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

在这项应用的第一个资助期，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Michael受体的天然产物的靶标。NFA可通过对半胱氨酸416和418进行共价修饰来抑制5-LO，从而在体内外抑制白三烯的生物合成。从机理上讲，在细胞和动物模型中，细胞内高浓度的一氧化氮通过半胱氨酸的亚硝化也观察到类似的抑制作用。因此，对5-LO半胱氨酸416和418的攻击可能是白三烯生物合成((病理性)生理开关)的基本调节机制，从而为抑制白三烯生物合成提供新的途径。NFA在小鼠疾病模型和早期临床研究中被证明耐受性良好，是开发新的抗炎疗法的创新支架。在这份后续提交的文件中，AIM 1寻求开发一种新的基于NFA的针对半胱氨酸416和418的5-LO抑制剂。目标2涉及新的细胞NFA靶标的鉴定和功能鉴定。为了实现目标1，将筛选新的结构相关的NFA衍生物，以抑制/激活已知的NFA靶标。其主要目的是提高NFA抑制5-LO的效率和效力。此外，这些研究应该表明，结构的变化是否会改变NFA衍生物对这些不同目标的选择性。目标2的重点是确定可能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结合伙伴可以成为新的共价候选药物的靶点。为了实现目标2，将并行执行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和使用特定的NFA探针鉴定新的NFA目标蛋白质，策略2)。对于质谱分析，我们使用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。与未修饰的多肽相比，9-NOA修饰多肽的质量增加了327.24 Da。为了覆盖尽可能广泛的潜在靶蛋白，并能够评估NFA对蛋白质修饰的一致性，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、初级粒细胞)进行实验。为执行这些项目，除消耗品资源外，还需要为一名博士后候选人提供36个月的人力资源。

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.