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

基于细胞的 TLR3 激活剂高通量筛选分析

• 批准号: 7993022
• 负责人: KRISTIINA VUORI
• 金额: $ 19.1万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993022
• 关键词: Adverse effects; Animal Model; Biological Assay; Biological Process; Cell Line; Cells; Chemicals; Clinical Trials; Communicable Diseases; Development; Double Stranded RNA Virus; Double-Stranded RNA; Embryo; Firefly Luciferases; Generations; Genome; Goals; Human; Immune; Immune response; Immunologic Adjuvants; Infection; Inflammatory; Interferons; Kidney; Lead; Libraries; Luciferases; Microbe; Molecular Bank; Molecular Weight; Oral; Poly I-C; Procedures; Process; Prodrugs; Production; Reporter; Reporter Genes; Reproducibility; Resistance; Screening procedure; Specificity; Structure; System; TLR1 gene; Therapeutic; Therapeutic Agents; Therapeutic Effect; Toll-like receptors; United States National Institutes of Health; Viral; Virus; Virus Diseases; Work; base; cancer cell; cancer therapy; cellular targeting; cytokine; eIF-2 Kinase; expression vector; high throughput screening; human TLR3 protein; interest; molecular size; novel; pathogen; promoter; public health relevance; receptor; stable cell line; tumor growth

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.

描述（由申请人提供）：toll样受体（TLRs）是人类先天免疫细胞中关键的病原体识别受体，可以从微生物中检测各种病原体，从而保护身体免受感染。在人类细胞中，已经鉴定出10种tlr (TLR1至TLR10)，每种tlr都有自己的特异性。TLR3识别来自dsRNA病毒基因组或在许多病毒复制过程中产生的病毒双链RNA。TLR3的激活诱导强烈的Th1免疫反应，包括炎症细胞因子和干扰素的分泌增加，随后导致免疫细胞的成熟、分化和增殖。由于这些有效的免疫佐剂作用有助于清除体内的病毒和恶性细胞，因此开发用于治疗的TLR3激活剂已经引起了人们的强烈兴趣。合成双链rna (dsRNAs)，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已显示出增加对病毒感染的抵抗力和抑制肿瘤生长的作用，其治疗效果正在临床试验中进行评估。然而，尽管它们具有有益的作用，但这些dsrna进一步发展为更有效的治疗剂首先受到它们的大小的限制；dsRNAs的分子大小大于8000 Da。其次，由于它们的大小，很难进一步将这些dsrna修饰成口服前药物格式。第三，这些dsRNAs缺乏特异性，除了TLR3外，还被多种细胞靶标识别，包括rig - i样受体和蛋白激酶PKR。这至少可以部分解释这些dsrna的不良影响。最后，到目前为止，尚未发现TLR3的小分子量激活剂，这使得阐明TLR3及其激活剂之间的结构-功能关系变得困难。由于这些原因，发现新的小分子量TLR3激活剂不仅对于开发更安全、更有效的基于TLR3的治疗剂是必要的，而且对于开发更特异性的TLR3激活剂以探索其生物学功能也是必要的。针对TLR3筛选化合物文库是鉴定先导化合物以开发新型TLR3活化剂的最佳方法之一。在这个项目中，我们计划开发一种稳定的基于细胞系的TLR3激活实验，并在384孔板格式中验证该实验的稳健性、可重复性和灵敏度，以便该实验适应于高通量筛选的自动化处理程序。开发的检测将提交给NIH分子文库生产中心中心网络（MLPCN）实施。我们相信这项工作是发现TLR3激活的新型先导化合物的第一步，这将导致开发高度特异性的激活剂来探测TLR3的生物学功能，以及开发新一代基于TLR3的治疗药物，用于治疗癌症和传染病。