效的AR拮抗剂。DL3抑制双氢睾酮(DHT)刺激的所有前列腺癌细胞系的生长和基因表达，包括雄激素非依赖性细胞和对抗雄激素、氟他胺(Flut)或尼鲁特胺(Nilut)耐药的细胞。DL3的抑制作用强于比卡鲁胺(Bical)、氟尿嘧啶和Nilut。它既不抑制AR的核定位，也不抑制DHT诱导的AR NH2末端和COOH末端的相互作用，也没有检测到的AR激动剂活性。DL3降低AR的稳定性，下调AR蛋白的表达。它与双羟色胺竞争与细胞的结合，但不与雌二醇竞争。利用AR配体结合域(LBD)的蛋白质晶体结构进行对接分析表明，DL3可以与LBD结合。这些观察结果促使董博士假设DL3是一种新型的AR拮抗剂，它与AR结合，诱导不活跃的AR转录机制的形成和AR的降解，从而阻断AR信号。提出了三个特定的目标来验证这一假设，并在动物身上研究DL3疗法对人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，并确定DL3结合部位的氨基酸残基。他将确定结合的生化性质，调查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在蛋白质体介导AR降解和AR转录复合体组装中的作用。他将确定DL3和Bical对AR稳定性、泛素化以及与E3泛素连接酶结合的影响。通过使用染色质免疫沉淀(ChIP)分析，他将调查DL3和Bical治疗是否会在前列腺特异性抗原基因的启动子区域诱导转录不活跃的AR复合体的形成，并确定该复合体中的辅助因素。在具体目标3中，董博士建议研究DL3对小鼠前列腺癌细胞的治疗作用。他将确定和比较DL3和Bical单独或与去势联合使用对雄激素依赖和非依赖细胞以及对氟尿嘧啶耐药的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AR和AR靶基因在肿瘤中的表达相关联。这些研究将确定DL3是一种新的AR拮抗剂，并将丰富对Bical阻断AR信号的机制的理解。项目简介我们的初步研究已经确定了一种新颖的合成化合物6-氨基-2-[2-(4-叔基-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one，)，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，但不抑制雌二醇与人乳腺癌细胞的结合。它下调雄激素受体的表达，抑制雄激素诱导的细胞生长和前列腺癌细胞中的基因表达。这项拟议的研究将确定DL3是一种新的AR拮抗剂，将阐明DL3阻断AR信号的机制，并将确定DL3对小鼠人前列腺癌细胞的治疗效果。与公共健康相关：我们的初步研究已经确定了一种新的合成化合物6-amino-2-[2-(4-tertbutyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one，，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，但不抑制雌二醇与人乳腺癌细胞的结合。它下调雄激素受体的表达，抑制雄激素诱导的细胞生长和前列腺癌细胞中的基因表达。这项拟议的研究将确定DL3是一种新的AR拮抗剂，将阐明DL3阻断AR信号的机制，并将确定DL3对小鼠人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.