Design of Novel Rho Kinase Inhibitors Using Energy Based Pharmacophore Modeling, Shape-Based Screening, in Silico Virtual Screening, and Biological Evaluation

Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound <b>8</b> exhibited an IC₅₀ value of 1.5 μM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI₅₀ value of 0.27 μM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound <b>8</b> with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 μM CC₅₀ (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor