Indole and Coumarin Derivatives Targeting EEF2K in Aβ Folding Reporter Cells

Misfolding and accumulation of amyloid-β (Aβ) in the brains of patients with Alzheimer's disease (AD) lead to neuronal loss through various mechanisms, including the downregulation of eukaryotic elongation factor 2 (EEF2) protein synthesis signaling. This study investigated the neuroprotective effects of indole and coumarin derivatives on Aβ folding and EEF2 signaling using SH-SY5Y cells expressing Aβ-green fluorescent protein (GFP) folding reporter. Among the tested compounds, two indole (NC009-1, -6) and two coumarin (LM-021, -036) derivatives effectively reduced Aβ misfolding and associated reactive oxygen species (ROS) production. Additionally, these compounds decreased acetylcholinesterase and caspase-3/-6 activities while promoting neurite outgrowth. NC009-1 increased active phosphorylation of extracellular-signal regulated kinase (ERK) (T202/Y204), leading to an increase in inactive eukaryotic elongation factor 2 kinase (EEF2K) phosphorylation (S366). LM-021 decreased the active phosphorylation of AMP-activated protein kinase (AMPK) (T172) and EEF2K (S398), while LM-036 exhibited dual effects, increasing inactive phosphorylation and decreasing active phosphorylation of EEF2K. These changes in EEF2K phosphorylation led to decreased EEF2K activity and a subsequent reduction in inactive phosphorylation of EEF2 (T56). This cascade further promoted the phosphorylation of transcription factor cAMP-response-element binding protein (CREB) (S133) and the expression of brain-derived neurotrophic factor (BDNF), and reduced BCL-2 associated X-protein (BAX)/B-cell lymphoma 2 (BCL2) ratio. Knockdown of EEF2 abolished the effects of NC009-1, LM-021, and LM-036 on CREB phosphorylation, BDNF expression, caspase-3 activity, and neurite outgrowth. These findings demonstrate that NC009-1, LM-021, and LM-036 exert their neuroprotective effects through modulation of EEF2K signaling, highlighting their potentials as therapeutic candidates for AD.