Discovery of 1,3-disubstituted prop-2-en-1-one derivatives as inhibitors of neutrophilic inflammation via modulation of MAPK and Akt pathways

Abstract

Targeting neutrophil function has gained attention as a propitious therapeutic strategy for diverse inflammatory diseases. Accordingly, a series of enone-based derivatives were developed to inhibit neutrophil-mediated inflammation, showing promise for treating inflammatory diseases. These compounds fall into two clusters with distinct effects: one inhibits neutrophilic superoxide (SO) anion production and elastase release triggered by N-formyl-Met-Leu-Phe (fMLF), with compound 6a being most effective (IC₅₀ values of 1.23 and 1.37 μM, respectively), affecting c-Jun N-terminal kinase (JNK) and Akt phosphorylation. The second cluster suppresses formation of SO anion without affecting elastase levels, surpassed by compound 26a (IC₅₀ of 1.56 μM), which attenuates various mitogen-activated protein kinases (MAPKs) with minimal Akt impact. Notably, none of the tested compounds showed cytotoxicity in human neutrophils, underscoring their potential as therapeutic agents against inflammatory diseases.

Keywords: Enone derivatives; elastase; human neutrophils; inflammation; superoxide.

Graphical abstract

Figure 1.

fMLF-induced signalling cascade for neutrophil activation. Chemoattractants such as formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF), are “formyl peptide receptor 1” (FPR1) agonists that orchestrate various neutrophil-activating signalling cascades. The activation of phosphoinositide-specific phospholipase C (PLC) leads to the production of inositol triphosphate (IP3) and diacylglycerol (DAG), which in turn triggers the activation of protein kinase C (PKC) and the release of calcium. Moreover, FPR activation modulates mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/Akt signalling axes which play pivotal roles in elastase release, chemotaxis, NADPH oxidase activation, and superoxide anion generation (Self-created image).

Figure 2.

The adopted structural diversification for the synthesised prop-2-en-1-one derivatives.

Scheme 1.

Synthesis of prop-2-en-1-one derivatives (1a–26a).

Figure 3.

Summary for the SAR of screened and synthesised prop-2-en-1-one derivatives. EW: electron-withdrawing; ED: electron-donating.

Figure 4.

Potent prop-2-en-1-one derivatives do not impact human neutrophils survival. Human neutrophils were subjected to DMSO (0.1%), or tested compound (10 μM) for 15 min. LDH levels in the cell-free supernatant were used for estimating cell viability. Cells were lysed with 0.1% Triton X-100 for 30 min at 37 °C in order to assess total LDH release. All data are provided as the mean ± SEM (n = 3).

Figure 5.

Compound 6a diminishes JNK and Akt phosphorylation in neutrophils activated by fMLF. Human neutrophils were subjected to either DMSO (0.1%) or compound 6a (10 μM) for 5 min, followed by stimulation with or without fMLF (0.1 μM) for 30 s. Subsequently, the cells were lysed and underwent immunoblotting analysis. The phosphorylation of (A) JNK, (B) ERK, (C) p38 MAPK, and (D) Akt was evaluated using antibodies that recognise both the total and phosphorylated forms of these proteins. The data were normalised to the levels of the total assessed protein and are presented as the mean ± SEM. ***p < 0.001 in comparison with DMSO + fMLF (n = 3).

Figure 6.

Compound 26a diminishes JNK, ERK, and p38 MAPK phosphorylation in neutrophils activated by fMLF. Human neutrophils were subjected to either DMSO (0.1%) or compound 26a (10 μM) for 5 min, followed by stimulation with or without fMLF (0.1 μM) for 30 s. Subsequently, the cells were lysed and underwent immunoblotting analysis. The phosphorylation of (A) JNK, (B) ERK, (C) p38 MAPK, and (D) Akt was evaluated using antibodies that recognise both the total and phosphorylated forms of these proteins. The data were normalised to the levels of the total assessed protein and are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 in comparison with DMSO + fMLF (n = 3).