One of the major key organelles for neuronal survival is the mitochondria. To maintain synaptic functions and neurotransmission, these cells rely upon high ATP levels and the regulated process of mitochondrial dynamics (fission and fusion) and mitophagy. Dysregulation in any of the mitochondrial processes, such as excessive fission and/or impaired removal of damaged mitochondria, can lead to damage to nerve cells and cause neuroinflammation, a major cause of various neurodegenerative diseases. Rho-associated coiled-coil kinase isoforms (ROCK1 and ROCK2) have emerged as critical upstream regulators of mitochondrial dynamics, where ROCK1 promotes Drp1-mediated mitochondrial fission. On the other hand, ROCK2 suppresses PINK1-Parkin-dependent mitophagy. Overexpression of both ROCK isoforms leads to increased ROS production and reduced ATP generation, compromising neuronal health. Although synthetic ROCK inhibitors show neuroprotective potential, their long-term clinical use is limited due to their toxicity, poor pharmacokinetics, and suboptimal BBB penetration. In contrast, natural compounds may offer a safer, cheaper and more effective inhibitory effect. The current study follows an integrated in-silico approach to screen natural phytochemicals derived from Indian medicinal plants, known for their neuroprotective effects. Based on neuroprotective properties and less explored mechanistic potential,11 plants were selected. From these plants, a library of 1,223 natural product compounds was downloaded, and a further 428 unique phytocompounds were obtained after curation with the known inhibitor for reference. These compounds were screened against key proteins ROCK1 and ROCK2 using molecular docking, followed by pharmacokinetic and toxicity class, MM-GBSA free binding energy, and molecular dynamics simulations for 200 ns. Several natural phytocompounds showed comparable binding affinities with respect to known inhibitors. Based on all the in-silico observations, Scopolin and Lycorine emerged as promising ROCK1 inhibitors, while Cytisoside and N-Methyltyramine demonstrated strong ROCK2 inhibition. Notably, Coumarin exhibited stable and persistent interactions with both ROCK1 and ROCK2, with good binding energies, high blood-brain barrier permeability, and strong dynamic stability across simulations. Collectively, all these findings indicate the natural phytochemicals as viable modulators of mitochondrial quality control. Particularly, Coumarin emerges as a promising dual ROCK inhibitor; its potential to restore mitochondrial fission and mitophagy balance and mitigate neurodegenerative diseases may be further validated using in vitro assays.
