Alzheimer's disease (AD) is the most common form of dementia and a progressive neurodegenerative disorder characterized by severe cognitive decline, memory impairment, personality disturbances, and deficits in language and executive function. Despite being extensively studied, the precise etiology and pathogenesis of AD remain incompletely understood, and no definitive diagnostic test exists for its early detection. Clinical diagnosis currently relies on a combination of cognitive assessments, neurological examinations, neuroimaging, and laboratory analyses, but these methods often fail to detect the disease in its preclinical or prodromal stages [[1], [2], [3]].

AD pathology is primarily driven by the abnormal accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein [[4], [5], [6], [7]]. Tau is a microtubule-associated protein that, under pathological conditions, becomes hyperphosphorylated, loses its affinity for microtubules, and aggregates into toxic fibrils that impair neuronal integrity and synaptic function. These tau aggregates are strongly correlated with disease severity and progression. In the early stages of AD, patients typically present with episodic and spatial memory deficits, while long-term semantic memory is affected in more advanced stages.

Despite the high global burden of AD, current pharmacological interventions are limited in efficacy. As of now, only five FDA-approved drugs are available for symptomatic treatment - four cholinesterase inhibitors and the NMDA receptor antagonist memantine [8,9]. These treatments provide modest symptomatic relief but fail to address the underlying molecular pathology, highlighting the urgent need for disease-modifying therapies that can prevent or slow neurodegeneration.

A growing interest has been directed toward the therapeutic potential of medicinal plants rich in neuroprotective phytochemicals. Bacopa monnieri is a perennial herb belonging to the Scrophulariaceae family and has been a cornerstone of Indian ayurvedic medicine for over 3000 years [10,11]. Traditionally used to enhance memory, attention, and learning, Bacopa has also demonstrated a wide spectrum of biological activities, including antioxidant, anti-inflammatory, antipyretic, antiepileptic, antidiabetic, and anticancer effects [[12], [13], [14], [15]]. Preclinical studies suggest that Bacopa monnieri may support neuronal regeneration, restore synaptic activity, and improve cognitive function, making it a promising candidate for AD therapy [16,17].

Among emerging molecular targets, microtubule afffinity-regulating kinase 4 (MARK4), a member of the serine/threonine kinase family, has gained considerable attention. MARK4 plays a major role in phosphorylating tau at KXGS motifs (e.g., Ser262), thereby promoting its dissociation from microtubules and facilitating aggregation. MARK4 overexpression has been observed in AD brains, and its inhibition has been shown to reduce tau hyperphosphorylation and neurotoxicity, making it an attractive therapeutic target [18,19].

In this study, we aimed to systematically investigate the neuroprotective potential of phytoconstituents derived from Bacopa monnieri by targeting MARK4. We initially extracted 25 compounds from Bacopa and subjected them to ADME and toxicity filtering. Among these, five compounds - oroxindin, cucurbitacin B, cucurbitacin E, ebalin lactone, and bacosine - exhibited the most favorable binding affinities. Subsequent computational analyses validated the stability of these ligand-MARK4 interactions and further characterized the conformational changes and energetics associated with binding. In particular, oroxindin demonstrated the highest binding affinity and conformational stabilization of the MARK4 catalytic site. All five compounds were also found to be non-cytotoxic in SH-SY5Y cells up to 10 μM, as confirmed by MTT assays. Furthermore, Tau-GFP-expressing SH-SY5Y cells revealed a significant reduction in intracellular tau aggregates following treatment with these compounds, suggesting functional inhibition of MARK4 in a cellular context. Taken together, our integrative computational, biochemical, and cellular approach identifies promising lead compounds from Bacopa monnieri, particularly oroxindin, as potential natural inhibitors of MARK4.