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Zikra Zulfiqar,1 Fawad Ali Shah,1 Shagufta Shafique,2 Abdullah Alattar,3 Tahir Ali,4 Arooj Mohsin Alvi,1 Sajid Rashid,2 Shupeng Li5 1Department of Pharmacology, Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan; 2National Center for Bioinformatics, Quaid-I-Azam University, Islamabad, Pakistan; 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia; 4Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; 5State Key Laboratory of Oncogenomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, People’s Republic of ChinaCorrespondence: Fawad Ali Shah; Shupeng Li Tel +92-51-2891835-8Fax +92-51-2890690Email fawad.shah@riphah.edu.pk; lisp@pkusz.edu.cnBackground: Stress-associated kinases are considered major pathological mediators in several incurable neurological disorders. Importantly, among these stress kinases, the c-Jun NH2-terminal kinase (JNK) has been linked to numerous neuropathological conditions, including oxidative stress, neuroinflammation, and brain degeneration associated with brain injuries such as ischemia/reperfusion injury. In this study, we adopted a drug repurposing/reprofiling approach to explore novel JNK3 inhibitors from FDA-approved medications to supplement existing therapeutic strategies.Materials and Methods: We performed in silico docking analysis and molecular dynamics simulation to screen potential candidates from the FDA approved drug library using the standard JNK inhibitor SP600125 as a reference. After the virtual screening, dabigatran, estazolam, leucovorin, and pitavastatin were further examined in ischemic stroke using an animal rodent model of focal cerebral ischemia using transient middle cerebral artery occlusion (t-MCAO). The selected drugs were probed for neuroprotective effectiveness by measuring the infarct area (%) and neurological deficits using a 28-point composite score. Biochemical assays including ELISA and immunohistochemical experiments were performed.Results: We obtained structural insights for dabigatran, estazolam, and pitavastatin binding to JNK3, revealing a significant contribution of the hydrophobic regions and significant residues of active site regions. To validate the docking results, the pharmacological effects of dabigatran, estazolam, leucovorin, and pitavastatin on MCAO were tested in parallel with the JNK inhibitor SP600125. After MCAO surgery, severe neurological deficits were detected in the MCAO group compared with the sham controls, which were significantly reversed by dabigatran, estazolam, and pitavastatin treatment. Aberrant morphological features and brain damage were observed in the ipsilateral cortex and striatum of the MCAO groups. The drugs restored the anti-oxidant enzyme activity and reduced the levels of oxidative stress-induced p-JNK and neuroinflammatory mediators such as NF-kB and TNF-ɑ in rats subjected to MCAO.Conclusion: Our results demonstrated that the novel FDA-approved medications attenuate ischemic stroke-induced neuronal degeneration, possibly by inhibiting JNK3. Being FDA-approved safe medications, the use of these drugs can be clinically translated for ischemic stroke-associated brain degeneration and other neurodegenerative diseases associated with oxidative stress and neuroinflammation.Keywords: re-purposing, in silico docking, JNK3 kinase, MCAO stroke, JNK3 inhibitors, neuroinflammation, brain degeneration |
