Methyl 2-[3-(4-hydroxyphenyl)prop-2-enoylamino]-3-phenylpropanoate Is a Potent Cell-Permeable Anti-Cytokine Compound To Inhibit Inflammatory Cytokines in Monocyte/Macrophage-Like Cells.

Abstract

Cytokines are signaling molecules involved in inflammation process. Interleukin (IL)-6 is one of pivotal inflammatory cytokines associated with many human diseases. Therefore, there are on-going efforts to find a therapeutic to inhibit IL-6 and other cytokines. Methyl 2-[3-(4-hydroxyphenyl)prop-2-enoylamino]-3-phenylpropanoate (MHPAP) is a phenolic amide ester, transported better than its non-ester form (NEF) in monocyte/macrophage-like cells. However, there is no information about the effects of their cell permeability on cytokines. Therefore, the effects of MHPAP and NEF on cytokines were investigated in lipopolysaccharide (LPS)-stimulated THP-1 and human peripheral blood mononuclear cells (PBMCs). In the THP-1 cells, MHPAP significantly inhibited IL-6, IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha (P < 0.05), but NEF showed no effects. MHPAP also inhibited nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation in the THP-1 cells (P < 0.05), without significant effects on c-FOS, ATF-2, and JUN phosphorylations. Because NF-κB p65 is phosphorylated by IκB kinase (IKK), in silico analysis was performed on IKK. MHPAP was found to bind to IKK better than an IKK inhibitor ((E)-2-fluoro-4'-methoxystilbene). Furthermore, MHPAP inhibited the luminescence increased in the LPS-stimulated NF-κB-Luc2 THP-1 cells. As anticipated, MHPAP was also found to inhibit IL-6, IL-1beta, IL-8, and TNF-alpha significantly in LPS-stimulated PBMCs (P < 0.05). Especially, MHPAP inhibited IL-6 and IL-1beta with an IC50 of 0.85 and 0.87 µM, better than IL-8 (1.58 µM) and TNF-alpha (1.22 µM) in the cells. Altogether, the data suggest that cell permeability may have a significant impact on MHPAP's ability to inhibit cytokines and MHPAP may be used as a potent cell-permeable compound to inhibit inflammatory cytokines in monocyte/macrophage-like cells. SIGNIFICANCE STATEMENT: Potential effects of MHPAP and NEF on inflammatory cytokines (IL-6, IL-8, IL-1beta, and TNF-alpha) were investigated in LPS-stimulated THP-1 and PBMCs. Cell transport had a great impact on cytokine inhibition in the cells. MHPAP was also found to inhibit NF-κB pathway, which was supported by in silico and NF-κB reporter (Luc)-THP-1 data. Also, in LPS-stimulated PBMCs, MHPAP significantly inhibited IL-6, IL-1beta, IL-8, and TNF-alpha, suggesting that MHPAP may be a potent cell-permeable compound to inhibit inflammatory cytokines in monocyte/macrophage-like cells.