Machine Learning-Integrated Pharmacophore, DFT Analysis, and molecular dynamics of Diosmetin as a potent ache inhibitor with neuroprotective activity in a Scopolamine-Induced alzheimer's zebrafish model.

Alzheimer's Disease (AD) is a degenerative disorder of the brain that causes a gradual loss of cognitive function. The cholinergic hypothesis suggests that acetylcholine deficiency is the main cause of AD, which explains why blocking acetylcholinesterase (AChE) is the most effective way to treat AD. Nevertheless, there are some drawbacks to the currently available AChE inhibitors; thus, new molecules with better therapeutic effects and fewer side effects are needed. In this study, the anti-Alzheimer activity of diosmetin, a natural flavonoid, was investigated via an integrated computational and experimental approach. Pharmacophore mapping revealed that the essential chemical features of diosmetin are responsible for AChE inhibition, and density functional theory calculations were employed to investigate its electronic properties and chemical behavior. Molecular docking experiments indicated that diosmetin could bind firmly to AChE with a binding energy of -9.49 kcal/mol. Molecular dynamics simulations strengthened this hypothesis by showing that the diosmetin-AChE complex remained stable over time. In vivo verification using a scopolamine-induced zebrafish model of Alzheimer's disease revealed that diosmetin administration notably enhanced learning and memory abilities in zebrafish. Various behavioral paradigms, including the light/dark preference test, novel tank diving test, T-maze test, and novel object recognition test, have been used to assess cognitive function. Biochemistry revealed that diosmetin counteracted the scopolamine-induced increase in AChE activity, increased oxidative stress, increased myeloperoxidase inflammatory markers, decreased antioxidant activity, and restored normal histology in the brains of the zebrafish. Most importantly, high-dose diosmetin demonstrated comparable neuroprotective efficacy to donepezil in behavioral and biochemical assays while exhibiting weaker molecular binding affinity toward AChE, as indicated by MM-PBSA analysis, underscoring that similar in vivo outcomes do not necessarily imply molecular equivalence at the binding level.