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

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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.