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