Identification of REV-ERB inverse agonists for cancer immunotherapy

项目总结 虽然免疫疗法是我们抗击癌症的一项突破，但只有一小部分患者表现强劲 以及持久的反应，突出了单独使用或结合使用新方法的迫切需要 目前的治疗方案。Th17细胞，表达定义转录因子RORgt的谱系， 已成为增强抗肿瘤免疫的靶点，RORg激动剂目前处于1/2期临床 审判。转录抑制因子Rev-Erba和Rev-ErbB经常在同一组织中共表达 作为RORg(T)，并结合相同的DNA反应元件，这表明相互串扰和共同调节 它们的目标基因。我们的初步数据表明，REV-ERB是受配体调控的转录 因子并在TH17反应中发挥关键作用，包括通过竞争调节IL-17A的表达 与RORgt在其共有的DNA共识序列(S)，以及RORgt的表达本身的调控。我们有 产生增强REV-ERBS抑制功能并抑制TH17细胞的小分子配体 体外和体内发育。因此，我们假设抑制REV-ERB的配体的识别 抑制活性将增强TH17反应和抗肿瘤免疫。为了识别抑制的配体 REV-ERB活性，我们设计了一种与高通量筛选(HTS)兼容的初级检测方法，可以检测到 直接与每个受体结合的配基。我们的目标是使用Scripps实现完整的HTS-Campaign 机构药物发现文库(SDDL)，用于识别、验证和表征有效的小分子反义药物 REV-ERB活性激动剂。为了实现我们的目标，我们将把我们的测试微型化成1,536孔板的格式 在满足HTS自动化标准后，开展一项全甲板HTS活动，以筛选&gt；640,000 SDDL中的化合物(目标1)。“Hits”的化学信息学分析将有助于识别最有希望的线索 通过结构聚类、生物信息学分析确定化合物的混杂，并通过支架分析来确定 确定化学合成的简易性和易处理性，以便进一步进行药物化学工作，以执行结构- 活动关系研究。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REV-ERB介导的转录作用机制 活动。最后，在目标3中，验证的HTS HITS将被推进到早期药物化学以进行领先优化 以及对REV-Erba/b的新型负调控因子的表征。我们预计该申请的完成 将提供多种结构不同的REV-ERB反向激动剂，这些激动剂表现出适当水平的细胞活性， 效力和选择性。我们的合作研究团队在执行高吞吐量方面有着良好的记录 筛选、支架的选择性优化以及化合物的体外和体内表征。总而言之， 我们的筛选方法使我们处于识别、验证和表征新的REV-ERB的独特位置- 选择性小分子反向激动剂研究REV-ERB在增强TH17反应中的作用 并确定以这些受体为靶点是否是免疫治疗的可行选择。

PROJECT SUMMARY While immunotherapy is a breakthrough in our fight against cancer, only a subset of patients display robust and long-lasting responses highlighting the critical need for novel approaches to be used alone or in combination with current therapeutic regimens. TH17 cells, which express the lineage defining transcription factor RORgt, have emerged as targets to enhance antitumor immunity, with RORg agonists currently in Phase 1/2 clinical trials. The transcriptional repressors REV-ERBa and REV-ERBb are often co-expressed in the same tissues as RORg(t) and bind the same DNA response elements, which suggests mutual cross talk and co-regulation of their target genes. Our preliminary data demonstrates that the REV-ERBs are ligand-regulated transcription factors and play critical roles in TH17 responses, including regulation of IL-17A expression through competition with RORgt at their shared DNA consensus sequence(s), and regulation of RORgt expression itself. We have generated small molecule ligands that enhance the REV-ERBs repressive function and inhibit TH17 cell development in vitro and in vivo. Thus, we hypothesize that identification of ligands that inhibit the REV-ERBs repressive activity will enhance TH17 responses and antitumor immunity. In order to identify ligands that inhibit REV-ERB activity, we have designed a high-throughput screening (HTS) compatible primary assay that detects direct ligand binding to each receptor. 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 inverse agonists of REV-ERB activity. To achieve our goal, we will miniaturize our assay into a 1,536-well plate format and upon meeting HTS automation criteria, 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 REV-ERB-mediated transcriptional activity. Finally, in Aim 3, validated HTS hits will be advanced to early medicinal chemistry for lead optimization and characterization of novel negative regulators of REV-ERBa/b. We expect that completion of this application will deliver multiple structurally distinct REV-ERB inverse agonists that exhibit suitable levels of cellular activity, potency, and selectivity. 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 REV-ERB- selective small molecule inverse agonists for the study of the REV-ERB’s function in enhancing TH17 responses and to determine whether targeting these receptors is a viable option for immunotherapy.