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

针对神经炎症的小分子 RGS10 调节剂的测定开发和筛选

• 批准号: 9977073
• 负责人: Shelley B Hooks
• 金额: $ 18.6万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977073
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Anti-Inflammatory Agents; Binding; Biological Assay; CRISPR/Cas technology; Cardiac Myocytes; Cell Line; Cell Surface Receptors; Cells; Central Nervous System Diseases; Chronic; Chronic Disease; Clinical; Development; Dinoprostone; Disease; Engineering; Epigenetic Process; Future; GTP-Binding Protein Regulators; GTP-Binding Proteins; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; HDAC1 gene; Histone Deacetylase; Histone Deacetylase Inhibitor; Individual; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interleukins; Intervention; Lead; Luciferases; Malignant neoplasm of ovary; Mediating; Mediator of activation protein; Medical; Methods; Microglia; Mus; Noise; Outcome Study; Parkinson Disease; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Physiological; Production; Prostaglandins; Proteins; RGS Proteins; RGS2 gene; Receptor Activation; Recurrent disease; Regulation; Reporter; Research; Rest; Role; Series; Signal Pathway; Signal Transduction; Signaling Protein; Specificity; System; TLR4 gene; TNF gene; TNFRSF1A gene; Therapeutic; Toll-like receptors; Translating; Tumor Necrosis Factor Receptor; Up-Regulation; Validation; assay development; chemokine; chronic neurologic disease; chronic pain; clinical efficacy; cyclooxygenase 2; cytokine; effective therapy; epigenetic regulation; high throughput screening; improved; innovation; knock-down; macrophage; nano; nanoluciferase; nervous system disorder; neuroinflammation; neuron loss; neurotoxicity; new therapeutic target; novel; novel strategies; outcome forecast; overexpression; painful neuropathy; pathogen; promoter; receptor; recruit; response; screening; small molecule; treatment strategy

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.

项目摘要 小胶质细胞，CNS居民巨噬细胞，通过病原体和内源性损伤信号激活 细胞表面受体，包括收费受体（TLR）和TNF受体，从而培养基炎症 反应包括上调炎症基因表达和产生炎症前列腺素。 这些介体直接引起神经毒性，从而进一步激活小胶质细胞，引发了自我 慢性神经炎症的传播周期。这种现象直接与发病机理和 多种慢性神经系统疾病的进展，包括帕金森氏病，阿尔茨海默氏病和 神经性疼痛。这些疾病也普通缺乏有效的，疾病修饰的疗法，导致 在慢性和继电器疾病中。这是紧急未满足的医疗需求，以识别新的疗法。 靶标和铅化合物以改善治疗选择和提示。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。但是，策略调节个人炎症 信号通路缺乏临床效率。拟议研究的长期目标是针对多个 中枢神经系统中的炎症途径将在临床上更有效，并且可能会改变疾病 潜在的慢性神经炎症的疾病。该建议的理由是小胶质细胞蛋白 G蛋白信号传导的调节剂10（RGS10）抑制TLR4和TNF下游的促炎信号传导 受体，并防止炎症引起的神经毒性。 RGS10表达被强烈抑制 通过小胶质激活后的表观遗传机制和这种沉默放大器的小胶质细胞信号传导 喂养机制，导致炎症信号失调，并导致慢性 神经炎症。中心假设是阻止激活的RGS10表达的抑制 小胶质细胞会减少多种炎症途径并限制神经炎症。总体目标 建议是开发无偏见的高通量筛选（HTS）测定法，以识别和验证小 RGS10表达的分子调节剂通过三个目标在静息和激活的小胶质细胞中。 1）开发 使用crispr/cas9，稳定的小胶质细胞系表示用nanoluc®荧光素酶标记的内源性RGS10 技术，并优化纳米-Glo®荧光素酶测定法。 2）为其执行30,000个小分子的HT a）阻断TLR4介导的RGS10抑制作用或b）在小胶质细胞中增强基本RGS10水平的能力。 3）验证 确定命中以确定最有前途的进一步发展候选人。建议的方法是 创新性是因为它针对内源性RGS10表达在物理相关的调节 状况。此外，靶向RGS10的表观遗传调节是控制钥匙的高度新方法 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 多个系统（巨噬细胞，心肌细胞和卵巢癌）。那是重要的 因为它引入了一种新型方法，以调节具有广泛治疗意义的新型目标。