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

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.

总结 内体Toll样受体（TLR）激活和晚期内体启动的信号传导是细胞凋亡的中心机制。 先天免疫、炎症和自身免疫。虽然核酸感应内体TLR激活是一个重要的机制， 内体TLR的不受限制的激活启动了促炎性反应， 在人类包括缺血在内的几种疾病的发展中起核心作用的途径， 再灌注损伤，类风湿性关节炎，系统性红斑狼疮，幼年特发性关节炎和1型 糖尿病内体TLR活化需要内体TLR部分消化成其活性形式， 这是一个依赖于晚期内体（LE）成熟的过程。我们最近描述了一种新的机制， 原发性炎症细胞内体成熟晚期，涉及钙传感器的直接结合 Munc 13 -4与晚期内体SNARE蛋白syntaxin 7（STX 7）（一种膜融合调节因子）的结合。钙- Munc 13 -4与STX 7的依赖性结合调节内体成熟和TLR信号传导。重要的是 在Munc 13 -4缺陷细胞中观察到的晚期内体缺陷表型被野生型Munc 13 -4挽救， 而不是通过削弱STX 7-Munc 13 -4相互作用的钙结合缺陷型Munc 13 -4突变体。我们的数据 确定Munc 13 -4与syntaxin 7的相互作用是调节内体TLR的重要过程 activation.我们认为，干扰Munc 13 -4与STX 7的相互作用可以阻止晚期内体细胞凋亡。 通过损害TLR 7和TLR 9依赖性信号传导途径，促进细胞成熟并减少炎症。这是 我们的初步数据支持，表明对内体激发的炎症反应， TLR 9配体，但不与TLR配体，通过质膜受体的运作减少， Munc 13 -4缺陷小鼠。该提案的目的是利用高通量筛选来识别小- 由Munc 13 -4和突触融合蛋白7形成的复合物的分子抑制剂，其用于 导致全身性炎症。我们还旨在通过建立的二级结构来验证这些化合物。 分析和基于细胞的方法。我们的具体目标是：1）利用高通量筛选小- syntaxin 7-Munc 13 -4结合的分子抑制剂，使用一种创新的方法， 2）为了进行正交确认试验，基于细胞的 活性系列的分子相似性的次要方法和分析，以识别和优先考虑活性 探针和3）使用内体成熟和核内体成熟的机制分析来验证活性探针。 在初级免疫细胞中的酸敏感TLR启动的信号传导。本研究的意义在于， 选择性和特异性抑制突触融合蛋白7-Munc 13 -4复合物的新的小分子抑制剂， 核酸传感TLR信号传导，将导致新的治疗前的治疗线索的发展， 包括自身免疫性疾病在内的全身性炎症被上调的疾病。