Repurposing FDA Approved Drugs as JNK3 Inhibitor for Prevention of Neuroinflammation Induced by MCAO in Rats

Abstract

Background: 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: JNK3 inhibitors; JNK3 kinase; MCAO stroke; brain degeneration; in silico docking; neuroinflammation; re-purposing.

Figure 1

The 3D structure of protein and ligand molecules (A) JNK3 (PDB ID: 3TTI). Structures of the selected FDA approved drugs (B) dabigatran, (C) estazolam, (D) leucovorin, and (E) pitavastatin.

Figure 2

Schematic plot for the in vivo study design. Rats were first acclimatized to the environment before stroke induction. Following drug administration after MCAO, rats were subjected to neurobehavioral analysis, and subsequently, tissues were collected for biochemical analysis or for preparing brain sections for 2,3,5-triphenyl tetrazolium chloride staining, followed by their histological analysis.

Figure 3

The refined 3D structure of JNK3. The 3D structural architecture of JNK3 is shown. α-helices and β-sheets are colored in green and yellow, and the linker region is colored in gray.

Figure 4

Time-dependent analysis of MD trajectories of apo and inhibitor bound states of JNK3. (A) RMSD plots were made via the least square fitting method using the backbone of the α-carbons. (B) Relative RMSF plot of apo-JNK3 (blue), JNK3^Dabigatran (green), JNK3^Estazolam (orange), and JNK3^Pitavastatin (pink). (C) Time versus intermolecular hydrogen bonding pattern for the 50 ns MD simulation.

Figure 5

Time versus binding energy plots for 50 ns MD simulation. The LJ-SR binding energy profile of the (A) dabigatran-JNK3, (B) estazolam-JNK3, and (C) pitavastatin-JNK3 complexes.

Figure 6

Surface view of RBX1 binding cavity orientation. (A) SP600125-JNK3, (B) Dabigatran-JNK3, (C) Estazolam-JNK3, and (D) Pitavastatin-JNK3. (E–H) SP600125, dabigatran, estazolam, and pitavastatin are indicated in red, green, orange, and pink colors respectively.

Figure 7

Docking and superimposed orientation of FDA drugs and SP600125 on JNK3. The post-docking study was pictured using DSV in 2D and 3D poses. The superimposed pose analysis and 2D depictions for dabigatran (green, B), estazolam (orange, D), pitavastatin (pink, F), and leucovorin (blue, H), while 3D images are shown (A, C, E, G) respectively for dabigatran, estazolam, pitavastatin, and leucovorin. SP600125 is demarcated by yellow colour in 2D images.

Figure 8

The effect of test drugs on MCAO induced neurodegeneration (A) The 28 point composite scoring. The test drugs significantly reduced neurological deficits. ***Indicates p<0.001 relative to sham group and ^#p<0.05, ^##p < 0.01 compared to the MCAO rats. Data are presented as mean ± SEM and were analyzed using a grouped two-way ANOVA (n = 10/group). SP600125 treated rats had a higher cumulative score whereas leucovorin treated rats had the least scores. The composite scores of dabigatran, pitavastatin, and estazolam treatment were comparable after 72 h of MCAO. (B) Brain coronal sections were stained with TTC after 72 h of MCAO. Data are presented as mean ± SEM and were analyzed by one-way ANOVA (n = 10/group). *** or ^###Indicates p < 0.001, and ^##Represents p < 0.01. The TTC sections were made from the same cohort as the behavioral (first cohort). (C) Coronal sections were separated by the frontal cortex (1), parietal cortex and insular cortex (2), and the piriform cortex (3). The region of interest is indicated by the square 1 and F. (D) Representative photomicrograph of H&E staining showing the extent of the surviving neurons in the cortex and striatum. H&E stained slides from coronal sections were prepared (first cohort). Arrowhead indicates relatively intact neurons in the treated groups while the open arrows indicate the swelled and vacuolated forms. Magnification 40x, scale bar = 20 μm, (n = 7/group). ***Indicates p < 0.001, and the symbols ^## and ^#Represent p < 0.01 and p < 0.05.

Figure 9

The effects of FDA approved drugs on endogenous antioxidant enzymes and oxidative stress markers (A and B) Histograms showing the results of the GST and GSH assays in the cortical and striatum homogenates from rat brains. (C) Histograms showing the CAT level in the cortical and striatum homogenates from rat brains. (D) Histograms showing the results of the LPO assay and MDA analyses in the cortical and striatum homogenates from the rat brain. The samples were collected from the second cohort. The results are represented as the means ± SEM (n = 7 rats/group) for three independent and reproducible experiments. The data were analyzed by one-way ANOVA followed by the post hoc Bonferroni multiple comparison test. The symbols *** or ^###Indicate significant differences at p < 0.001, whereas ^# and ^##Indicate significant difference at p < 0.05 and p < 0.01, respectively. (E) The effect of dabigatran, estazolam, leucovorin, and pitavastatin on HO-1 in the cortex and striatum was analyzed via immunohistochemistry. Scale bar = 20 μm, magnification 40x, (n = 7/group). HO-1 exhibited nuclear localization. Arrowhead indicated either HO-1 expression (MCAO group) in the nucleus while double open arrow indicated magnified cells. The slides were prepared from the first cohort of animals. *** or **Indicate p < 0.001 or p <0.01 respectively, while ^#Indicate p < 0.05.

Figure 10

The effect of dabigatran, estazolam, leucovorin, and pitavastatin on activated JNK3 in the cortex and striatum. (A) Histograms representing the JNK levels assayed via ELISA using extracts from the cortex and striatum. A one way ANOVA was used for the analysis (n = 7/group). The samples were collected from the 2nd cohort of animals.*** indicates p < 0.001, ^##Represents p < 0.01, ^#Indicates p < 0.05. (B) The effect of dabigatran, estazolam, leucovorin, and pitavastatin on p-JNK in the cortex and striatum was analyzed via immunohistochemical analysis. Scale bar = 20 μm. p-JNK exhibited cytoplasmic localization; the double open arrow shows magnified cells, while the arrowheads show p-JNK expression and the arrow show no expression. The samples were collected from the first cohort (n = 7/group). ***Indicates p < 0.001, ^##Represent p < 0.01, ^#Indicates p < 0.05.

Figure 11

The effect of the dabigatran, estazolam, leucovorin, and pitavastatin neuroinflammatory mediators. (A) The representative immunohistochemical staining of p-NF-κB in the cortex and striatum are shown. p-NF-κB exhibited nuclear localization; the arrowhead indicates no or less expression in the neuronal nucleus and the thin arrow indicates localization to the nucleus. Scale bar = 20 μm. One-way ANOVA was used to analyze the data (n = 7/group). *** and ^###Indicate p < 0.001, ^##Represents p < 0.01, ^#Indicates p < 0.05. The slides were prepared from the first cohort of animals (B) Representative immunohistochemical analysis of TNF-α in the cortex and striatum. Scale bar = 20 μm. One-way ANOVA was used to analyze the data (n = 7/group) ***Indicates p < 0.001, ^##Represents p < 0.01, ^#Indicates p < 0.05.

Figure 12

The graphical representation illustrates the underlying antioxidant and anti-inflammatory mechanisms of the re-profiled drugs against MCAO-induced brain injury using JNK3 as a target.