Investigation of novel cysteine-reactive nitro fatty acids binding covalently to drug targets with relevance for inflammatory reactions

研究与炎症反应相关的与药物靶标共价结合的新型半胱氨酸反应性硝基脂肪酸

• 批准号: 224877466
• 负责人: Professor Dr. Thorsten Jürgen Maier
• 金额: --
• 依托单位: Paul-Ehrlich-Institut - Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/224877466?language=en
• 关键词: Investigation; novel; cysteine; reactive; nitro

During the first funding period of this application we were able to show that 5-lipoxygenase (5-LO), which is the key enzyme of leukotriene biosynthesis, is a target for anti-inflammatory nitro fatty acids (NFA) and some natural products with Michael acceptor. 5-LO can be inhibited by NFA using a covalent modification to cysteine 416 and 418 causing suppression of leukotriene biosynthesis in vitro and in vivo. Mechanistically, similar inhibition was also observed with high intracellular concentrations of nitric oxide via nitrosylation of cysteines in both cellular and animal models. The attack on cysteines 416 and 418 of 5-LO could thus be a fundamental regulatory mechanism of leukotriene biosynthesis ((patho) physiological "on-off switch"), which can provide a novel approach to inhibiting leukotriene biosynthesis. NFAs, which proofed to be well-tolerated in murine disease models and early clinical studies, are innovative scaffolds for the development of new anti-inflammatory therapeutics. In this follow-up submission, Aim 1 seeks to develop a new class of 5-LO inhibitors based on NFA targeting cysteines 416 and 418. Aim 2 involves the identification and functional characterization of novel cellular NFA targets. To achieve Aim 1, novel structurally related NFA derivatives will be screened for inhibition / activation of known NFA targets. The primary goal is to increase the efficiency and potency of inhibition of 5-LO by NFA. In addition, these studies should show whether variations of the structure can change the selectivity of NFA derivatives to these different targets. Goal 2 focuses on the identification of NFA target proteins that could play a potential regulatory role in inflammatory processes. Furthermore, these potential binding partners of NFAs can be targets for novel covalent drug candidates. For the implementation of objective 2, two proteomics approaches (direct detection of posttranslationally modified proteins, strategy 1 and use of specific NFA probes for the identification of new NFA target proteins, strategy 2) will be carried out in parallel. For the mass-spectrometric analysis we use the ability of NFAs to chemically modify cysteine and histidine residues by a Michael-addition. 9-NOA-modified peptides, in contrast to the unmodified peptides, show a 327.24 Da higher mass. In order to cover the broadest possible range of potential target proteins and to be able to assess the consistency of protein modification by NFAs, we aim using different cell species (monocytes, epithelial cells, fibroblasts, primary granulocytes) for the experiments. For the implementation of the projects, personnel resources for a postdoctoral candidate for 36 months are requested in addition to resources for consumables.

在本申请的第一个资金期间，我们能够证明5-脂氧合酶（5-lo）是白三烯生物合成的关键酶，是抗炎硝基脂肪酸（NFA）的靶标，有些天然产物与迈克尔接受者。 NFA可以使用对半胱氨酸416和418的共价修饰来抑制5-lO，从而导致体外和体内抑制白三烯生物合成。从机理上讲，在细胞和动物模型中，通过硝基化的半胱氨酸的硝基化，也观察到具有高细胞内氧化物的相似抑制作用。因此，对5-LO的半胱氨酸416和418的攻击可能是白三烯生物合成的基本调节机制（（病原）生理“开关开关”），这可以提供一种新的方法来抑制白细胞生物合成的新方法。 NFA在鼠类疾病模型和早期临床研究中证明了良好的耐受性，是开发新的抗炎疗法的创新脚手架。在此后续提交中，AIM 1试图基于NFA靶向半胱氨酸416和418的新类5-LO抑制剂。AIM2涉及新型细胞NFA靶标的鉴定和功能表征。为了达到目标1，将筛选新型的结构相关的NFA衍生物，以抑制 /激活已知的NFA靶标。主要目标是提高NFA对5-LO抑制的效率和效力。此外，这些研究应表明结构的变化是否可以改变NFA衍生物对这些不同目标的选择性。目标2的重点是鉴定NFA靶蛋白，这些蛋白可能在炎症过程中起潜在的调节作用。此外，NFA的这些潜在结合伙伴可能是新型共价候选者的靶标。为了实施目标2，两种蛋白质组学方法（直接检测翻译后修饰的蛋白质，策略1，以及使用特定NFA探针鉴定新的NFA靶蛋白，策略2），将同时进行。对于质谱分析，我们使用NFA通过迈克尔的化学修改半胱氨酸和组氨酸残基的能力。与未修饰的肽相比，9-NOA修饰的肽表现出327.24 da的质量。为了涵盖潜在靶蛋白的最广泛范围，并能够评估NFA的蛋白质修饰的一致性，我们旨在使用不同的细胞种类（单核细胞，上皮细胞，成纤维细胞，原发性粒细胞）进行实验。为了实施项目，除了用于消耗品的资源外，还要求还要求提供36个月的博士后候选人的人事资源。