Drug Repurposing by Virtual Screening: Identification of New Already Approved ROCK Inhibitors as Promising Drugs to...

ROCK kinases are key players in neurodegenerative diseases such as Alzheimer's disease (AD), making them attractive therapeutic targets. In this study, we developed a pharmacophoric map of ROCK inhibitors and highlighted the key affinity sites in ROCK1/2 through molecular modeling. Virtual screening led to the identification of six approved drugs as ROCK inhibitors: ruxolitinib (36), baricitinib (37), ponatinib (38), tivozanib (39), nialamide (40), and tucatinib (41). Ruxolitinib (36) (hROCK1 IC₅₀ = 0.025 μM; hROCK2 IC₅₀ = 0.007 μM) and baricitinib (37) (hROCK1 IC₅₀ = 0.019 μM; hROCK2 IC₅₀ = 0.011 μM) showed the highest potency, while tivozanib (39) displayed 15-fold selectivity for ROCK2 over ROCK1. Molecular dynamics revealed that ruxolitinib (36) forms stable bidentate hydrogen bonds with the ROCK hinge region and has selectivity across the AGC kinase family. Biological assays confirmed ruxolitinib's (36) safety in neuronal and glial cells and its ability to reduce C3 immunolabeling, a glial inflammation marker, in LPS-treated astrocytes. These findings not only highlight ruxolitinib (36) as a promising candidate for AD but also provide a structural basis for designing novel dual JAK-ROCK inhibitors and pave the way for further in vitro and in vivo studies. Moreover, the validated pharmacophoric map for ROCK inhibition highlights the identification of an affinity pocket that can be useful for the design of new ROCK inhibitors.