Drug Repurposing by Virtual Screening: identification of new already approved ROCK inhibitors as promising drugs to target neurodegeneration

Rho-associated protein kinases (ROCK) play a pivotal role in various cellular processes and have emerged as therapeutic targets for neurodegenerative diseases such as Alzheimer's disease (AD). Inhibition of ROCK affects the production of key AD pathophysiological markers and reduces neuroinflammation. This study aims to accelerate the identification of effective candidates for neurodegeneration by drug repurposing. By conducting chemical space and crystallographic structure analyses, we developed a pharmacophoric map and optimized it to perform a virtual screening campaign. This approach led to the discovery of novel 6 already approved ROCK inhibitors: ruxolitinib (hROCK1IC50 = 0.025 µM; hROCK2IC50 = 0.007 µM), baricitinib (hROCK1IC50 = 0.019 µM; hROCK2IC50 = 0.011 µM), ponatinib (hROCK1IC50 = 0.197 µM; hROCK2IC50 = 0.056 µM), tivozanib (hROCK1IC50 = 12.86 µM; hROCK2IC50 = 0.856 µM), nialamide (hROCK1IC50 = 18.8 µM; hROCK2IC50 = 29.2 µM) and tucatinib (hROCK1IC50 = 51.45 µM; hROCK2IC50 = 18.62 µM). The results of this study highlight ruxolitinib and baricitinib as potent ROCK inhibitors with IC50 values in the low nanomolar range. Tivozanib showed 15-fold selectivity for ROCK2 over ROCK1. Molecular dynamics simulations with ruxolitinib revealed a tight binding with the ATP-binding site of ROCK1 and ROCK2, with stable bidentate hydrogen bonding interactions with the main chain of amino acid residues in the hinge region of ROCK1 and ROCK2. In addition, ruxolitinib showed selectivity for ROCK enzymes across AGC kinase family, highlighting its interesting ROCK/JAK multitarget profile, which can open new avenues in the discovery of novel multitarget-directed ligands for the treatment of neurodegenerative disorders. Biological validation revealed ruxolitinib’s safety profile against different neuronal and glial cell lines. Further, ruxolitinib reduced the immunolabeling for C3, a glial inflammation marker, in LPS-treated astrocytes. This work highlights ruxolitinib as a promising therapeutic option for Alzheimer's disease and other neurodegenerative conditions and pave the way for the new in vitro/in vivo studies for its repurposing.