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

概括 色氨酸 (Trp) 分解代谢是一条复杂的途径，以细胞特异性方式产生 50 多种代谢物。 除了是血清素和褪黑激素的前体之外，色氨酸还会产生一系列已知的代谢物 作为犬尿氨酸。犬尿氨酸代谢物被认为是最强大的免疫介质之一 规定。该应用的主旨源于我们最初的观察：（i）淋巴内皮细胞 (LEC) 和树突状细胞 (DC) 分泌一种先前未鉴定的生物胺 3HKA，它源自 色氨酸分解代谢的侧向途径，其功能目前未知； (ii) 3HKA 表现出明显的抗 通过抑制小鼠和人类树突状细胞 (DC) 中的 STAT1/NF-κB 通路来调节炎症特征 促炎趋化因子和细胞因子的释放随之减少；最值得注意的是，IL-6， IL12p70 和 TNFα； (iii) 在体内，3HKA 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，3HKA 有所改善 蛋白尿和血清尿素氮，总体改善免疫介导的肾小球肾炎和肾病 功能障碍。因此，本应用的最终目标是充分表征3HKA的生物活性。 在目标 1 中，将采用一系列生化和生物物理分析来鉴定负责的酶。 3HKA的产生并研究其合成在生理和病理条件下的调控 状况。在目标2中，通过使用3HKA的生物素化衍生物，以及一系列的生化和生物物理 实验中，我们将鉴定3HKA的受体靶点及其表达谱。在目标 3 中，使用有针对性的和 非靶向磷酸化蛋白质组学方法、激酶文库筛选和 CRISPR/Cas9 敲除 我们将描述 3HKA 信号转导途径的策略。另外，我们还要分析一下效果 3HKA 关于 T 细胞激活和分化为 TH1、TH2、TH17 表型、幼稚 T 细胞与效应细胞和记忆 T 细胞的关系 细胞转变和 3HKA 对不同 APC（DC、pDC、MΦ）成熟/激活的影响。免疫表型分析 将使用银屑病或肾毒性狼疮作为动物模型在体外和体内进行。最后，在目标 4 我们利用我们对 3HKA 的了解，生成了类似 3HKA 的化合物，预计具有抗- 炎症作用。这些类似物将在一系列体外和体内测定中进行筛选，以充分评估其 作为新型免疫调节分子的潜力