Phytochemicals from Bacopa monnieri as small molecule modulators of MARK4: A multi-modal strategy for preventing Alzheimer's disease-causing tau aggregation.

Neurodegenerative tauopathies, such as Alzheimer's disease, are closely associated with the dysregulation of tau phosphorylation, a process regulated in part by the serine/threonine kinase MARK4. In this study, we explored phytochemicals derived from Bacopa monnieri as potential natural inhibitors of MARK4. Using pressurized liquid extraction with an ethanol-water mixture, we efficiently extracted bioactive compounds from Bacopa leaves. LC-MS analysis identified 25 distinct phytoconstituents spanning flavonoids, triterpenoids, cucurbitacins, sterols, and alkaloids. In silico analysis revealed that several compounds, including oroxindin, cucurbitacin B, and bacosine, bind strongly to the catalytic pocket of MARK4. Molecular dynamics simulations confirmed their stability within the MARK4 active site, with oroxindin demonstrating the most favorable thermodynamic and conformational profile. Principal component and free energy landscape analyses further supported their capacity to stabilize MARK4 in low-energy conformations. Microscale thermophoresis further validated high-affinity binding of MARK4 with oroxindin, while other four compounds also show strong interaction with MARK4. MTT assays in SH-SY5Y cells confirmed the non-cytotoxic nature of all five lead compounds across a concentration range of 10 nM to 10 μM. Cellular assays revealed a significant reduction in Tau-GFP aggregates upon treatment with the compounds, particularly oroxindin. These results highlight oroxindin and other Bacopa monnieri phytochemicals as promising natural inhibitors of MARK4, with potential to attenuate tau pathology in neurodegenerative diseases.