High throughput screens for modulators of inflammatory cytakine gene expression

描述(由申请人提供)：炎症有助于对微生物病原体的正常免疫反应。然而，长期的炎症会在感染过程中促进组织损伤，并与多种疾病密切相关，包括癌症、动脉粥样硬化和几种炎症性自身免疫性疾病。尽管有许多抗炎药可用，但由于各种原因，包括靶向性不足，没有一种被认为是理想的。因此，需要新的策略来开发促炎症基因和蛋白的选择性抑制剂。寻找抑制特定促炎基因转录的药物的一个主要限制是，我们对负责选择性基因调控的分子机制的了解令人惊讶地有限。已知的信号通路，如核因子？B和AP-1通路，有助于许多促炎基因的激活。然而，由于其广泛的功能，这些途径并不是个别基因选择性调控的合适靶点。由于事实证明，通过使用传统的实验策略很难揭示选择性调节的机制，我们已经开始使用一种新的策略来解决选择性问题，这应该会导致对这个问题的更广泛的认识，并有可能识别治疗性的先导化合物。具体地说，我们正在从小鼠培养巨噬细胞系，其中荧光蛋白报告基因由其天然染色质环境中的细胞因子基因控制区调节。然后将进行高通量筛选，以确定差异改变关键细胞因子基因表达的小分子，包括编码IL-12 p40、IL-12 p35、IL-23 p19和IL-10的基因。将荧光蛋白报告基因插入到天然染色质环境中的基本原理是，我们过去的研究表明，传统的启动子-报告载体通常用于高通量筛选，在稳定转染后无法组装成生理相关的染色质结构。此外，荧光蛋白报告试验比监测内源性细胞因子分泌的酶联免疫吸附试验更可取，因为酶联免疫吸附试验容易受到细胞因子翻译、加工和分泌的误导性影响。通过测试分子靶标已知的小分子文库，我们希望对有助于选择性基因调控的信号通路有前所未有的了解。然后将更深入地研究已确定的信号通路，以阐明选择性机制。还将筛选目标未知的更大化合物库，以进一步了解选择性调控的潜力，并有可能识别治疗性先导化合物。 公共卫生相关性拟议研究的目标是探索一种新的高通量筛查策略的可行性，该策略可能导致发现能够调节参与炎症的蛋白质表达的小分子。识别出的小分子将有助于我们正在进行的调控炎症基因选择性表达的信号通路的研究。此外，拟议的筛查可能导致发现治疗性先导化合物，用于治疗与异常炎症相关的疾病，包括动脉粥样硬化、癌症和一些炎症性自身免疫性疾病。

DESCRIPTION (provided by applicant): Inflammation can be beneficial for a normal immune response to microbial pathogens. However, prolonged inflammation can promote tissue damage during infection and has been closely linked to a diverse range of diseases, including cancer, atherosclerosis, and several inflammatory autoimmune diseases. Although a number of anti-inflammatory drugs are available, none of them are considered to be ideal for a variety of reasons, including insufficient target specificity. Therefore, new strategies are needed for the development of selective inhibitors of pro-inflammatory genes and proteins. One major limitation in pursuing pharmaceuticals that inhibit the transcription of specific pro-inflammatory genes is that our understanding of the molecular mechanisms responsible for selective gene regulation is surprising limited. Signaling pathways such as the NF-?B and AP-1 pathways are known to contribute to the activation of many pro-inflammatory genes. However, because of their broad functions, these pathways are not appropriate targets for the selective modulation of individual genes. Because it has proved to be difficult to uncover the mechanisms of selective regulation through the use of conventional experimental strategies, we have begun to attack the selectivity question using a new strategy that should lead to a much broader appreciation of this issue, with the possibility of identifying therapeutic lead compounds. Specifically, we are generating macrophage cell lines from mice in which fluorescent protein reporter genes are regulated by cytokine gene control regions in their native chromatin environment. High-throughput screens will then be performed to identify small molecules that differentially alter the expression of key cytokine genes, including the genes encoding IL-12 p40, IL-12 p35, IL-23 p19, and IL-10. The rationale for inserting fluorescent protein reporter genes into a native chromatin environment is that our past studies have revealed that conventional promoter-reporter plasmids, often used for high-throughput screens, fail to assemble into physiologically relevant chromatin structures upon stable transfection. Furthermore, the fluorescent protein reporter assay is preferable to an ELISA assay that monitors endogenous cytokine secretion because the ELISA is susceptible to misleading effects on cytokine translation, processing, and secretion. By testing small-molecule libraries in which the molecular targets are known, we hope to gain unprecedented insight into the signaling pathways that contribute to selective gene regulation. The signaling pathways identified will then be examined in greater depth to elucidate selectivity mechanisms. Larger libraries of compounds whose targets are unknown will also be screened to gain further insight into the potential for selective regulation, with the possibility of identifying therapeutic lead compounds. PUBLIC HEALTH RELEVANCE The objective of the proposed research is to explore the feasibility of a novel high-throughput screening strategy that may lead to the discovery of small molecules capable of modulating the expression of proteins involved in inflammation. The small molecules identified will facilitate our ongoing studies of the signaling pathways that regulate the selective expression of inflammatory genes. Furthermore, the proposed screens may lead to the discovery of therapeutic lead compounds for the treatment of diseases associated with aberrant inflammation, including atherosclerosis, cancer, and a number of inflammatory autoimmune disorders.