Small Molecule inhibitors of late endosomal pro-inflammatory signaling

摘要 内小体Toll样受体(TLR)激活和晚期内小体启动的信号转导是高血压的主要机制 先天免疫、炎症和自身免疫。尽管核酸敏感的内体TLR激活是 对于正确的感染反应很重要，不受限制地激活内体TLRs启动促炎反应 在包括缺血在内的人类几种疾病的发展中发挥核心作用的通路- 再灌注损伤、类风湿关节炎、系统性红斑狼疮、幼年特发性关节炎和1型 糖尿病。内体TLR的激活需要将内体TLR部分消化成其活性形式，a 依赖于晚内体(LE)成熟的过程。我们最近描述了一种新的机制 与钙感受器直接结合有关的初级炎症细胞的晚期内体成熟 Munc13-4与膜融合调节因子--晚期内体SNARE蛋白合成素7(STX7)结合。钙- Munc13-4与STX7的依赖结合调节内体成熟和TLR信号转导。重要的是， 在Munc13-4缺陷细胞中观察到的晚期内体缺陷表型可被野生型Munc13-4拯救，但 而不是通过钙结合缺陷的Munc13-4突变体来破坏STX7-Munc13-4的相互作用。我们的数据 确定Munc13-4与Synaxin 7的相互作用是调节内体TLR的重要过程 激活。我们认为，干扰Munc13-4与STX7的相互作用可以防止晚期内吞 通过损害TLR7和TLR9依赖的信号通路，成熟并减轻炎症。这是 由我们的初步数据支持的是，炎症反应对体内内毒素的挑战 TLR9配体而不是通过质膜受体作用的TLR配体减少 Munc13-4缺陷小鼠。这项提议的目标是利用高通量筛查来识别小的- Munc13-4和Synaxin 7形成的复合体的分子抑制剂，用于初级免疫细胞， 导致全身性炎症。我们还打算通过已建立的二级结构来验证这些化合物 化验和基于细胞的方法。我们的具体目标是：1)利用高通量筛查 使用一种创新的方法分析合成素7-Munc13-4结合的分子抑制剂的激活 完整的细胞内内体上的复合体；2)以细胞为基础进行正交确认分析 活性系列分子相似性的二次方法和分析以确定活性并优先排序 探针和3)通过分析内体成熟和核的机制来验证活性探针 初级免疫细胞酸敏TLR启动的信号转导。提出这项研究的意义在于 新的小分子抑制剂选择性和特异性地抑制Synaxin 7-Munc13-4复合体和 核酸感应TLR信号，将导致新的治疗前导联的发展 包括自身免疫性疾病在内的全身性炎症被上调的疾病。

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.