SAR-407899

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na⁺/K⁺-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

Recovery after ischemic stroke is slow and highly variable.Activated ROCK (Rho-associated coiled-coil kinase) pathway hampers recovery of impaired neurons.Though inhibiting ROCK pathway has shown therapeutic effects in vitro, the selectivity of most of the ROCK inhibitors is still not investigated.Present study aims to investigate the binding affinity in silico of nine widely used ROCK inhibitors with brain-specific ROCK2 isoform.Three-dimensional structures of ROCK2 and eight drugs were taken from Protein Data Bank and PubChem Chem. Compound Database, resp., whereas, FSD-C10 structure was generated based on Xin et al., 2015.In docking, ROCK2 was set to be rigid and drugs were free to rotate.All simulations were carried out using Autodock-4.2.This study demonstrated strong complexation between all ligands and ROCK2.All ROCK inhibitors, except FSD-C10, were able to bind to ROCK2 more strongly [Binding constant (Ka) between 2.6 - 36.7 x 10⁵ M^-1] than fasudil (Ka = 2.5 x 10⁵ M^-1).SLx-2119 (KD-025) had the highest binding constant (Ka = 36.7 x 10⁵ M^-1) thus succeeding as a better ROCK2 specific inhibitor.Selectivity of ROCK inhibitors (in silico) towards ROCK2 can be an indicative measure to estimate therapeutic benefits or adverse effects prior to in vitro study.