Abstract
Fatty acid nitroalkene derivatives (NO2-FA) activate Nrf2-regulated antioxidant gene expression and inhibit NF-κB-dependent cytokine expression. To better define NO2-FA structure-function relationships, a series of 22 new chemical entities (NCEs) containing an electrophilic nitroalkene functional group were synthesized and screened for both Nrf2- and NF-κB activities using luciferase-based assays. The structural variables were acyl chain length (11 to 24 carbons) and position of the electrophilic nitroalkene group. In luciferase-based reporter assays, Nrf2 was maximally activated by omega-12 nitroalkene fatty acids while TNFα stimulated NF-κB-inhibition was maximal for omega-5 nitroalkenes. The top pathway-modulating NO2-FAs were a) evaluated for an ability to activate Nrf2-dependent signaling and inhibit NF-κB-dependent inflammatory responses of RAW264.7 cells and b) compared to electrophilic compounds in clinical development. These findings revealed that 8/9-nitro-eicos-8-enoic acid (NCE−10) was collectively the most effective NCE and that both the α and ω acyl chain lengths influence nitroalkene activation of Nrf2 and inhibition of NF-κB signaling. This insight will guide development of more effective non-natural homologs of endogenously-detectable fatty acid nitroalkenes as anti-inflammatory and anti-fibrotic drug candidates.
Highlights
Nitro-fatty acids (NO2-FAs) are endogenously formed during metabolism and inflammation through the non-enzymatic reaction of unsaturated fatty acids with secondary products of nitric oxide (NO) and nitrite (NO2−) oxidation, in particular nitrogen dioxide (NO2)[1,2]
The potential cytotoxicity of all new chemical entities (NCEs) was first determined in HepG2 antioxidant response element (ARE) reporter cells and HEK293 NK-κB reporter cells
There was no cytotoxicity observed, using the MTT assay, in the HepG2 cells treated with all 22 NCEs for 18 hr up to a maximal concentration of 10 μM compared to media alone (Table 3)
Summary
Nitro-fatty acids (NO2-FAs) are endogenously formed during metabolism and inflammation through the non-enzymatic reaction of unsaturated fatty acids with secondary products of nitric oxide (NO) and nitrite (NO2−) oxidation, in particular nitrogen dioxide (NO2)[1,2]. NO2-FAs covalently adduct functionally significant cysteine residues (reacting with Cys[273] and 288) of Kelch-like ECH-associated protein (Keap)-1 in the cytoplasm, inducing stabilization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear translocation and transactivation of gene signaling. Fatty acid nitroalkenes alkylate functionally-significant cysteines of the p65 (Cys 38) and p50 (Cys 62) subunits of nuclear factor kappa B (NF-κB) leading to the inhibition of pro-inflammatory transcriptional activity This inhibits NF-κB translocation and DNA binding, suppression of vascular cell adhesion molecule 1 expression and the inhibition of monocyte rolling, adhesion and tissue infiltration[10,11,12].
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