Cell-based assay for high-throughput screening of TLR3 activators

描述(申请人提供)：Toll样受体(TLRs)是人类先天性免疫细胞中的关键病原体识别受体，可以从微生物中检测不同的病原体，从而保护身体免受感染。在人类细胞中，已经鉴定了10个TLRs(TLR1到TLR10)，每个都有自己的特异性。TLR3识别来自dsRNA病毒基因组的病毒双链RNA，或在许多病毒复制过程中产生的双链RNA。TLR3的激活可诱导强烈的Th1免疫应答，包括炎性细胞因子和干扰素的分泌增加，进而导致免疫细胞的成熟、分化和增殖。由于这些强大的免疫佐剂作用有助于清除体内的病毒和恶性细胞，因此开发用于治疗应用的TLR3激活剂一直受到人们的强烈兴趣。合成双链RNA(DsRNA)，如多肌苷-多胞苷(Poly(I：C))模拟病毒dsRNA激活TLR3。在动物模型中，这些合成的dsRNAs已被证明可以增强对病毒感染的抵抗力，并抑制肿瘤生长，其治疗效果正在临床试验中进行评估。然而，尽管这些dsRNA具有良好的效果，但其进一步发展成为更有效的治疗剂首先受到其大小的限制；dsRNAs的分子尺寸大于8000Da。其次，由于它们的大小，很难进一步将这些dsRNA修饰成口服亲药物形式。第三，这些dsRNAs缺乏特异性，除了TLR3外，还能被多种细胞靶点识别，包括RIG-I样受体和蛋白激酶PKR。这至少在一定程度上可以解释这些dsRNA的不利影响。最后，到目前为止，还没有发现TLR3的小分子激活剂(S)，这给阐明TLR3与其激活剂之间的结构-功能关系带来了困难。因此，发现新的小分子TLR3激活剂不仅是开发更安全、更有效的TLR3类药物的必要条件，也是开发更具特异性的TLR3激活剂以探讨其生物学功能的必要手段。靶向TLR3筛选化合物文库是开发新型TLR3激活剂寻找先导化合物的最佳途径之一。在这个项目中，我们计划开发一种稳定的基于TLR3激活试验的细胞系，并在384孔板格式中验证该试验的稳健性、重复性和灵敏度，以便将该试验适应于高通量筛选的自动化处理程序。开发的分析将提交给NIH分子图书馆生产中心网络(MLPCN)进行实施。我们相信，这项工作是发现新的TLR3激活先导化合物的第一步，这将导致开发高特异性激活剂来探索TLR3的生物学功能，以及开发新一代基于TLR3的治疗癌症和感染性疾病的药物。 公共卫生相关性：Toll样受体3(TLR3)识别病毒双链(DS)RNA。TLR3的激活可诱导强烈的Th1免疫应答。由于这种强大的免疫佐剂作用有助于清除体内的病毒和恶性细胞，因此开发TLR3激活剂用于治疗一直是人们的浓厚兴趣。在这个项目中，我们计划开发一种基于细胞的高通量筛选方法，以发现新的TLR3激活先导化合物，这将导致开发高特异性激活剂来探索TLR3的生物学功能，以及开发新一代基于TLR3的治疗癌症和感染性疾病的药物。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) are key pathogen-recognition receptors in human innate immune cells that detect diverse pathogens from microbes and thus protect the body against infection. In human cells, ten TLRs (TLR1 to TLR10) have been identified, each with their own specificities. TLR3 recognizes viral double-stranded (ds) RNA derived from the genomes of dsRNA viruses, or generated during the replication of many viruses. Activation of TLR3 induces a strong Th1 immune response, including increased secretion of inflammatory cytokines and interferons, and subsequently leads to the maturation, differentiation, and proliferation of immune cells. Because these potent immune adjuvant effects facilitate eradication of viruses and malignant cells in the body, there has been intense interest in developing TLR3 activators for therapeutic applications. Synthetic double-stranded RNAs (dsRNAs) such as polyinosinic-polycytidylic (Poly (I: C) mimics viral dsRNA in activation of TLR3. These synthetic dsRNAs have been shown to increase resistance to viral infection and inhibit tumor growth in animal models, and their therapeutic effects are being evaluated in clinical trials. However, despite their beneficial effects, further development of these dsRNAs into more effective therapeutic agents is restricted firstly by their size; dsRNAs have a molecular size greater than 8,000 Da. Secondly, because of their size, it is difficult to further modify these dsRNAs into an oral pro-drug format. Thirdly, these dsRNAs lack specificity and are recognized by multiple cellular targets including RIG-I-like receptors and protein kinase PKR, in addition to the TLR3. This could explain, at least in part, the adverse effects of these dsRNAs. Finally, to date no small molecular weight activator(s) has been identified for TLR3, which has made it difficult to elucidate the structure-functional relationship between TLR 3 and its activators. For these reasons, discovery of novel small molecular weight TLR3 activators is necessary not only for the development of safer and more effective TLR3-based therapeutic agents but also for the development of a more specific activator of TLR3 to probe its biological function. Targeting TLR3 to screen chemical compound libraries is one of the best approaches to identify lead compounds for development of novel TLR3 activators. In this project, we plan to develop a stable cell line based TLR3 activation assay, and validate the robustness, reproducibility, and sensitivity of this assay in a 384-well plate format in order for this assay to be adapted into the automated processing procedures of a high throughput screening. The developed assay will be submitted to the NIH Molecular Libraries Production Center Centers Network (MLPCN) for implementation. We believe that this proposed work is a first step to discover novel lead compounds for TLR3 activation, which will lead to the development of highly specific activators to probe the biological function of TLR3, as well as the development of a new generation of TLR3-based therapeutic agents for treatment of cancers and infectious diseases. PUBLIC HEALTH RELEVANCE: Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA. Activation of TLR3 induces a strong Th1 immune response. Because this potent immune adjuvant effect facilitates eradication of viruses and malignant cells in body, there has been intense interest in developing TLR3 activators for therapeutic applications. In this project, we plan to develop a cell-based assay for high throughput screening to discover novel lead compounds for TLR3 activation, which will lead to the development of highly specific activators to probe the biological function of TLR3, as well as the development of a new generation of TLR3-based therapeutic agents for treatment of cancers and infectious diseases.