High throughput screens for modulators of inflammatory cytakine gene expression

描述（由申请人提供）：炎症可能有利于对微生物病原体的正常免疫应答。然而，长期的炎症可以在感染期间促进组织损伤，并且与多种疾病密切相关，包括癌症，动脉粥样硬化和几种炎性自身免疫性疾病。尽管有许多抗炎药物可用，但由于各种原因，包括靶点特异性不足，它们都不被认为是理想的。因此，需要新的策略来开发促炎基因和蛋白的选择性抑制剂。在寻求抑制特定促炎基因转录的药物方面的一个主要限制是，我们对负责选择性基因调控的分子机制的理解令人惊讶地有限。信号通路，如NF-？已知B和AP-1途径有助于许多促炎基因的激活。然而，由于其广泛的功能，这些途径不是选择性调节单个基因的合适靶点。因为它已被证明是很难发现的机制，通过使用传统的实验策略的选择性调节，我们已经开始攻击的选择性问题，使用一种新的策略，应该导致更广泛的赞赏这个问题，与可能性确定治疗的先导化合物。具体来说，我们正在从小鼠中产生巨噬细胞系，其中荧光蛋白报告基因在其天然染色质环境中受细胞因子基因控制区的调控。然后进行高通量筛选以鉴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天然染色质环境中的基本原理是，我们过去的研究已经揭示，通常用于高通量筛选的常规启动子-报告质粒在稳定转染后不能组装成生理相关的染色质结构。此外，荧光蛋白报告基因测定优于监测内源性细胞因子分泌的ELISA测定，因为ELISA对细胞因子翻译、加工和分泌的误导性影响敏感。通过测试已知分子靶点的小分子文库，我们希望对有助于选择性基因调控的信号通路获得前所未有的洞察力。然后将更深入地研究所确定的信号通路，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节的潜力，并有可能确定治疗性先导化合物。 公共卫生相关性拟议研究的目的是探索一种新的高通量筛选策略的可行性，该策略可能导致发现能够调节炎症相关蛋白质表达的小分子。这些小分子的鉴定将有助于我们正在进行的调节炎症基因选择性表达的信号通路的研究。此外，所提出的筛选可能导致发现用于治疗与异常炎症相关的疾病的治疗性先导化合物，包括动脉粥样硬化、癌症和许多炎性自身免疫性疾病。

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.