Small Molecule inhibitors of late endosomal pro-inflammatory signaling

晚期内体促炎症信号传导的小分子抑制剂

• 批准号: 9217039
• 负责人: Sergio Daniel Catz
• 金额: $ 42.57万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9217039
• 关键词: Activation Analysis; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biological Assay; Calcium; Calcium Binding; Cell membrane; Cells; Chemistry; Chronic Childhood Arthritis; Clinical; Complex; Data; Development; Digestion; Disease; Endosomes; Fingerprint; Fluorescence Resonance Energy Transfer; Goals; Human; Immune; Impairment; Infection; Inflammation; Inflammatory; Inflammatory Response; Insulin-Dependent Diabetes Mellitus; Knowledge; Ligands; Measures; Membrane; Membrane Fusion; Molecular; Molecular Analysis; Mus; Natural Immunity; Nucleic Acids; Organelles; Pathologic Processes; Pathway interactions; Phenotype; Play; Preparation; Process; Proteins; Protocols documentation; Receptor Activation; Receptor Signaling; Regulation; Reperfusion Injury; Research; Rheumatoid Arthritis; Role; SNAP receptor; Series; Signal Pathway; Signal Transduction; Specificity; Stimulus; Systemic Lupus Erythematosus; TLR7 gene; Technology; Testing; Therapeutic; Time; Toll-like receptors; Toxic effect; Validation; base; cytokine; high throughput screening; human disease; in vivo; inhibitor/antagonist; innovation; late endosome; mutant; novel; prevent; rab GTP-Binding Proteins; receptor; response; screening; sensor; small molecule; small molecule inhibitor; syntaxin; tool; trafficking; Activation Analysis; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Binding; Biological Assay; Calcium; Calcium Binding; Cell membrane; Cells; Chemistry; Chronic Childhood Arthritis; Clinical; Complex; Data; Development; Digestion; Disease; Endosomes; Fingerprint; Fluorescence Resonance Energy Transfer; Goals; Human; Immune; Impairment; Infection; Inflammation; Inflammatory; Inflammatory Response; Insulin-Dependent Diabetes Mellitus; Knowledge; Ligands; Measures; Membrane; Membrane Fusion; Molecular; Molecular Analysis; Mus; Natural Immunity; Nucleic Acids; Organelles; Pathologic Processes; Pathway interactions; Phenotype; Play; Preparation; Process; Proteins; Protocols documentation; Receptor Activation; Receptor Signaling; Regulation; Reperfusion Injury; Research; Rheumatoid Arthritis; Role; SNAP receptor; Series; Signal Pathway; Signal Transduction; Specificity; Stimulus; Systemic Lupus Erythematosus; TLR7 gene; Technology; Testing; Therapeutic; Time; Toll-like receptors; Toxic effect; Validation; base; cytokine; high throughput screening; human disease; in vivo; inhibitor/antagonist; innovation; late endosome; mutant; novel; prevent; rab GTP-Binding Proteins; receptor; response; screening; sensor; small molecule; small molecule inhibitor; syntaxin; tool; trafficking

SUMMARY Endosomal Toll-like receptor (TLR) activation and late endosomal-initiated signaling are central mechanisms in innate immunity, inflammation and autoimmunity. Although nucleic acid-sensing endosomal TLR activation is important for a proper response to infection, unrestricted activation of endosomal TLRs initiates pro-inflammatory pathways that play a central role in the development of several disorders in humans including ischemia- reperfusion injury, rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis and type 1 diabetes. Endosomal TLR activation requires the partial digestion of endosomal TLRs into their active forms, a process that depends on late endosome (LE) maturation. We have recently described a novel mechanism of late endosomal maturation in primary inflammatory cells that involves the direct binding of the calcium sensor Munc13-4 to the late endosomal SNARE protein syntaxin 7 (STX7), a regulator of membrane fusion. Calcium- dependent binding of Munc13-4 to STX7 regulates endosomal maturation and TLR signaling. Importantly, the late endosomal defective phenotype observed in Munc13-4-deficient cells is rescued by wild type Munc13-4 but not by a calcium-binding-deficient Munc13-4 mutant that impairs the STX7-Munc13-4 interaction. Our data identify the interaction of Munc13-4 with syntaxin 7 as an essential process for the regulation of endosomal TLR activation. We propose that interference with the interaction of Munc13-4 with STX7 prevents late endosomal maturation and decreases inflammation by impairing TLR7 and TLR9-dependent signaling pathways. This is supported by our preliminary data showing that the inflammatory response to in vivo challenge with endosomal TLR9 ligands but not with TLR ligands that operate through plasma membrane receptors is decreased in Munc13-4-deficient mice. The objective of this proposal is to utilize high-throughput screening to identify small- molecule inhibitors of the complex formed by Munc13-4 and syntaxin 7 for use in primary immune cells that contribute to systemic inflammation. We also aim to validate these compounds through established secondary assays and cell-based approaches. Our specific Aims are: 1) To utilize high-throughput screening for small- molecule inhibitors of syntaxin 7-Munc13-4 binding using an innovative approach that analyzes the activation of the complex on intact intracellular endosomes; 2) To perform orthogonal confirmation assays, cell-based secondary approaches and analysis of the molecular similarity of the active series to identify and prioritize active probes and 3) To validate active probes using analysis of mechanisms of endosomal maturation and nucleic acid-sensing TLR-initiated signaling in primary immune cells. The significance of the research proposed is that new small-molecule inhibitors that selectively and specifically inhibit the syntaxin 7-Munc13-4 complex and nucleic acid-sensing TLR signaling, will lead to the development of novel pre-therapeutic leads for the treatment of diseases in which systemic inflammation is upregulated including autoimmune diseases.

概括 内体收费受体（TLR）激活和晚期内体启动信号传导是中心机制 先天免疫，炎症和自身免疫性。虽然核酸 - 感应内体TLR激活是 对于对感染的适当反应很重要，内体TLR的无限制激活会引发促炎性 在人类的多种疾病发展中起着核心作用的途径，包括缺血 - 再灌注损伤，类风湿关节炎，全身性红斑狼疮，青少年特发性关节炎和1型 糖尿病。内体TLR激活需要将内体TLR部分消化为活性形式 取决于晚期内体（LE）成熟的过程。我们最近描述了一种新颖的机制 涉及钙传感器直接结合的原发性炎症细胞中内体成熟晚期的成熟 Munc13-4到晚期内体核蛋白语法7（STX7），膜融合的调节剂。钙- MUNC13-4与STX7的依赖性结合调节内体成熟和TLR信号传导。重要的是， 在Munc13-4缺陷型细胞中观察到的晚期内体缺陷表型被野生型Munc13-4挽救，但 不通过钙结合缺陷的Munc13-4突变体损害STX7-MUNC13-4相互作用。我们的数据 识别Munc13-4与语法7的相互作用，作为调节内体TLR的必要过程 激活。我们提出对Munc13-4与STX7的相互作用的干扰可防止内体晚期 通过损害TLR7和TLR9依赖性信号通路来成熟并减少炎症。这是 在我们的初步数据的支持下，表明对体内挑战的炎症反应与内体挑战 TLR9配体但没有通过质膜受体作战的TLR配体在 MUNC13-4缺陷小鼠。该提案的目的是利用高通量筛选来识别小型 由Munc13-4和语法7形成的复合物的分子抑制剂用于原代免疫细胞， 有助于系统性炎症。我们还旨在通过已建立的二级验证这些化合物 测定和基于细胞的方法。我们的具体目的是：1）利用高通量筛选 使用创新方法分析语法7-MUNC13-4的分子抑制剂，该方法分析了激活的激活 完整的细胞内内体的复合物； 2）进行正交确认测定，基于细胞 次要方法和分析活动序列的分子相似性，以识别和优先级 探针和3）使用内体成熟和核酸的机理验证活性探针 原代免疫细胞中的酸性TLR启动信号传导。提出的研究的意义是 新的小分子抑制剂，有选择地，特异性地抑制语法7-MUNC13-4复合物和 核酸 - 敏感性TLR信号传导将导致新型治疗前铅的发展进行处理 全身性炎症的疾病上调，包括自身免疫性疾病。