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

项目摘要 小胶质细胞，CNS驻留的巨噬细胞，被病原体和内源性损伤信号激活， 细胞表面受体，包括Toll样受体（TLR）和TNF α受体，从而介导炎症 包括炎性基因表达上调和产生炎性胰高血糖素的应答。 这些介质直接引起神经毒性，进而进一步激活小胶质细胞，启动自我调节。 慢性神经炎症的传播周期。这种现象直接涉及发病机制， 多种慢性神经系统疾病的进展，包括帕金森病、阿尔茨海默病和 神经性疼痛这些疾病的共同点还在于缺乏有效的疾病修饰疗法， 慢性和复发性疾病。因此，迫切存在未满足的医学需求，以确定新的治疗方法。 靶点和先导化合物，以改善治疗选择和预后。靶向失调的炎症 小胶质细胞中的信号传导是一种有前途的治疗策略;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，这将是更临床有效的，并可能改善疾病，在中枢神经系统， 潜在慢性神经炎症的疾病。这一提议的基本原理是， G蛋白信号调节因子10（RGS10）抑制TLR4和TNF α下游的促炎信号传导 受体，并防止炎症诱导的神经毒性。RGS10表达被强烈抑制 通过小胶质细胞激活后的表观遗传机制，这种沉默放大了小胶质细胞的信号传导， 一种前馈机制，有助于炎症信号的失调，并有助于慢性炎症反应。 神经炎症中心假设是阻断RGS10表达的抑制， 小胶质细胞将减少多种炎症途径并限制神经炎症。本报告的总体目标 建议是开发一种无偏倚的高通量筛选（HTS）试验，以识别和验证小的 RGS10在静息和激活的小胶质细胞中表达的分子调节剂通过三个目的。1)开发一个 使用CRISPR/Cas9表达标记有Nanoluc®荧光素酶的内源性RGS10的稳定小胶质细胞系 技术，并优化Nano-Glo®荧光素酶测定。2)对30，000个小分子进行HTS测试， a）阻断TLR 4介导的RGS10抑制或B）增强小胶质细胞中的基础RGS10水平的能力。3)验证 确定了命中，以确定最有前途的候选人，以进一步发展。所提出的方法是 创新，因为它靶向调节内源性RGS10表达， 条件此外，靶向RGS10的表观遗传调控是一种非常新颖的控制关键基因表达的方法。 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 多系统（巨噬细胞、心肌细胞和卵巢癌）。因此，本研究具有重要的意义 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.