Interaction of vernodalin with p38 mitogen-activated protein kinase (p38 MAPK) and subsequent effects in lung cancer cell model

The p38 mitogen-activated protein kinase (p38 MAPK) is a key regulator of numerous cellular processes relevant to Cancer therapy. Apart from Reactive Oxygen Species (ROS)-mediated activation, the p38 pathway can also be activated through direct interactions between the p38 MAPK protein which stabilize an open conformation of the activation loop, exposing the phosphorylation sites. Therefore, assessing p38 MAPK expression and the direct binding of Anticancer agents with p38 MAPK can provide critical insights for advancing the development of innovative Anticancer therapies. In this research, following the incubation of A549 lung Cancer cells with vernodalin (VN), a sesquiterpene lactone, several cellular assays were conducted. It was noted that VN suppressed the proliferation of A549 cells with IC₅₀ values of 65.80 μM, 39,90 μM, and 25.85 μM at 24 h, 48 h, and 72 h, respectively, while, IC₅₀ values were higher for VN in BEAS-2B human bronchial epithelial cells. Moreover, we discovered that VN leads to excessive ROS and MDA production by downregulating the activities of SOD/CAT/GPX, reducing GSH levels, and decreasing both HO-1 mRNA expression and activity, along with Nrft mRNA expression. We subsequently found that VN boosted the expression of p38 MAPK, Bax/Bcl-2 mRNA, and Caspase-3/-9 mRNA and activity in A549 cells. Molecular docking analysis revealed strong binding affinity between VN and p38 MAPK (-9.97 kcal/mol) near the ATP-binding site (Asp-150 and Arg-149) and the activation loop (Gly-170 and Leu-171) facilitated by hydrophobic as well as hydrogen bond interactions. The fluorescence spectroscopy analysis demonstrated that the spontaneous interaction between p38 MAPK and one molecule VN occurred with logK_b = 5.02 ± 0.18 and ΔG^∘ = -28.49 ± 1.91 kJ/mol. Multi-spectroscopy measurements revealed minor alterations in the p38 MAPK conformation following interaction with VN. We hypothesize that the pro-apoptotic effect of VN on lung Cancer cells may be mediated by two mechanisms: (1) ROS-dependent modulation of p38 pathway, and/or (2) direct ligand-receptor interaction between VN and critical residues near the ATP-binding pocket or activation loop of p38 MAPK, thereby stabilizing its catalytically active conformation. However, these preliminary findings necessitate further experimental validation and confirmation in subsequent studies.