Aryl Hydrocarbon Receptor Modulators for the Treatment of Hepatocellular Carcinom

描述（由申请人提供）：芳烃受体（AhR）是一种配体激活的转录因子。在与配体结合后，AhR从细胞质转移到细胞核，在那里它介导广泛的基因靶标的转录。除了AhR作为各种二恶英毒性介质的作用外，最近的研究已经确定了AhR在细胞凋亡，细胞周期调节和肿瘤抑制中的作用，所有这些都使AhR成为开发新型癌症治疗药物的潜在有吸引力的分子靶点。我们假设，某些选择性AhR调节剂（SAhRMs）能够启动对预防和/或治疗肝细胞癌有用的生物反应。我们建议通过基于硅结构的虚拟配体筛选（VLS）结合基于细胞的AhR转录激活试验来鉴定SAhRMs。将进一步筛选SAhRMs诱导AhR依赖性细胞死亡和/或抑制癌细胞增殖的能力。在我们的实验室中，许多具有良好特征的表达AhR和不表达AhR的细胞培养模型的可用性将使我们能够评估AhR激活的功能后果和sAhRMs在肝癌细胞中的抗癌作用。在此应用程序中提出的具体目标如下所述。1. 鉴定具有治疗癌症潜力的新型AhR调节剂我们将使用基于硅结构的VLS来鉴定新的AhR替代配体。VLS利用蛋白质的三维结构或同源模型来识别目标蛋白质的潜在结合物。AhR的per - art - sim (PAS)-B结构域对配体结合至关重要。由于AhR配体结合袋尚未结晶，我们基于AhR与已有的PAS蛋白家族成员的同源性，生成并完善了小鼠和人类AhR的结构模型。这些AhR的同源性模型将用于VLS筛选合成和天然化合物数据库，以识别AhR的潜在结合物。新发现的化合物对AhR激活的影响将通过AhR依赖的报告基因测定和评估AhR依赖的内源性靶基因诱导来测试。利用上述AhR PAS结构域同源性模型，VLS对化合物文库进行了初步筛选，已经鉴定出一些感兴趣的化合物，包括临床使用的抗雄激素氟他胺，它可以促进AhR的转录激活，诱导AhR调节的靶基因，并抑制AhR依赖性的癌细胞生长。这些结果有力地支持了本探索性研究的可行性。2. 测试VLS鉴定的化合物对AhR的选择性和潜在的抗癌作用VLS鉴定的化合物将测试其直接与AhR结合的能力。能够与AhR结合的分子将在肝癌细胞中测试其对AhR DNA结合和诱导AhR特异性转录活性的影响。AhR配体在肝癌细胞中以AhR依赖的方式诱导细胞死亡和/或抗增殖作用，将优先进行进一步评估。3. 我们将比较SAhRMs诱导的基因表达，以区分它们与经典AhR配体（如TCDD）诱导的转录程序，使用人类和小鼠微阵列。表达谱与TCDD显著不同的化合物，如我们最初的先导化合物（氟他胺和C-24），通过AhR诱导生长抑制/细胞凋亡，将优先用于未来的体内研究。基因表达谱与TCDD高度相似的SAhRMs将不会被用作抗癌药物。在通过一个有趣的分子靶点AhR开发预防和/或治疗肝癌的治疗干预的背景下，提出的研究是及时和重要的。令人兴奋的是，这些研究将确定的sahrm也可能对其他癌症有效。我们将在未来的小鼠异种移植肿瘤研究中评估一些入围的SAhRMs对ahr依赖性肿瘤生长的抑制作用。

DESCRIPTION (provided by applicant): The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcription factor. Upon binding to a ligand, the AhR translocates from the cytosol to the nucleus, where it mediates the transcription of a wide range of gene targets. Aside from the AhR's role as the mediator of toxicity of various dioxins, recent studies have identified roles for the AhR in apoptosis, cell cycle regulation, and tumor suppression, all of which make the AhR a potentially attractive molecular target for development of novel cancer therapeutics. We hypothesize that certain selective AhR modulators (SAhRMs) are capable of initiating biological responses that should prove useful for prevention and/or treatment of hepatocellular carcinoma. We propose to identify SAhRMs by in silico structure-based Virtual Ligand Screening (VLS) coupled with cell- based AhR transcriptional activation assays. SAhRMs will be screened further for their ability to induce AhR- dependent cell death and/or inhibition of cancer cell proliferation. The availability of numerous well- characterized AhR expressing and non-expressing cell culture models in our lab will enable us to evaluate the functional consequences of AhR activation and the anticancer effects of sAhRMs in hepatoma cells. The Specific Aims proposed in this application are described below. 1. Identify novel AhR modulators that have the potential for treatment of cancer We will use in silico structure-based VLS for identifying novel alternative ligands of the AhR. VLS utilizes a three dimensional structure of a protein or homology model to identify potential binders of a target protein. The Per-Arnt-Sim (PAS)-B domain of AhR is essential for ligand binding. As the AhR ligand binding pocket is not yet crystallized, we have generated and refined mouse and human AhR structural models based on the AhR's homology with existing PAS protein family members whose three-dimensional structures have been solved. These homology models of the AhR will be used to screen both synthetic and natural compound databases by VLS to identify potential binders of the AhR. The effects of the newly identified compounds on AhR activation will be tested by AhR-dependent reporter assays and evaluation of AhR- dependent induction of endogenous target genes. Preliminary screening of a compound library by VLS using the AhR PAS domain homology model described above has already resulted in the identification of some compounds of interest including the clinically used antiandrogen flutamide that promoted transcriptional activation of AhR, induction of AhR- regulated target genes and AhR-dependent inhibition of cancer cell growth. These results strongly support of the feasibility of this exploratory research. 2. Test the compounds identified by VLS for AhR selectivity and potential anticancer effects The compounds identified by VLS will be tested for their ability to directly bind to the AhR. Molecules that are able to bind to the AhR will be tested for their effects on AhR DNA binding and inducing AhR-specific transcriptional activity in liver cancer cells. AhR ligands that induce cell death and/or antiproliferative effects in an AhR-dependent manner in liver cancer cells will be prioritized for further evaluation. 3. Shortlist compounds for future in vivo testing by gene expression profiling We will compare the gene expression induced by SAhRMs to distinguish them from the transcriptional program induced by the classical AhR ligands such as TCDD using human and mouse microarrays. Compounds with expression profiles that differ significantly from that of TCDD, such as our initial lead compounds (flutamide and C-24), and that induce growth inhibition/apoptosis via AhR will be given priority for future in vivo studies. SAhRMs with gene expression profiles highly similar to that of TCDD will not be pursued as anti-cancer agents. The proposed studies are timely and important in the context of developing therapeutic intervention for the prevention and/or treatment of hepatoma via an intriguing molecular target, AhR. Excitingly, the SAhRMs that will be identified by these studies may also be effective against other cancers. We will evaluate some of short-listed SAhRMs for AhR-dependent inhibition of tumor growth in mouse xenografts tumor studies in the future.