Steered molecular dynamics and free-energy analysis of withanolide unbinding from tau kinases TTBK1 and GSK-3β

Tau hyperphosphorylation is identified as a critical process in microtubule destabilisation, neurodegeneration and synaptic dysfunction. Considering the kinases involved in this process, Tau Tubulin Kinase 1 (TTBK1) and Glycogen Synthase Kinase-3β (GSK-3β) have emerged as potential therapeutic targets. Here, we employed an integrated molecular simulation approach to investigate the binding thermodynamics and unbinding mechanisms of five Ashwagandha-derived withanolides sominone, withanolide A, withanolide D, withanone and withaferin A targeting TTBK1 and GSK-3β. Principal component analysis and free-energy landscape mapping identified distinct conformational basins and representative bound-state structures for each protein-ligand complex. Steered molecular dynamics simulations, followed by umbrella sampling and weighted histogram analysis, were used to characterise ligand dissociation pathways and determine the potentials of mean force. The calculated binding free energies ranged from −9 to −13 kcal mol−1, indicating favourable ligand binding affinity for both kinases. MM-GBSA analyses complemented these findings and identified van der Waals interactions as the primary contributors to protein-ligand complex stabilisation. Collectively, the results provide detailed molecular insights into ligand recognition, binding stability and dissociation energetics in tau-associated kinases. These findings highlight withanolidebased scaffolds as promising candidates for structure guided optimisation and the development of kinase targeted therapeutics against tau-related neurodegenerative disorders.Communicated by Kavitha J.