Androgen Receptor-JunD Complex Inhibitors to Prevent Prostate Cancer Progression

描述（由申请人提供）：大多数常规癌症疗法对CRPCa仅适度有效。因此，有必要开发新的有效疗法来治疗早期前列腺癌（PCa），以防止PCa进展为CRPCa。我们的合作者Wilding博士和他的同事对PCa进展的可能机制的基础科学研究导致了一类天然癌症致病因子，细胞活性氧（ROS），如超氧化物，羟基自由基，过氧化氢等。他们发表的强有力的初步数据显示，在人类PCa细胞中，过量的细胞ROS激活转录因子NF-κ B，其在雄激素不存在的情况下防止凋亡并驱动PCa细胞增殖。提出PCa特异性ROS产生途径的靶向抑制剂在预防PCa复发和进展为CRPCa方面将比化学和/或膳食抗氧化剂更有效。我们发表的数据显示，雄激素诱导亚精胺/精胺乙酰转移酶（SSAT），启动亚精胺和精胺氧化途径，特别是在天然富含精胺和亚精胺的PCa细胞中产生大量的ROS。由于JunD蛋白表达在前列腺癌细胞中由雄激素特异性诱导，因此假设JunD与活化的雄激素受体（AR）复合以诱导前列腺癌细胞中SSAT基因表达和ROS产生。我们和其他人还表明NF-κ B可以诱导SSAT表达，从而为SSAT活化、ROS产生和PCa进展建立了一个前馈回路。因此，提出JunD-AR复合物的特异性抑制剂应该阻断SSAT诱导、ROS产生、NF-κ B活化并停止帮助CRPCa进展的自分泌前馈环。到目前为止，使用Gaussia荧光素酶重建试验进行原位蛋白质-蛋白质相互作用的高通量筛选，鉴定了七种可以阻断JunD-AR相互作用的分子。这些化合物中的两种在雄激素依赖性PCa细胞中阻断雄激素诱导的ROS产生，并在亚微摩尔至低微摩尔水平下显示出对雄激素依赖性和雄激素非依赖性人PCa细胞的生长抑制作用。使用计算机辅助分子对接来确定抑制剂的结合位点，我们建议设计两种先导化合物的类似物，它们应该更有效地阻断雄激素诱导的ROS生成和细胞生长。我们的具体目标是：1）使用计算机对接开发小分子与JunD-AR复合物相互作用的模型，并设计和合成JunD-AR相互作用的小分子抑制剂的类似物; 2）根据它们破坏JunD-AR相互作用的相对能力和它们减少细胞内ROS和抑制培养的人PCa细胞生长的功效来鉴定有希望的试剂; 3)鉴定目标2研究中的先导候选药物，并检测其在裸鼠异种移植物中抑制人前列腺癌生长的能力，以及在自发性前列腺癌发生的转基因小鼠模型中的抑制能力。 公共卫生相关性：晚期激素难治性转移性前列腺癌（CRPCa）是美国男性癌症死亡的第二大原因。大多数常规癌症疗法对CRPCa仅适度有效。因此，有必要开发新的有效疗法来预防前列腺癌（PCa）复发和/或进展为CRPCa。我们的合作者Wilding博士和他的同事对PCa复发和进展的可能机制的研究导致了一类天然的癌症致病因子，细胞活性氧（ROS）。PCa细胞中的ROS水平通常高于其正常对应物。雄性激素睾酮在前列腺癌细胞中特异性地诱导大量的ROS产生。最近的研究表明，前列腺ROS的主要来源是精胺和亚精胺的氧化，在前列腺中发现非常高的水平。也已经证明，激活的雄激素受体与蛋白质JunD结合以诱导精胺/亚精胺氧化。因此，提出JunD-AR复合物的特异性抑制剂应抑制精胺/亚精胺氧化，并因此应阻断PCa细胞中的ROS产生并防止CRPCa增殖。我们使用高通量筛选来鉴定JunD-AR相互作用的抑制剂，并鉴定了几种可以抑制JunD-AR相互作用的化合物。这些化合物之一阻断了雄激素依赖性前列腺癌细胞中雄激素诱导的ROS生成，并显示出对雄激素依赖性和雄激素非依赖性人前列腺癌细胞的生长抑制作用。科尔比已经为这些化合物申请了临床前和临床开发专利，用于治疗早期进展性前列腺癌患者。使用计算机辅助分子设计来确定抑制剂的结合位点，在这里，我们建议开发先导化合物的新类似物，其应该更有效地阻断雄激素诱导的ROS生成和细胞生长。由此鉴定的最具活性的药剂可以进一步开发用于临床前和临床测试，以防止疾病的进展。 早期雄激素依赖性PCa转化为CRPCa。

DESCRIPTION (provided by applicant): Most conventional cancer therapies are only modestly effective against CRPCa. Therefore, the development of new and effective therapies to treat early-stage prostate cancer (PCa) to prevent progression of PCa to CRPCa is warranted. Our collaborator Dr. Wilding and his coworkers' basic science investigation of probable mechanism(s) of PCa progression led to a class of natural cancer causative agents, cellular reactive oxygen species (ROS) such as superoxide, hydroxyl radical, hydrogen peroxide, etc. Their published and strong preliminary data show that in human PCa cells, excess cellular ROS activate the transcription factor NF-kappa B that prevents apoptosis and drives PCa cell proliferation in the absence of androgen. It is proposed that targeted inhibitors o PCa specific ROS generation pathway(s) will be more effective than are chemical and/or dietary anti-oxidants in preventing PCa recurrence and progression to CRPCa. Our published data show androgen induces spermidine/spermine acetyl transferase (SSAT) that initiates a spermidine and spermine oxidation pathway to generate copious amounts of ROS specifically in PCa cells that are naturally rich in spermine and spermidine. As JunD protein expression is induced by androgen specifically in PCa cells, it is hypothesized that JunD complexes with the activated androgen receptor (AR) to induce SSAT gene expression and ROS production in PCa cells. We and others have also shown that NF-kappa B can induce SSAT expression, thus setting up a feed-forward loop for SSAT activation, ROS production and PCa progression. Therefore, it is proposed that specific inhibitor(s) of JunD-AR complex should block SSAT induction, ROS production, NF-kappa B activation and stop the autocrine feed-forward loop that helps CRPCa progression. A high-throughput screen using Gaussia luciferase enzyme reconstitution assay for in situ protein-protein interaction, thus far, identified seven inhibitorsthat can block JunD-AR interaction. Two of these compounds blocked androgen-induced ROS generation in androgen-dependent PCa cells and showed growth inhibitory effects against both androgen- dependent and androgen-independent human PCa cells at sub-micromolar to low micromolar levels. Using computer-aided molecular docking to determine the binding site(s) of the inhibitor, we propose to design analogs of the two lead compounds that should more efficiently block androgen-induced ROS generation and cell growth. Our Specific Aims are: 1) To develop models for small molecule interaction with JunD- AR complex using in silico docking and design and synthesize analogs of small molecule inhibitors of JunD- AR interaction; 2) To identify promising agents from their relative abilities to disrupt JunD-AR interaction and their efficacies in reducing intracellular ROS and inhibit cultured human PCa cell growth.; 3) To identify the lead drug candidate from the studies in Aim 2 and test its ability to inhibit human PCa growth in nude mouse xenografts and the same in a transgenic mouse model developing spontaneous PCa. PUBLIC HEALTH RELEVANCE: Advanced hormone refractory metastatic prostate cancer (CRPCa) is the second leading cause of cancer deaths among US men. Most conventional cancer therapies are only modestly effective against CRPCa. Therefore, the development of new and effective therapies to prevent prostate cancer (PCa) recurrence and/or progression to CRPCa is warranted. Our collaborator Dr. Wilding and his coworkers' investigation of probable mechanism(s) of PCa recurrence and progression led to a class of natural cancer causative agents, cellular reactive oxygen species (ROS). ROS levels are generally high in PCa cells as compared to their normal counterpart. Male hormone testosterone induces copious amounts of ROS generation specifically in PCa cells. Recent discoveries show that the main source of the prostatic ROS is oxidation of spermine and spermidine that is found in very high level in the prostate. It has also been demonstrated that activated androgen receptor binds with a protein JunD to induce the spermine/spermidine oxidation. Therefore, it is proposed that specific inhibitor(s) of JunD-AR complex should inhibit spermine/spermidine oxidation and should thus block ROS production in PCa cells and prevent CRPCa proliferation. We used a high throughput screen to identify inhibitors of JunD-AR interaction and identified several compounds that can inhibit JunD-AR interaction. One of these compounds blocked androgen-induced ROS generation in androgen-dependent PCa cells and showed growth inhibitory effects against both androgen-dependent and androgen-independent human PCa cells. Colby has patented these compounds for preclinical and clinical development for treatment of early-stage progressing PCa patients. Using computer-aided molecular design to determine the binding site(s) of the inhibitor, here, we propose to develop new analogs of the lead compound that should more efficiently block androgen-induced ROS generation and cell growth. The most active agent, thus identified, can be further developed for preclinical and clinical testing to prevent progression of early-stage androgen-dependent PCa to CRPCa.