Assay development and screening for small molecule RGS10 regulators to target neuroinflammation

项目摘要 小胶质细胞是驻留在中枢神经系统的巨噬细胞，通过病原体激活和内源性损伤信号 细胞表面受体包括Toll样受体和肿瘤坏死因子受体，从而介导炎症 反应包括炎性基因表达上调和产生炎性前列腺素。 这些介质直接导致神经毒性，进而进一步激活小胶质细胞，启动一种自身的 慢性神经炎的传播循环。这一现象直接牵涉到发病机制和 多种慢性神经系统疾病的进展，包括帕金森氏病、阿尔茨海默病和 神经性疼痛。这些疾病也有一个共同之处，即缺乏有效的疾病修饰疗法，导致 慢性和复发性疾病。因此，迫切需要确定新的治疗方法，这一需求尚未得到满足。 目标和先导化合物，以改善治疗选择和预后。靶向调节失调的炎症 在小胶质细胞中传递信号是一种很有前途的治疗策略；然而，调节个体炎症的策略 信号通路缺乏临床疗效。拟议研究的长期目标是针对多个 炎症途径，这将是临床上更有效的，并可能改变疾病，在中枢神经系统 有潜在慢性神经炎的疾病。这项提议的基本原理是，小胶质细胞蛋白 G蛋白信号转导调节因子10抑制TLR4和肿瘤坏死因子下游的促炎信号 受体，并防止炎症引起的神经毒性。RGS10的表达被强烈抑制 通过表观遗传机制激活小胶质细胞，这种沉默放大了小胶质细胞的信号转导 一种前馈机制，有助于炎症信号的失调，并有助于慢性 神经炎。中心假设是阻断激活的RGS10表达的抑制 小胶质细胞将减少多种炎症途径，限制神经炎症。这样做的总体目标是 建议开发一种无偏高通量筛选(HTS)方法来识别和验证小分子 RGS10在静息和活化小胶质细胞中表达的分子调控1)开发 用CRISPR/Cas9技术稳定表达纳米荧光素酶标记的内源性RGS10小胶质细胞系 技术，并优化了Nano-Glo®荧光素酶检测。2)对其进行3万个小分子的高温超导 A)阻断TLR4介导的RGS10抑制或b)提高小胶质细胞基础RGS10水平的能力。3)验证 确定热门项目，以确定最有希望进一步发展的候选者。建议的方法是 创新是因为它针对生理相关条件下内源性RGS10表达的调节 条件。此外，靶向RGS10的表观遗传调控是控制密钥的一种非常新的方法 小胶质细胞中的炎症信号通路。此外，RGS10表达的抑制也发生在 多种系统(巨噬细胞、心肌细胞和卵巢癌)。因此，本研究具有重要的现实意义。 因为它引入了一种新的方法来调节具有广泛治疗意义的新靶点。

Project Summary Microglia, CNS-resident macrophages, get activated by pathogens and endogenous damage signals through cell surface receptors including toll like receptors (TLRs) and TNF receptors and thereby mediate inflammatory responses including upregulation of inflammatory gene expression and generating inflammatory prostaglandins. These mediators directly cause neurotoxicity, which in turn further activates microglia, initiating a self- propagating cycle of chronic neuroinflammation. This phenomenon is directly implicated in the pathogenesis and progression of multiple chronic neurological diseases, including Parkinson’s disease, Alzheimer’s disease and neuropathic pain. These diseases also have in common a lack of effective, disease modifying therapies, resulting in chronic and relapsing disease. Thus, there is an urgent unmet medical need to identify novel therapeutic targets and lead compounds to improve treatment options and prognosis. Targeting dysregulated inflammatory signaling in microglia is a promising therapeutic strategy; however, strategies regulating individual inflammatory signaling pathways have lacked clinical efficacy. The long-term goal of the proposed studies is to target multiple inflammatory pathways, which would be more clinically efficacious, and possibly disease modifying, in CNS diseases with underlying chronic neuroinflammation. The rationale for this proposal is that the microglial protein Regulator of G protein Signaling 10 (RGS10) inhibits pro-inflammatory signaling downstream of TLR4 and TNF receptors, and protects against inflammation-induced neurotoxicity. RGS10 expression is strongly suppressed through epigenetic mechanisms following microglial activation and this silencing amplifies microglial signaling in a feed-forward mechanism that contributes to dysregulation of inflammatory signaling and contributes to chronic neuroinflammation. The central hypothesis is that blocking the suppression of RGS10 expression in activated microglia will diminish multiple inflammatory pathways and limit neuroinflammation. The overall objective of this proposal is to develop a non-biased high-throughput screening (HTS) assay to identify and validate small molecule regulators of RGS10 expression in resting and activated microglia through three aims. 1) Develop a stable microglial cell line expressing endogenous RGS10 tagged with Nanoluc® luciferase, using CRISPR/Cas9 technology, and optimize a Nano-Glo® luciferase assay. 2) Perform HTS of 30,000 small molecules for their ability to a) block TLR4-mediated RGS10 suppression or b) enhance basal RGS10 levels in microglia. 3) Validate identified hits to identify the most promising candidates for further development. The proposed approach is innovative because it targets regulation of endogenous RGS10 expression under physiologically relevant conditions. In addition, targeting epigenetic regulation of RGS10 is a highly novel approach to control key inflammatory signaling pathways in microglia. Furthermore, suppression of RGS10 expression also occurs in multiple systems (macrophages, cardiac myocytes, and ovarian cancer). Thus, this research is significant because it introduces a novel approach to modulate a novel target with broad therapeutic implications.