Identification of Chemical Probes for the Orphan Nuclear Receptor NR2F6

项目摘要 免疫检查点疗法被证明是治疗各种癌症的有效方法。 然而，只有一部分患者表现出持久的反应，这突出了对药物治疗的迫切需要。 确定增强效果的新办法。配体调节的核受体（NR）超家族 转录因子已被证明是用于治疗性干预广泛疾病的靶点的极好来源。 一系列疾病。NR的NR 2F亚家族，COUP-TF 1（NR 2F 1）、COUP-TFII（NR 2F 2）和COUP-TFIII （NR 2F 6）被认为是孤儿受体，因为它们的内源性配体尚未被鉴定。NR2F6 最近作为细胞内T细胞免疫检查点出现; NR 2F 6缺陷小鼠自发排斥 肿瘤和发展宿主保护性免疫记忆。然而，关于NR 2F 6的知之甚少 转录活性，这部分是由于缺乏特异性分子探针， 它的功能。为了鉴定调节NR 2F 6活性的配体，我们设计了高通量的 筛选（HTS）兼容的初步测定，其特异性测量NR 2F 6在 体外我们的目标是利用斯克里普斯机构药物发现图书馆实施一个完整的HTS活动 （SDDL）来鉴定、验证和表征NR 2F 6活性的有效小分子调节剂。实现 我们的目标是将我们的分析小型化到1，536孔板格式中。使用LOPAC和10，000 化合物库HTS筛选我们已经验证了我们的测定，符合HTS自动化标准，并建议 开展“全套”高温超导活动，筛选SDDL中的> 640，000种化合物（目标1）。 “命中”的化学信息学分析将有助于通过结构聚类来识别最有希望的线索， 化合物的生物信息学分析以确定混杂性，以及支架分析以确定 化学合成和可处理性，用于进一步的药物化学工作，以执行结构-活性 关系研究。在目标2中，我们将使用一系列后续检测来验证筛选命中，确定 特异性，并开始理解NR 2F 6介导的转录活性的作用机制。最后， 在目标3中，经过验证的HTS命中将被推进到早期的药物化学中，以进行先导物优化， 新型NR 2F 6分子探针的表征。我们预计，完成这一申请将提供 多种结构上不同的NR 2F 6调节剂，其表现出合适水平的细胞活性、效力， 选择性进一步的药物化学努力将使先导化合物的开发，以确定 对NR 2F 6的效力和特异性，同时使毒性最小化。我们的合作研究团队拥有强大的 进行高通量筛选、支架选择性优化以及体外和体内研究的跟踪记录 化合物的表征。总的来说，我们的筛选方法使我们处于一个独特的位置， 验证和表征新型NR 2F 6选择性小分子，用于研究受体在 疾病的动物模型。

PROJECT SUMMARY Immune checkpoint therapy is proving to be an effective approach for the treatment of a variety of cancers. However, only a subset of patients exhibits long-lasting responses highlighting the critical need for the identification of novel options to augment effects. The nuclear receptor (NR) superfamily of ligand-regulated transcription factors has proven to be an excellent source of targets for therapeutic intervention of a broad range of diseases. The NR2F subfamily of NRs, COUP-TF1 (NR2F1), COUP-TFII (NR2F2), and COUP-TFIII (NR2F6), are considered orphan receptors since their endogenous ligands have yet to be identified. NR2F6 has recently emerged as an intracellular T-cell immune checkpoint; NR2F6-deficient mice spontaneously reject tumors and develop host-protective immunological memory. However, little is known about NR2F6's transcriptional activity, which is due, in part, to lack of specific molecular probes that would enable interrogation of its function. In order to identify ligands that modulate NR2F6 activity, we have designed a high-throughput screening (HTS) compatible primary assay that specifically measures the transcriptional activity of NR2F6 in vitro. Our goal is to implement a full HTS-campaign using the Scripps Institutional Drug Discovery Library (SDDL) to identify, validate, and characterize potent small molecule modulators of NR2F6 activity. To achieve our goal, we have miniaturized our assay into a 1,536-well plate format. Using LOPAC and a 10,000 compound library HTS screen we have validated our assay, meeting HTS automation criteria, and propose to carry out a “full-deck” HTS campaign to screen the >640,000 compounds in the SDDL (Aim 1). Cheminformatic analysis of “hits” will help identify the most promising leads by structural clustering, bioinformatics analysis of compounds to determine promiscuity, and scaffold analysis to determine ease of chemical synthesis and tractability for further medicinal chemistry efforts to perform structure-activity relationship studies. In Aim 2, we will use a cascade of follow up assays to validate screening hits, determine specificity, and begin to understand mechanism of action of NR2F6-mediated transcriptional activity. Finally, in Aim 3, validated HTS hits will be advanced to early medicinal chemistry for lead optimization and characterization of novel NR2F6 molecular probes. We expect that completion of this application will deliver multiple structurally distinct NR2F6 modulators that exhibit suitable levels of cellular activity, potency, and selectivity. Further medicinal chemistry efforts will enable development of lead compounds to determine potency and specificity for NR2F6 while minimizing toxicity. Our collaborative research team has a strong track record of performing high-throughput screens, selective optimization of scaffolds, and in vitro and in vivo characterization of compounds. Collectively, our screening approach puts us in a unique position to identify, validate, and characterize novel, NR2F6-selective small molecules for the study of the receptor's function in animal models of disease.