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助期间，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）和一些具有迈克尔受体的天然产物的靶标。 NFA 可以通过对半胱氨酸 416 和 418 进行共价修饰来抑制 5-LO，从而抑制体外和体内的白三烯生物合成。从机制上讲，在细胞和动物模型中，通过半胱氨酸的亚硝基化，细胞内高浓度一氧化氮也观察到类似的抑制作用。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（（病理）生理“开关”），这可以提供抑制白三烯生物合成的新方法。 NFA 在小鼠疾病模型和早期临床研究中被证明具有良好的耐受性，是开发新型抗炎疗法的创新支架。在这份后续提交的文件中，目标 1 寻求基于针对半胱氨酸 416 和 418 的 NFA 开发一类新型 5-LO 抑制剂。目标 2 涉及新型细胞 NFA 靶标的识别和功能表征。为了实现目标 1，将筛选结构相关的新型 NFA 衍生物，以抑制/激活已知的 NFA 靶点。主要目标是提高 NFA 抑制 5-LO 的效率和效力。此外，这些研究应该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 侧重于鉴定可在炎症过程中发挥潜在调节作用的 NFA 靶蛋白。此外，这些潜在的 NFA 结合伴侣可以成为新型共价候选药物的靶点。为了实现目标 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 和使用特定 NFA 探针鉴定新的 NFA 靶蛋白，策略 2）将并行进行。对于质谱分析，我们利用 NFA 通过迈克尔加成对半胱氨酸和组氨酸残基进行化学修饰的能力。与未修饰的肽相比，9-NOA 修饰的肽显示出更高的 327.24 Da 质量。为了覆盖尽可能广泛的潜在目标蛋白，并能够评估 NFA 修饰蛋白的一致性，我们的目标是使用不同的细胞种类（单核细胞、上皮细胞、成纤维细胞、原代粒细胞）进行实验。为实施这些项目，除了消耗品资源外，还需要36个月的博士后候选人的人力资源。