Identification of Chemical Probes for the Orphan Nuclear Receptor NR2F6

项目总结 事实证明，免疫检查点疗法是治疗各种癌症的一种有效方法。 然而，只有一小部分患者表现出长期的反应，这突显了 确定新的选项以增强效果。配体调节的核受体超家族 转录因子已被证明是广泛的治疗干预的良好靶点来源。 一系列疾病。NRS、COUP-TF1(NR2F1)、COUP-TFII(NR2F2)和COUP-TFIII的NR2F子家族 (NR2F6)被认为是孤儿受体，因为它们的内源性配体尚未确定。NR2F6 最近作为细胞内T细胞免疫检查点出现；NR2F6缺陷小鼠自发排斥 肿瘤和发展宿主保护性免疫记忆。然而，人们对NR2F6的S知之甚少 转录活性，部分原因是缺乏能够进行讯问的特定分子探针 它的功能。为了确定调节NR2F6活性的配体，我们设计了一种高通量 特异性检测NR2F6转录活性的(HTS)兼容初筛试验 体外培养。我们的目标是利用斯克里普斯机构药物发现库实施全面的HTS活动 (SDDL)以识别、验证和表征有效的NR2F6活性小分子调节剂。要实现 我们的目标是，我们已经将我们的检测微型化为1,536孔板格式。使用Lopac和10,000 化合物文库HTS筛选我们已经验证了我们的方法，符合HTS自动化标准，并建议 开展“全甲板”高温超导活动，筛选SDDL中的640,000个化合物(目标1)。 “HITS”的化学信息学分析将通过结构聚类来帮助识别最有希望的线索， 对化合物进行生物信息学分析以确定混杂情况，并进行支架分析以确定 化学合成和易操纵性为进一步的药物化学工作执行结构活性 关系研究。在目标2中，我们将使用一系列后续分析来验证筛查命中率，确定 特异性，并开始了解NR2F6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 HITS将被推进到早期药物化学，以进行先导优化和 新型NR2F6分子探针的表征。我们希望这项申请的完成将交付 多种结构不同的NR2F6调节剂，表现出适当水平的细胞活性、效力和 选择性。进一步的药物化学努力将使先导化合物的开发能够确定 NR2F6的效力和特异性，同时将毒性降至最低。我们的合作研究团队拥有强大的 在高通量筛选、支架的选择性优化以及体外和体内试验方面的记录 化合物的表征。总体而言，我们的筛选方法使我们处于独特的地位来识别， 验证和表征新型的NR2F6选择性小分子，用于研究受体的功能 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.