Biochemical and functional characterization of a novel anti-inflammatory biogenic amine

新型抗炎生物胺的生化和功能表征

• 批准号: 10610183
• 负责人: LAURA SANTAMBROGIO
• 金额: $ 73.73万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-06 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10610183
• 关键词: Adenylate Cyclase; Amines; Animal Model; Anti-Inflammatory Agents; Antigen-Presenting Cells; Antiinflammatory Effect; Autoimmune Diseases; Binding; Biochemical; Biogenic Amines; Biological; Biological Assay; Biological Response Modifiers; Biophysics; Biotinylation; CD8-Positive T-Lymphocytes; CRISPR library; Catabolism; Cell secretion; Cells; Clinical; Complex; Cyclic AMP; Data; Dendritic Cells; Disease; Drug Kinetics; Endowment; Enzymes; Erythema; Essential Amino Acids; Exhibits; Experimental Models; Forskolin; G-Protein-Coupled Receptors; Generations; Genetic; Genetic Transcription; Glomerulonephritis; Goals; Human; IL17 gene; Immune; Immune Tolerance; Immunologics; Immunophenotyping; Immunoprecipitation; Immunosuppression; In Vitro; Incubated; Inflammatory; Interferon Type II; Interleukin-1 beta; Interleukin-6; Knowledge; Kynurenine; Lateral; Libraries; Ligands; Lupus; Lymphatic Endothelial Cells; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Melatonin; Memory; Metabolic Pathway; Modeling; Mus; Nephritis; Nuclear; Nuclear Translocation; Orphan; Pathologic; Pathway interactions; Pharmaceutical Preparations; Pharmacodynamics; Phenotype; Phosphotransferases; Physiological; Process; Production; Proteinuria; Psoriasis; Regulation; Reporting; STAT1 gene; Series; Serotonin; Serum; Signal Transduction; Signal Transduction Pathway; Skin; Stimulus; Systemic Lupus Erythematosus; T memory cell; T-Cell Activation; TNF gene; Testing; Therapeutic; Thick; Titrations; Toxic effect; Tryptophan; Tryptophan Metabolism Pathway; Urea Nitrogen; adaptive immune response; analog; biophysical analysis; chemokine; cytokine; experimental study; immunoregulation; improved; in vitro testing; in vivo; kidney dysfunction; knock-down; mouse model; nephrotoxicity; novel; phosphoproteomics; protective effect; receptor; screening; stem

Summary Tryptophan (Trp) catabolism is a complex pathway that generates over fifty metabolites in a cell-specific manner. Besides being the precursor of serotonin and melatonin, tryptophan generates a cascade of metabolites known as kynurenines. Kynurenine metabolites are regarded as one of the most powerful mediators of immune regulation. The thrust of this application stems from our original observations that: (i) lymphatic endothelial cells (LEC) and dendritic cells (DCs) secrete a previously unidentified biogenic amine, 3HKA, which derives from a lateral pathway of Trp catabolism, whose function is currently unknown; (ii) 3HKA exhibits a clear anti- inflammatory profile by inhibiting the STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, IL-6, IL12p70 and TNFα; (iii) in vivo, 3HKA exerts protective effects in the experimental model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring. In a model of nephrotoxic lupus, 3HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. As such, the ultimate goal of this application is to fully characterize the biological activity of 3HKA. In Aim 1, a series of biochemical and biophysical analyses will be employed to identify the enzyme responsible for 3HKA production and investigate the regulation of its synthesis under physiological and pathological conditions. In Aim 2, by using biotinylated derivatives of 3HKA, and a series of biochemical and biophysical experiments, we will identify the receptor target of 3HKA and its expression profile. In Aim 3, using targeted and untargeted phosphoproteomic approaches, screening of kinase libraries and CRISPR/Cas9 knock down strategies we will characterize the 3HKA signal transduction pathway. Additionally, we will analyze the effect of 3HKA on T cell activation and differentiation into a TH1, TH2, TH17 phenotype, naïve vs effector and memory T cell transition and 3HKA effect on maturation/activation of different APC (DC, pDC, MΦ). Immunophenotyping will be performed both in vitro and in vivo, using psoriasis or nephrotoxic lupus as animal models. Finally, in Aim 4 we leveraged our knowledge on 3HKA and generated 3HKA-like compounds, predicted to have an anti- inflammatory effect. These analogs will be screened in a series of in vitro and in vivo assay to fully assess their potential as novel immunomodulatory molecules

总结 色氨酸（Trp）催化剂是一种复杂的途径，以细胞特异性方式产生五十多种代谢产物。 色氨酸除了是血清素和褪黑激素的前体外，还能产生一系列代谢物， 犬尿氨酸犬尿氨酸代谢物被认为是免疫调节的最强有力的介质之一。 调控本申请的主旨源于我们最初的观察：（i）淋巴管内皮细胞 (LEC)树突状细胞（DC）分泌一种以前未鉴定的生物胺，3 HKA，它来自于一种 Trp催化剂的侧向途径，其功能目前未知;（ii）3 HKA表现出明显的抗- 通过抑制小鼠和人树突状细胞（DC）中的STAT 1/NF-κ B通路， 导致促炎趋化因子和细胞因子的释放减少;最值得注意的是，IL-6， IL 12 p70和TNFα;（iii）在体内，3 HKA通过以下方式在银屑病实验模型中发挥保护作用： 减少皮肤厚度、红斑、鳞屑和龟裂。在肾毒性狼疮模型中， 蛋白尿和血清尿素氮，全面改善免疫介导的肾小球肾炎和肾 功能障碍因此，本申请的最终目标是充分表征3 HKA的生物活性。 在目标1中，将采用一系列生物化学和生物物理分析来鉴定负责的酶 并研究了生理和病理条件下3 HKA合成的调控 条件在目的2中，通过使用3 HKA的生物素化衍生物，以及一系列生物化学和生物物理化学方法， 实验中，我们将确定3 HKA的受体靶点及其表达谱。在目标3中，使用有针对性和 非靶向磷酸蛋白质组学方法，激酶文库筛选和CRISPR/Cas9敲低 策略，我们将表征3 HKA信号转导通路。此外，我们还将分析 3 HKA对T细胞活化和分化为TH 1、TH 2、TH 17表型的作用，幼稚型与效应型和记忆型T细胞 细胞转化和3 HKA对不同APC（DC、pDC、MΦ）成熟/活化的影响。免疫分 将在体外和体内进行，使用银屑病或肾毒性狼疮作为动物模型。最后，在Aim 4我们利用我们对3 HKA的了解并产生了3 HKA样化合物，预测具有抗- 炎症效应。将在一系列体外和体内测定中筛选这些类似物，以充分评估它们的生物活性。 作为新型免疫调节分子的潜力