Identification of REV-ERB inverse agonists for cancer immunotherapy

项目摘要 虽然免疫疗法是我们对抗癌症的一个突破，但只有一部分患者表现出强大的免疫功能。 和持久的反应，强调迫切需要单独或联合使用新的方法 目前的治疗方案。表达谱系限定转录因子RORgt的TH 17细胞， 已经成为增强抗肿瘤免疫的靶点，RORg激动剂目前处于1/2期临床试验中。 审判转录抑制因子REV-ERBa和REV-ERBb通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 他们的目标基因。我们的初步数据表明，REV-ERBs是配体调控的转录 因子，并在TH 17应答中发挥关键作用，包括通过竞争调节IL-17 A表达 与RORgt在它们共有的DNA共有序列处的相互作用，以及RORgt表达本身的调节。我们有 产生的小分子配体增强REV-ERBs的抑制功能并抑制TH 17细胞 体外和体内发育。因此，我们假设，抑制REV-ERBs的配体的鉴定 抑制活性将增强TH 17应答和抗肿瘤免疫。为了鉴定出能够抑制 REV-ERB活性，我们设计了一种高通量筛选（HTS）兼容的初步测定， 直接配体与每个受体结合。我们的目标是使用Scripps实现一个完整的HTS活动 机构药物发现库（SDDL），用于识别、验证和表征有效的小分子逆 REV-ERB活性的激动剂。为了实现我们的目标，我们将把我们的检测方法应用到1，536孔板中 在满足HTS自动化标准后，开展“全套”HTS活动，以筛选> 640，000名 SDDL中的化合物（目标1）。化学信息学分析“命中”将有助于确定最有希望的线索 通过结构聚类、化合物的生物信息学分析以确定混杂性，以及支架分析以 确定化学合成的容易性和进一步药物化学工作的易处理性，以进行结构- 活动关系研究。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，并开始了解REV-ERB介导的转录调控的作用机制。 活动最后，在目标3中，经验证的HTS命中将被推进到早期药物化学，以进行铅优化 和REV-ERBa/B的新型负调节剂的表征。我们希望完成这项申请 将递送多种结构上不同的REV-ERB反向激动剂， 效力和选择性。我们的合作研究团队在执行高通量 筛选、支架的选择性优化以及化合物的体外和体内表征。总的来说， 我们的筛选方法使我们处于一个独特的位置，以确定，验证和表征新的REV-ERB， 选择性小分子反向激动剂用于研究REV-ERB在增强TH 17应答中的功能 并确定靶向这些受体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.