Tozasertib Attenuates Neuropathic Pain by Interfering with Aurora Kinase and KIF11 Mediated Nociception
Ankit Uniyal,# P. A. Shantanu,# Shivani Vaidya, Daria A. Belinskaia, Natalia N. Shestakova, Rajnish Kumar, Sanjay Singh, and Vinod Tiwari*
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ABSTRACT: Kinesins are the motor proteins that transport excitatory receptors to the synaptic membrane by forming a complex with receptor cargo leading to central sensitization causing neuropathic pain. Many regulatory proteins govern the transit of receptors by activating kinesin, and Aurora kinases are one of them. In this study, we have performed in silico molecular dynamics simulation to delineate the dynamic interaction of Aurora kinase A with its pharmacological inhibitor, tozasertib. The results from the molecular dynamics study shows that tozasertib- Aurora kinase A complex is stabilized through hydrogen bonding, polar interactions, and water bridges. Findings from the in vitro studies suggest that tozasertib treatment significantly attenuates lipopolysaccharide (LPS)-induced increase in oxidonitrosative stress and kif11 overexpression in C6 glial cell lines. Further, we investigated the regulation of kif11 and its modulation by tozasertib in an animal model of neuropathic pain. Two weeks post-CCI surgery we observed a signifi cant increase in pain
hypersensitivity and kif11 overexpression in DRG and spinal cord of nerve-injured rats. Tozasertib treatment signifi cantly attenuates enhanced pain hypersensitivity along with the restoration of kif11 expression in DRG and spinal cord and oxidonitrosative stress in the sciatic nerve of injured rats. Our fi ndings demonstrate the potential role of tozasertib for the management of neuropathic pain. KEYWORDS: Aurora kinase, Kif11, chronic constriction injury, neuropathic pain, molecular dynamics simulation, tozasertib
Chronic pain is accompanied by many neurological diseases such as brain or spinal cord injury, tumors, neurodevelopmen- tal disorders, neurodegeneration, neuroinfl ammation, etc. Neuropathic pain occurs when there is actual or potential damage to the somatosensory system. Due to this damage, various inflammatory mediators and toxins are released which causes nonspecific stimulation of nociceptors resulting in increased synaptic plasticity leading to both peripheral and central sensitization.1 Recent epidemiological studies suggest that about a quarter of the world’s population suffers from the pain of which 7-8% of cases are neuropathic in origin. Even for more than a century of research on nociception and analgesia, 35% of neuropathic pain patients do not respond to standard treatment and show serious side eff ects.2 The clinically available drugs fail to provide adequate pain relief as neuropathic pain progresses to a chronic state due to its dose-limiting side eff ects and the inability of current pharmacotherapy to counteract the pain progression as they fail to act on cellular and molecular mechanisms driving neuropathic pain which remains elusive.3 Thus, identifying the
potential targets and development of efficacious drugs with minimal side eff ects is of utmost clinical importance.4
Various receptors are involved in the development and maintenance of neuropathic including TRPV-1, Nav1.6, NMDA, and LPA1, etc.5 The pathogenesis of chronic neuropathic pain has been proven to include hyperactivation
of glutamatergic transmission. Prolonged activation of nociceptors or damage to the aff erent nerve leads to the central sensitization of the nociceptive system. The putative electrophysiological mechanism of this sensitization (wind-up phenomenon) depends on the activation of N-methyl-D- aspartate receptors (NMDAR).7 Blocking glutamate receptors has been shown to alleviate both acute and chronic pain in animal models. It has been proven that NMDA receptors play
Received: January 23, 2021 Accepted: May 17, 2021 Published: May 24, 2021
© 2021 American Chemical Society
ACS Chem. Neurosci. 2021, 12, 1948-1960
Figure 1. (A) The molecule of tozasertib bound in the binding site of human Aurora kinase A according to X-ray data (structure code 3E5A).18 The hydrogen atoms in the protein molecule were added using Nanoscale Molecular Dynamics (NAMD) molecular modeling software.25 (B) Comparative analysis of primary sequences of human and rat Aurora kinase A. The symbol # is displayed in the consensus sequence when a polar amino acid is replaced with a polar one (glutamine, glutamate, asparagine, aspartate); ! is when valine is replaced with isoleucine. The bottom line is the so-called consensus sequence. It is a generalized sequence that is obtained by comparing letters in the alignment columns. When homology is high, the consensus line represents the predominant amino acid, indicated by a red capital letter. When homology is low, the consensus line represents the predominant amino acid indicated by a lowercase blue letter.
a role in the development of chronic pain syndromes, and NMDA receptor antagonists can be considered as agents for the relief and prevention of the development of chronic neuropathic pain.8 However, direct NMDAR blockade is associated with a number of side eff ects; therefore, an indirect eff ect on NMDAR is preferable for the safe treatment of neuropathic pain. One of the indirect methods of exposure is the calcium-dependent desensitization of NMDAR, described in our previous study.9 An indirect eff ect on the conduct (blocking, relief) of pain by targeting the NMDA receptor can also occur at the stage of its synthesis, maturation, or transport of excitatory receptors to the synaptic membrane. Thus, NMDA receptor transportation to the synaptic membrane represents a potential therapeutic target for various chronic neurological conditions.
Kinesins are the motor proteins that deliver the newly synthesized receptors to the surface of the synaptic membrane making them functional. The delivery of excitatory receptors to the synaptic membrane is upregulated in neuropathic pain conditions. The nucleus and rough endoplasmic reticulum synthesize the receptors. Further the Golgi body packed them into the vesicles. Kinesins are localized into the cytoplasm in autoinhibited state. After their activation by various kinase enzymes (like cyclin-dependent kinase 5 (CDK-5) and Ca2+/
calmodulin kinase 2), kinesins attach to their respective cargos (vesicles containing receptors). The binding is facilitated by scaffolding adapter proteins, and fi nally the whole complex binds to the microtubule and movement occurs across the axon. On reaching the synaptic membrane the whole complex gets disassembled and the receptor is delivered to the dendritic surface and made functional. Researchers are targeting kinesins and enzymes responsible for the surface delivery of receptors
for pain relief. Kinesins-5 superfamily member 11 (kif11/
eg5) is a molecular motor protein responsible for the delivery of NR2b subunit of NMDA receptors to the synapse causing its upregulation.13,14 There are many regulatory proteins (like kinases) involved in the activation and regulation of kinesin for the delivery of receptors to form motor-protein complexes. Aurora kinase is a serine-threonine kinase that is involved in cell division. Recently it has been shown that Aurora kinase B
plays a critical role in spinal microgliosis during the neuropathic pain condition in nerve-injured rats.15 Aurora kinase A phosphorylates kif11/eg-5 during mitosis to form mitotic spindle, while Aurora kinase B is involved in ensuring correct attachment to centromeres, and Aurora kinase C is found in cells undergoing meiosis.16 Tozasertib is known for being one of the strongest inhibitors of Aurora kinase, with an inhibitory eff ect at nanomolar concentrations.17 Until now, interest in the study of tozasertib as an inhibitor of Aurora kinases has been determined in terms of its use in the treatment of cancer, including myelogenous leukemia.18 Hitherto, to the best of our knowledge, the role of Aurora kinase-mediated regulation of kif11 causing amelioration of pain is not known. Kinases are master switches that regulate multiple signaling and cellular responses; thus we dissected a novel mechanism involving the interactions of Aurora kinases and kinesin motor protein kif11 that might be involved in the suppression of pain-like behavior. In the presented work, for the fi rst time, an integrated approach was applied for investigating the possibility of using tozasertib for the management of neuropathic pain syndrome. We studied molecular features of the interaction of the drug with Aurora kinase using molecular modeling methods. Further, in in vivo experiments, the functional eff ect of the drug was studied using a chronic constriction injury (CCI) model. Finally, the mechanism of the analgesic eff ect of tozasertib was determined in an in vivo experiment on the dorsal root ganglion (DRG), spinal cord (SC), and isolated sciatic nerve of experimental rats and on a culture of C6 glial cells. Our results demonstrated the role of Aurora kinase-mediated regulation of kif11 causing amelioration of pain.
RESULTS AND DISCUSSION
In Silico Studies. Tozasertib is an anticancer drug that has undergone clinical trials for lymphoblastic leukemia, myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and colorectal cancer.19 The in vitro studies have suggested that tozasertib can regulate cell death via caspase-3, receptor-interacting serine/threonine-
Figure 2. (A) Protein-ligand RMSD for tozasertib and Aurora kinase A. Left Y-axis shows RMSD evolution for the protein. The stability of ligand is represented by right Y-axis. (B) Protein-ligand contacts for tozasertib and Aurora kinase A. Four types of interaction were categorized and studied, namely, hydrogen bonding, hydrophobic interactions, ionic or polar interactions, and water bridges.
Figure 3. Root mean square fluctuation for tozasertib and Aurora kinase A: (A) RMSF for Aurora kinase A; (B) RMSF for tozasertib; (C) timeline representation of the interactions and contacts for tozasertib and Aurora kinase A; (D) schematic of detailed ligand atom interactions with the protein residues.
protein kinase 1 (RIPK-1) and poly(ADP-ribose) polymerase, phase inducer phosphatase 3 enzyme.20,21 Further, the
mast cell responsiveness via NFkB signaling, epigenetic functioning via histone deacetylases, and other downstream cellular processes via cyclin B, ERK, and cdc25c which is an M-
neuroprotective eff ect of tozasertib is also acknowledged by acting through the DLK/JIP3/MA2K7/JNK signaling path- way.22 The main side eff ect of tozasertib is the prolongation of
Figure 4. Eff ect of tozasertib on in vitro cell viability and reactive oxygen species (ROS) quantifi cation. (A) Effect of tozasertib on % cell viability. Tozasertib at 1 nM, 10 nM, and 20 nM did not alter the cell viability of C6 glial cells (n = 6). (B) Eff ect of tozasertib on lipopolysaccharide (LPS) (10 μg/mL) induced ROS generation. The LPS induced C6 glial cells were further exposed to the diff erent concentrations of tozasertib. The fluorescent microscope was used for imaging (bar = 5 μM). The fi gure shows the phase contrast, fluorescence, and merged images of control, LPS (10 μg/mL), LPS+T (10 and 20 nM). (C) Bar graph showing the LPS-induced ROS production reversed by higher concentrations of tozasertib. The data are expressed as the mean ± SEM and analyzed for statistical signifi cance using one-way ANOVA followed by Tukey’s post hoc analysis for multiple comparisons (n = 3). Tozasertib dose: 1T, 1 nM; 10T, 10 nM; 20T, 20 nM.
the QT interval, which is why its clinical trials as an anticancer drug were stopped.23 Tozasertib has been identifi ed as a potential inhibitor of pan Aurora kinase enzymes. Although the crystal structure of Aurora kinase with cocrystallized tozasertib is reported earlier but as a general limitation of crystal structures, it captures only a single pose. However, the protein-ligand interactions are always dynamic in nature. To overcome this limitation and to investigate the architectural interplay of tozasertib with Aurora kinase, we performed classical molecular dynamics simulation of the complex obtained from the crystal structure (PDB code 3E5A).24 Figure 1A shows the conformation of tozasertib in the Aurora kinase A binding site according to the crystallographic data. Amino acids Leu139, Phe144, Lys162, Leu210, Tyr212, Thr217, Lys224, Leu263, and Asp274 are located in the immediate vicinity of the drug sorbed in the binding site. Amino acid residues Gly140, Val147, Leu194, Glu211, Pro214, Leu215 are positioned within 4 Å from the tozasertib molecule as well (not shown).
At the next stage, we performed the alignment of the primary sequences of human and rat Aurora kinase A (Figure 1B). According to the alignment result, the primary sequence identity between human and rat Aurora kinase A is 82.9%. All amino acids of the binding site for tozasertib are identical in the kinases of these organisms. For this reason, we suppose that the eff ectiveness of the interaction of the drug with human and rat Aurora kinase A will be similar; therefore, in vitro and ex vivo experiments are most likely to give similar results for these species. However, the pharmacokinetics of tozasertib may diff er due to the fact that rats and humans have diff erent concentrations of transport proteins in the blood and diff erent ratios of the concentrations of enzymes involved in
trials will help to evaluate the eff ectiveness of the drug for the management of neuropathic pain syndrome in humans.
For the first time we have conducted studies on the architectural interaction between tozasertib and Aurora kinase using molecular dynamics simulation. The MD results made it possible not only to determine the location and structure of the protein pockets for binding to tozasertib molecules but also to assess their degree of conformational fl uctuations. For tozasertib Figures 2 and 3 carry all the analysis data of molecular dynamics (MD) trajectory. The root-mean-square deviation (RMSD) plot (Figure 2A) shows that the protein RMSD remains between 1 and 2 Å indicating stable conformation of the protein during the simulation time frame. Tozasertib interacts with the ATP binding site of Aurora kinase A and inhibits the phosphorylation process. During the initial 15 ns the protein and ligand RMSD fl uctuates a little bit which indicated equilibration. Later, from 15 to 50 ns the ligand-protein complex is converged to a stable state and the RMSD remains between 1 and 2 Å and 2- 3.5 Å for Aurora kinase A and tozasertib, respectively.
The interaction of the ligand with the individual amino acid residues in the binding pocket of Aurora kinase A is quantified and depicted in Figure 2B. It can be seen from the plot that the ligand interacted with the Aurora kinase A active site with ALA_213 and GLU_211 subunits through hydrogen bonding. The hydrogen bonding played a major role in the interaction of this ligand with Aurora kinase A active site.
Further, the hydrophobic bonding with PHE_144 and water bridges with GLU_260 and LYS_143 were also observed. Finally, we found ligand-protein polar interaction or ionic interaction with the ARG_260 subunit. Furthermore, in order to examine the fl exibility of the structure of the protein, we have calculated the root-mean-square fluctuation (RMSF)
These diff erences might lead to failure when
(Figure 3A). The whole protein had an RMSF value between
studying the effi cacy of tozasertib in patients. Further clinical 0.4 and 2.2 Å except the N- and C-terminal amino acids which
showed moderate movement throughout the 50 ns long simulation. The RMSF plot of the ligand (Figure 3B) indicated that the ligand largely stayed bound to the active site amino acids as the residues were observed consistently throughout the simulation.
Finally, a timeline of protein-ligand interactions in the form of hydrogen bonds, hydrophobic interaction, polar interaction, and water bridges was plotted as shown in Figure 3C throughout the simulation time of 50 ns. The top panel indicates the total number of specific protein-ligand contacts, and the bottom panel indicates residue level interaction of the ligand. The results have demonstrated that there were a minimum of six contacts between ligand and protein throughout the simulation which is indicative of good ligand-protein interactions. Figure 3D shows specifi c inter- actions of the ligand with active site amino acid residues of Aurora kinase A. Thus, the information obtained by the molecular modeling methods reveals the details of the interaction between tozasertib and Aurora kinase and explains why tozasertib is such a potent inhibitor of this enzyme.
In Vitro Studies. 2.2.1. Eff ect of Tozasertib on Cell viability. Around 1.2 × 104 cells were seeded into a 96-well plate, and diff erent concentrations of tozasertib (1 nM, 10 nM, 20 nM) were exposed. Increasing the tozasertib concentration until 20 nM was found to be nontoxic as it did not significantly alter the cell viability (Figure 4A). We have also found that increasing the drug concentrations beyond 20 nM significantly reduced cell viability (data not shown). Therefore, 1, 10, and 20 nM concentrations were chosen for further biochemical studies.
Eff ect of Tozasertib on LPS Induced ROS Generation. It is a well-known fact that during pathological conditions excitatory receptors (such as NMDA) contribute to the generation of oxidative stress and thereby cause neuro- toxicity.28 Further, the excitatory synaptic transmission promotes the development and maintenance of neuropathic pain. The involvement of ROS and oxidonitrosative stress is well evident in promoting the pathophysiology of neuropathic pain. Thus, we used the cell line model of LPS induced inflammation. We observed a significant eff ect across the groups on ROS production (F(6,14) = 12.26; p < 0.001) using one-way ANOVA and Tukey’s post hoc analysis. 2′,7′- Dichlorofluorescin diacetate (DCFDA) assay revealed the total ROS generated by the C6 glial cell upon induction by LPS (10 μg/mL). LPS treatment to cells caused an increase in ROS generation leading to increased fl uorescence intensity. The fl uorescence intensity was found to be signifi cant compared to the control group (P < 0.001). Tozasertib at 10 and 20 nM concentration was found to significantly reduced ROS production compared to the LPS group (Figure 4B and Figure 4C; P < 0.001).
Eff ect of Tozasertib on LPS Induced Depletion of Glutathione Level. Glutathione is an important antioxidant responsible for scavenging free radicals. A significant effect was found across the groups on reduced glutathione levels (F(5,12) = 27.1; p < 0.001) using one-way ANOVA and Tukey’s post hoc analysis. The LPS (10 μg/mL) treatment to C6 glial cells induced significant depletion of glutathione levels as compared to the control group (p < 0.001). Following tozasertib treatment, a significant improvement in reduced glutathione levels was observed thereby ameliorating the oxidative stress (p
< 0.01 vs LPS group) (Figure S1A).
Eff ect of Tozasertib on LPS Induced Nitrosative Stress. Nitrite is another important marker for cellular stress that promotes the pathophysiology of neuropathic pain. A significant eff ect was observed across the groups on nitrite levels (F(5,18) = 12.64; p < 0.001) using one-way ANOVA followed by Tukey’s post hoc analysis. The LPS (10 μg/mL) treatment in C6 glial cells induced a signifi cant increase in nitrite level compared to the control group (p < 0.001). Tozasertib at a lower concentration (1 nM) did not produce any signifi cant reduction of nitrite level as compared to LPS. However, tozasertib at higher concentrations (10 and 20 nM) was found to be significant in reducing nitrite level after LPS (Figure S1B) (P < 0.01 and P < 0.001 vs LPS group).
Effect of Tozasertib on LPS Induced Malondialde- hyde (MDA) Level. MDA gives a direct prediction of lipid peroxidation which is again a striking feature of oxidative stress promoting the pathophysiology of chronic pain. There was a significant eff ect on nitrite levels across the groups (F(5,18) = 12.99; p < 0.001) using one-way ANOVA followed by Tukey’s post hoc analysis. The LPS (10 μg/mL) treatment caused a profound increase in the MDA level which was signifi cantly higher than the control group (P < 0.001, Figure S1C). Tozasertib at 20 nM but not at 10 nM concentration was found to decrease the MDA level significantly (P < 0.01 vs LPS) in LPS+C6 glial cells.
The induction of LPS has led to the activation of glial cells which progressively caused an increase in ROS, MDA, nitrite generation as well as a decrease in reduced glutathione levels, which are indicative of oxidonitrosative stress pathway activation. After treatment, tozasertib caused a decrease in LPS induced nitrite and MDA levels as well as an increase in reduced glutathione levels. The tozasertib has reduced the LPS induced ROS and oxidonitrosative stress in the glial cell. This could lead to the suppressed neuronal excitotoxicity and maintenance of local cellular functioning thereby promoting antinociception.
Eff ect of Tozasertib on Kif11 Expression. Kif11 transports excitatory receptors especially NMDA and increases its surface expression which promotes excitotoxicity. Fur- thermore, the Aurora kinase regulates the kif11; thus we evaluated the eff ect of tozasertib on kif11 expression. We found a signifi cant eff ect across the groups (F(6,14) = 5.371; p < 0.001). Expression of kif11 signifi cantly increased after LPS treatment in C6 glial cells (Figure 5A and Figure 5B) as compared to the control group (P < 0.01). Treatment with tozasertib at a higher concentration (20 nM) was found to be significant in reducing the kif11 expression with respect to LPS (P < 0.05).
In Vivo Studies. 2.3.1. Eff ect of Tozasertib on Mechanical Allodynia. The ispisilateral mechnical paw withdrawal threshold had a significant eff ect across the groups (F(5, 30) = 41.5; p < 0.001) and time points (F(6,180) = 142; p < 0.001) analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. On the 14th day after sciatic nerve injury, we observed a significant decrease in the mechanical paw withdrawal threshold in the ipsilateral paw (P < 0.001) but no significant changes in the contralateral paw was observed compared to the pre-CCI surgery threshold. The drug was administered intraperitoneally on the 14th day. The mechan- ical paw withdrawal threshold was measured at 30 min, 60 min, 120 min, 240 min, and 24 h following the drug administration. Tozasertib at both doses 10 and 20 mg/kg signifi cantly increased paw withdrawal threshold value at 60 and 120 min
Figure 5. Eff ect of tozasertib on kif11 expression in C6 glial cells. Kif11 expression was signifi cantly increased by LPS in C6 glial cells, and its expression was reversed after tozasertib treatment. Data were expressed as the mean ± SEM and analyzed by one-way ANOVA (post hoc analysis: Tukey’s test) (n = 3). ## represents signifi cance compared to the control group (p < 0.01); ∗ (p < 0.05) represents signifi cance compared to the LPS group. Tozasertib dose: 1T, 1 nM; 10T, 10 nM; 20T, 20 nM. CNTL, healthy animals. Perse, this group was used to study the effect of drug on eg-5 expression on normal cell lines (without inducing pathological conditions by LPS).
compared to the CCI group (P < 0.001; Figure 6A and Figure 6B), and tozasertib produced a similar pain-relieving eff ect as compared to standard drug gabapentin (30 mg/kg). No significant diff erence was observed in the paw withdrawal thresholds of the contralateral paw.
Eff ect of Tozasertib on Thermal Hyperalgesia. A significant eff ect was observed on ipsilateral paw withdwawal latancy across the groups (F(5,30) = 26.5; p < 0.001) and time points (F(6,180) = 62.7; p < 0.001) after two-way ANOVA followed by Bonferroni’s multiple comparisons test. There was a significant decrease in the thermal paw withdrawal threshold in the ipsilateral paw (P < 0.001) on the 14th day after CCI sciatic nerve injury. We did not observe any signifi cant changes in the contralateral paw as compared to the pre-CCI surgery threshold. The drug was administered on the 14th day, the thermal paw withdrawal threshold was measured at 30 min, 60 min, 120 min, 240 min, and 24 h following the intraperitoneal drug administration. Tozasertib at doses 10 mg/kg was found to attenuate thermal hyperalgesia at 120 min as compared to the CCI group (p < 0,05; (Figure 6C and Figure 6D), whereas at 20 mg/kg tozasertib signifi cantly increased the paw
Figure 6. Eff ect of tozasertib on pain-like behavior in nerve-injured rats. Mechanical allodynia in (A) ipsilateral paw and (B) contralateral paw of nerve-injured rats on day 14 post-CCI. The higher dose of tozasertib deceased mechanical allodynia induced by CCI. Thermal hyperalgesia in (C) ipsilateral paw and (D) contralateral paw of nerve-injured rats on day 14 post-CCI. Data were analyzed by repeated measure two-way ANOVA followed by Bonferroni multiple comparison test and expressed as the mean ± SEM (n = 6). ### represents signifi cance compared to control group (p < 0.001); ∗ (p < 0.05) and ∗∗ (P < 0.01) represent signifi cance compared to CCI group. Tozasertib dose: 5T, 5 mg/kg; 10T, 10 mg/kg; 20T, 20 mg/kg.
Figure 7. Eff ect of tozasertib on kif11 expression induced by CCI nerve injury in isolated (A, B) spinal cord and (C, D) DRG tissues. Tozasertib attenuates CCI-induced kif11 expression in the spinal cord and DRG of rats. Data were expressed as the mean ± SEM and analyzed by one-way ANOVA (post hoc analysis: Tukey’s post hoc analysis test) (n = 3). ## represents signifi cance compared to the control group (p < 0.01); ∗ (p < 0.05) represents signifi cance compared to the CCI group. Tozasertib dose: 5T, 5 mg/kg; 10T, 10 mg/kg; 20T, 20 mg/kg.
withdrawal threshold value at 60 and 120 min compared to the CCI group (P < 0.05, P < 0.01). The tozasertib produced a similar pain-relieving eff ect compared to the gabapentin (30 mg/kg). No significant diff erence was observed in the paw withdrawal thresholds of the contralateral paw.
In this study, we have used lower doses of tozasertib which was approx 1/10 of the dose corresponding to the cancer treatment. We observed a better antinociceptive effi cacy at lower doses of tozasertib which could provide a safe and potential therapeutics for the management of neuropathic pain by minimizing the risk of undesirable side eff ects and enhancing patient compliance. Further experimentation will help determine the applications and limitations of tozasertib.
Eff ect of Tozasertib on Biochemical Parameters in the Isolated Sciatic Nerve. 188.8.131.52. Eff ect of Tozasertib on Nerve-Injury-Induced Depletion of Glutathione Levels in Sciatic Nerve. There was a signifi cant eff ect on reduced glutathione levels across the groups (F(4,10) = 48.5; p < 0.001) analyzed by one-way ANOVA followed by Tukey’s post hoc analysis. CCI sciatic nerve injury model induced oxidative stress that resulted in rapid depletion of glutathione level. Reduced glutathione levels in sciatic nerve tissue samples of nerve-injured CCI (Figure S2A) rats were found to be significantly decreased as compared to the control group (p < 0.001). Tozasertib treatment increases the levels of reduced glutathione as compared to the CCI group. These results demonstrate the effi cacy of tozasertib to suppress oxidative stress pathways.
Eff ect of Tozasertib on Nerve-Injury-Induced Nitrosative Stress. A signifi cant eff ect was observed across the groups on nitrite levels (F(4,10) = 16.3; p < 0.001) using one-way ANOVA followed by Tukey’s post hoc analysis. Nitrite level was found to be significantly increased in sciatic nerve tissues isolated from nerve-injured rats (p < 0.001 vs control). Tozasertib at the highest dose of 10 and 20 mg/kg
was found to be eff ective in reducing the nitrite levels (P < 0.05 vs CCI) (Figure S2B).
Eff ect of Tozasertib on Nerve-Injury-Induced MDA Level. The one way ANOVA followed by Tukey’s post hoc analysis has suggested a significant eff ect across the groups (F(4,15) = 16.05; p < 0.001). CCI surgery caused a profound increase in MDA level which induced oxidative stress in isolated sciatic nerve tissue (P < 0.001vs control). Tozasertib at a higher dose of 10 and 20 mg/kg was found to be significant (P < 0.001 vs CCI) in decreasing the MDA levels (Figure S2C).
Eff ect of Tozasertib on Kif11 Expression in Isolated DRG and Spinal Cord. We observed a similar trend in in vivo studies as compared to the in vitro studies for kif11 expression. A signifi cant eff ect was observed on kif11 expression across the groups in isolated DRG (F(4,10) = 7.031; p < 0.001) and spinal cord (F(4,10) = 7.709; p < 0.001) using one-way ANOVA followed by Tukey’s test. The expression of kif11 was significantly increased 14 days after CCI nerve injury as compared to the control group (P < 0.01). Our fi ndings suggest the possible role of kif11 in the neurobiology of neuropathic pain. Tozasertib at 20 mg/kg dose was found to significantly reduce the kif11 expression with respect to the CCI group in DRG (Figure 7A and Figure 7B) and spinal cord (Figure 7C and Figure 7D) tissues (P < 0.05). The results demonstrated that inhibition of Aurora kinase depleted the levels of kif11 that has been increased in chronic pain condition.
A summary of this study has been depicted in Figure 8. For the fi rst time, the findings from the present study demonstrate that Aurora kinase inhibitor tozasertib exhibits the anti- nociceptive effect by reducing the kif11 expression in nerve- injured rats. This could have impacted the kif11 mediated transport of NR2b subunit to the dendritic endings and thus reduced the excitatory receptors mediated neuronal trans- mission. The suppressed oxidonitrosative stress and inhibition
Figure 8. Summary of the study. In chronic pain conditions, the synthesis and transportation of excitatory receptors to the synaptic membrane are upregulated. This further leads to the glial activation, and oxidative stress followed by central sensitization thus promotes the pathophysiology of neuropathic pain. Tozasertib inhibits the Aurora kinase which further regulates kif11 expression and inhibits the activated glial cells leading to the suppression of the oxidonitrosative stress pathway. Hence, tozasertib causes amelioration of pain-like behavior in neuropathic rats by interfering with kif11 and Aurora kinase-mediated nociception.
of glial cell response finally accounted for the amelioration of the pain-like behavior in nerve-injured rats (Figure 8). In a nutshell, the present findings suggest the involvement of kif11 in the progression of neuropathic pain, and their regulation by Aurora kinase represents a novel therapeutic strategy for the treatment of neuropathic pain. Therefore, we suggest that tozasertib and its derivatives may be used as a potential alternative therapy for the treatment of patients suffering from chronic neuropathic pain
Traffi cking of excitatory receptors (e.g., NMDA) to the synaptic membrane is essential for neuronal fi ring and excitability thereby contributing to the neurobiology of development and maintenance of chronic pain. Recent studies from various research groups suggest that anticancer drugs like monastrol and roscovitine at lower doses have a salvaging eff ect on cells. In the case of monastrol a kif11 inhibitor, lower doses were found to ameliorate chemotherapy-induced neuro- pathy.29 It was also observed that inhibition of kif11 led to the clearing of the path for slow-moving kinesins which delivered cargos responsible for the growth cone expansion, thus stimulating neurogenesis and recovery. A very interesting fact has been observed in studies conducted for anticancer activity
by targeting kif11, that the patient who underwent treatment with kif11 inhibitor did not develop any sign of peripheral neuropathy which is common among chemotherapeutic agents.19 In another study, roscovitine which is a CDK-5 inhibitor was found to prevent alloreactive T-cell expansion.30 This has led to a decreased anti-infl ammatory response, thus protecting acute graft versus host diseases. Later in a study done by Liu et al., they reported that roscovitine was able to reduce thermal hyperalgesia in CFA-induced rats.31 They demonstrated that roscovitine acted by inhibiting CDK-5 that further phosphorylates kif13b which is a typical member of the kinesin family. Further, the kif13b formed a complex with TRPV-1 and transported it along with the microtubule to the synaptic membrane, thus increasing surface expression of TRPV-1 receptors responsible for thermal hyperalgesia. Kinesin regulates the anterograde and retrograde traffi cking of various receptors from cell organelles to the synaptic membrane and vice versa. A study has reported that kinesin- mediated transport of Nav1.6 in sensory neurons promotes neuropathic pain.10 The pan Aurora kinase regulates a variety of cellular processes including the regulation of kinesin- mediated cargo transport and cell division. The findings from
Figure 9. Timeline for in vivo study. On day 0, the sciatic nerve injury was performed in rats. On day 14 post-CCI-surgery control and nerve injured rats were treated with tozasertib and pain behavior assessment (n = 6) was done. After completing the behavioral studies on day 14 post-CCI, animals were sacrifi ced and tissues were harvested (sciatic nerve, DRG, and spinal cord) and stored at -80 °C for biochemical (n = 3-4) and Western blot analyses (n = 3-4).
the present study demonstrated that tozasertib treatment reduced Eg-5/kif11 expression by inhibiting the Aurora kinase enzyme. This has further suppressed the activation of glial cells and ameliorated the pain-like behavior in nerve-injured rats (Figure 8). In summary, the present findings suggest the involvement of kif11 in the progression of neuropathic pain, and their regulation by Aurora kinase represents a novel therapeutic strategy for the treatment of neuropathic pain. Therefore, we suggest that tozasertib may be used as a potential alternative therapy for the treatment of patients suff ering from chronic neuropathic pain. Thus, targeting motor proteins for achieving neuroprotection is a novel and futuristic approach for understanding the pathophysiological processes and treatment of neuropathic pain.
The present study suggests a novel line of action for the development of therapeutic strategies that interfere with Aurora kinase and kinesin mediated nociception. Further, research is required to extrapolate the efficacy of tozasertib as a potent analgesic with the dissection of in-depth kinesin mediated mechanisms. At present most of the therapies for the management of neuropathic pain are having limitations due to undesirable eff ects. In this relevance, the kinesin targeted transport of receptors could provide a novel class of drugs with high effi cacy and safety margin. We believe that our research scheme (molecular modeling → experiments on cell culture → preclinical testing in rodents) can be used to develop this class of pharmaceuticals.
MATERIALS AND METHODS
Drugs and Reagents. C6 glial cells were procured from National Centre for Cell Science-Pune (NCCS-Pune, India) and cultured in Ham’s F12K (Gibco (Grand Island, NY, U.S.A.), 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, U.S.A), and LPS was purchased from Sigma-Aldrich and dissolved in phosphate buff er saline (PBS) solution (pH 7.2). Tozasertib was purchased from
Wuhan Goldenwing, China, and was prepared freshly in 1% dimethyl sulfoxide (DMSO; 0.5%). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT), Griess reagent, bicinchoninic acid assay (BCA) reagents, DCFDA, standard glutathione (GSH), protease cocktail inhibitors all were purchased from Sigma-Aldrich (St. Louis, Missouri, U.S.). Rabbit anti-eg-5 antibody was from Novus Biologicals (catalog no. NB500-181SS), rabbit anti-β-actin was from Abcam (catalog no. ab8227), and anti-rabbit secondary antibodies were from Sigma-Aldrich (catalog no. A0545-1ML).
In Silico Studies. Alignment of the amino acid sequences of human and rat Aurora kinase A was performed using the Multalin software32 and SIM alignment tool.33 The primary sequences from the UniProt database33 were used (the code O14965 for human and P59241 for rat Aurora kinase A).
Molecular Dynamics Simulation Study. The general limitation of any grid-based docking algorithm is that it treats the receptor as a rigid entity, and therefore it provides a still picture of the protein-ligand interaction.34 However, in the physiological system, this interaction is dynamic in nature. Therefore, to better understand the interaction between the protein-ligand complex of tozasertib and Aurora kinase A, we performed molecular dynamics simulation using the Desmond molecular dynamics program.35 In general, the protein-ligand complex was placed into an orthorhombic box of size 10 Å × 10 Å × 10 Å and solvated explicitly with TIP3P. OPLS3e was used as a force field for the system. The system was neutralized by Na+ and Cl- ions at a fi nal concentration of 0.15 M. The system was minimized and pre-equilibrated using the standard equilibration protocol implemented in Desmond which contains several steps before the final production run, beginning with a Brownian dynamics NVT simulation for 100 ps at 10 K temperature, with small timesteps and restraints on solute heavy atoms followed by NVT simulation for 12 ps at 10 K temperature with small timesteps and restraints on solute heavy atoms. Then a 12 ps NPT simulation at 10 K temperature with restraints on solute heavy atoms and a 12 ps NPT simulation with restraints on solute heavy atoms were done, and fi nally a 24 ps NPT simulation without any restraints was done. After all these equilibration steps, with default relaxation settings presimulation, a fi nal production simulation was performed for 50 ns using NPT (normal pressure and temperature) ensemble at 300 K and 1.013 bar. The pressure and temperature were maintained using Martyna-Tobias-Klein barostat and Nose-Hoover chain thermostat,
respectively.36 The RESPA integrator was used with a time step of 2 fs during the simulation with a smooth PME method for the calculation of long-range electrostatic interaction. The energy and coordinates were saved in the trajectory at every 10 ps. The fi nal simulation trajectory was analyzed using a simulation interaction diagram available in Maestro.
In Vitro Studies. Cells were cultured in Ham’s F12K medium containing 10% FBS, 100 U/mL penicillin and streptomycin at 37 °C at 5% CO2, and the medium was replenished every alternate day. On reaching 80-90% confluency, cells were subcultured and seeded in 96-well plates for MTT or 6-well plates for changes in biochemical and protein expression parameters. Approximately 4 × 106 cells/well were seeded in 6-well plate. After 24 h of incubation, cells were treated with LPS (10 μg/mL) followed by drug treatment (1, 10, 20 nM tozasertib). After drug treatment, cell lysates were prepared by adding RIPA buff er followed by scrapping out cells and centrifuged at 12 000g for 20 min at 4 °C. The supernatant was collected and stored at -80 °C for further estimation.
Cell Viability Assay. C6 glial cells were seeded into 96-well plates and incubated for 24 h with medium containing 10% FBS in 5% CO2. Then in order to measure cytotoxicity, the medium was replaced with diff erent concentrations of tozasertib (1, 10, 20 nM) followed by incubation for 24 h. After washing, cells were provided with 200 μL of fresh medium and 20 μL of MTT solution (5 mg/mL in PBS) followed by incubation for 4 h at 37 °C. Following 4 h of incubation, the MTT solution was replaced with 200 μL DMSO to dissolve formazan crystals and incubated for 30 min at room temperature with shaking. Absorbance was recorded at 570 nm using a microplate reader (Thermo Scientific Multiscan GO, USA), and % cell viability was calculated.37
In Vivo Studies. Adult male Sprague Dawley rats weighing 250-300 g were used for in vivo studies. Animals were housed three per cage under standard laboratory conditions. Food and water were given ad libitum. Animals were kept at a constant room temperature of 21 ± 2 °C and in a 12 h light and dark cycle to maintain their circadian rhythm. All the procedures and protocols were approved by the animal ethics committee of the institute. The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) were followed for conducting animal studies. Animals were assigned into diff erent groups (n = 6/
group): control, healthy animals; CCI, nerve-injured animals; and treatment groups (tozasertib and gabapentin). The experiment commenced with 3 days of habituation period, where animals were transferred to elevated Von Frey mesh and Hargreaves apparatus. During this habituation period, their hind paws were poked and irradiated with Von Frey hairs and IR beam to measure the threshold of both mechanical and thermal paw withdrawal, respectively. Baseline paw withdrawal threshold was taken a day before the conduction of CCI surgery to induce neuropathic pain in rats. After surgery on the 14th day (Figure 9) nerve-injured rats were treated with vehicle (1% DMSO, 30% PEG), tozasertib (5, 10, and 20 mg/kg), and gabapentin (30 mg/kg) and paw withdrawal thresholds/latencies were measured at 30, 60, 120, 240 min and 24 h after drug administration.
Animal Model of Neuropathic Pain. Neuropathic pain was induced by CCI surgery in rats. The procedure used for CCI is already reported in our previous publications.38 Briefly, rats were anesthetized using ketamine (80 mg/kg) and xylazine (10 mg/kg) ip. An incision was made to the bicep femoris muscle. Further, the sciatic nerve was exposed and approximately 10 mm of the nerve was made free from connective tissue. With a 1 mm space, three ligatures (silk 4.0) were tied proximal to the trifurcation of the sciatic nerve. Finally, the muscle layer was sutured (6-0 silk) and the animal was kept under postoperative care.
Assessment of Neuropathic Pain Behavior. 184.108.40.206. Me- chanical Allodynia. On 14th day after CCI diff erent doses of tozasertib (5, 10, 20 mg/kg) and gabapentin (30 mg/kg) were administered intraperitoneally followed by measurement of mechan- ical paw withdrawal thresholds at 30, 60, 120, 240 min and 24 h after drug administration. The mechanical sensitivity was assessed using Von Frey fi laments (0.40-13 g). In accordance with the top-down
method, we applied each fi lament to the plantar surface of hind paw of rats.39 The test has started with 1.8 g fi lament. If the response was negative, we used fi lament with a higher force, whereas if the test was positive, then we used fi lament with a smaller force. The assessment was continued until fi ve responses are recorded after the crossing of the fi rst withdrawal threshold or lower/upper end of filament reached before a negative or positive response has obtained. Paw withdrawal, shaking of paw, or licking was considered as positive pain response. Finally, the withdrawal threshold was calculated.
220.127.116.11. Thermal Hyperalgesia. The Hargreaves apparatus (Ugo Basile, Italy) was used to assess thermal hyperalgesia.39 Briefly, animals were kept under a plastic chamber placed on the glass fl oor and allowed to habituate. Radiant heat with a cutoff time of 20 s was applied to the plantar surface of rat’s hind paw with an interval of 3-5 min thrice. Paw withdrawal, flinching, licking were recorded as a positive response for the thermal hyperalgesia. The presurgery baseline was recorded, followed by induction of sciatic nerve injury. Following 14 days after CCI surgery, different doses of tozasertib (5, 10, 20 mg/kg) and gabapentin (30 mg/kg) were administered intraperitoneally and paw withdrawal latency (30, 60, 120, 240 min and 24 h after drug administration) was recorded.40 Data of three trials were used for further analysis.
Biochemical and Molecular Biology Estimations.
Tissue Harvest and Lysate Preparation of DRG, SC, and Sciatic Nerve from Nerve-Injured Rats. After behavioral tests, lumbar L4-L5 DRG, SC, and sciatic nerve from nerve-injured rats were isolated and subjected to several biochemical and molecular biology estimations. A deep cut is made on the abdominal wall lateral to the spinal cord and is continued until the spinal cord is exposed completely. While cutting deep into abdominal musculature a caution was made not to cut spinal nerves. The muscular, fat layer, vertebras connected to L4, L5, L6, and spinal cord were removed and cleaned.41 DRGs, sciatic nerve, and spinal cord were identifi ed and isolated and were placed in PBS followed by immediate transfer of all the isolated tissues to -80 °C. Tissue lysate was prepared by using RIPA buff er. Tissues were triturated, homogenized, and sonicated until a concentrated homogeneous white solution was observed followed by centrifugation at 12 000g at 4 °C. After that, supernatants were collected and stored at -80 °C.42
Nitrite Estimation. Nitrite assay was done by the Griess method of nitrite estimation. The detailed procedure is described in our previous studies.43,44 Briefly, 100 μL of cell supernatant was mixed with 100 μL of Griess reagent (1% p-aminobenzene sulfonamide, 0.01% naphthylethylenediamine in 5% v/v phosphoric acid) and kept for 20 min in the dark at room temperature. Absorbance was then measured at 540 nm using the microplate reader (Thermo Scientific Multiscan GO, USA). Nitrite levels were expressed in μM/mg of protein. The same procedure was followed for the tissue samples.
Glutathione Estimation. Glutathione estimation was done by using Ellman’s assay.45,46 In the presence of DTNB (5,5′-dithio bis(2-nitrobenzoic acid), GSH forms GSH-TNB adducts. Buff ers were prepared using sodium hydrogen phosphate and sodium dihydrogen phosphate. DTNB solution was prepared in pH 8.0 phosphate buffer. 400 μL of phosphate buff er, pH 7.4, and 1.5 mL of DTNB dissolved in the phosphate buff er (pH 8) were added to the 100 μL of lysate. Further, 15 min incubation was performed on water bath maintained at 38 °C. The absorbance was taken at 412 nm using a UV plate reader (Thermo Scientifi c Multiscan GO, USA). Reduce glutathione levels were expressed in μM/mg protein.
Lipid Peroxidation Estimation. MDA levels were measured by the method of refs 47 and 48 with slight modifi cations. In a microcentrifuge tube, 100 μL of cell lysate or tissue lysate from the sciatic nerve and 100 μL of 0.1 M TrisHCl were added and further incubated for 2 h at 37 °C. Following incubation, 200 μL of ice-cold 10% w/v trichloroacetic acid and 200 μL of 0.67% of TBA (thiobarbituric acid) were added and then put it in a water bath at 95 °C for 10 min. After heating in a water bath, the mixture was rapidly cooled by adding 200 μL of double distilled water and 1 mL of butanol/pyridine (15:1) mixture. Following the addition, the sample was centrifuged at 3000 rpm for 10 min and the upper pink layer was
transferred to 96-well plates. Absorbance was read at 532 nm. MDA levels were expressed in μM/mg protein.
Intracellular ROS Estimation Using DCFDA Dye. The complete method for the test is described somewhere else.49 Briefly, 3 × 105 cells were seeded in 6-well plates and were allowed to grow for 24 h. After that, cells were treated with LPS and diff erent concentrations of tozasertib (1, 10, 20 nM). After 24 h of incubation, the medium was removed and cells were washed with PBS solution. C6 glial cells were then treated with DCFDA solution prepared in medium and incubated for 2-3 h. After 2-3 h of incubation, cells were washed 2-3 times with PBS and then images were taken under a fluorescent microscope and fluorescence was quantifi ed using ImageJ software (NIH; Bethesda, MD).49
Western Blot Analysis. DRG and spinal cord tissue lysates from rats and cell lysates from C6 glial cells were prepared as per the above-mentioned method in tissue harvesting and lysate preparation of dorsal root ganglion, spinal cord, and sciatic nerve from the nerve- injured paragraph. The estimation of protein in each tissue sample was done by the BCA method. After loading an equal amount, proteins were separated using SDS-PAGE, transferred to PVDF membranes. Blocking was done using 3% BSA solution prepared in TBST (Tris buff er saline-Tween-20) for 2 h and incubated it with primary antibodies of rabbit kif11 (1:1000), rabbit β-actin (1:1000), at 4 °C for overnight. Following incubation, membranes were washed with TBST and were incubated with their respective secondary antibodies for 1 h. Immune complexes formed were detected using ECL (chemiluminescent agent) (Invitrogen, Carlsbad, California, United States). Blots were visualized in the gel documentation system (ChemiDoc, BioRad, Hercules, California, United States) and quantifi ed using ImageJ software.50
Statistical Analysis. Data obtained from in vitro and in vivo experiments were analyzed through one-way and two-way analysis of variance (ANOVA) followed by post hoc Newman-Kuels test. The statistical analysis was done using the Prism software of version 5.1, and values were expressed as SEM ± mean.
■ ASSOCIATED CONTENT
sı* Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.1c00043.
Figure S1 showing the effect of tozasertib on LPS induced oxidonitrosative stress in vitro and Figure S2 showing the eff ect of tozasertib on CCI induced oxidative stress in vivo (PDF)
■ AUTHOR INFORMATION
Vinod Tiwari - Neuroscience and Pain Research Laboratory, Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India; National Institute of Pharmaceutical Education & Research (NIPER)Ahmedabad, 382355 Gandhinagar, India; orcid.org/0000-0002-8491-2255; Email: [email protected], [email protected] gmail.com
Ankit Uniyal - Neuroscience and Pain Research Laboratory, Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India
P. A. Shantanu - National Institute of Pharmaceutical
Education & Research (NIPER)Ahmedabad, 382355 Gandhinagar, India
Shivani Vaidya - National Institute of Pharmaceutical Education & Research (NIPER)Ahmedabad, 382355 Gandhinagar, India
Daria A. Belinskaia - Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg 194223, Russia
Natalia N. Shestakova - Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg 194223, Russia
Rajnish Kumar - Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India; orcid.org/0000- 0002-7021-6996
Sanjay Singh - Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India; Baba Saheb Bhim Rao Ambedkar Central University (BBAU), Lucknow 226025, Uttar Pradesh, India
Complete contact information is available at: https://pubs.acs.org/10.1021/acschemneuro.1c00043
#A.U. and P.A.S. contributed equally. Conceptualization was by V.T. Data curation was by A.U., P.A.S., D.A.B., N.N.S, R.K., and V.T. Formal analysis was done by A.U., P.A.S, and V.T. Funding acquisition was done by N.N.S and V.T. Investigation was done by A.U., P.A.S., S.V., D.A.B., R.K., S.S., and V.T. Methodology was by A.U., P.A.S., S.V., N.N.S., R.K., S.S., and V.T. Project administration was done by V.T. Resources were handled by N.N.S, S.S., and V.T. Software was handled by A.U., P.A.S., D.A.B., R.K., S.S., and V.T. Supervision was done by N.N.S., R.K., S.S., and V.T. Validation was done by A.U., P.A.S, N.N.S., R.K., S.S., and V.T. Visualization was done by A.U. and V.T. Writing of the original draft was done by A.U., P.A.S,. and V.T. Manuscript review and editing were done by N.N.S., R.K., S.S., and V.T.
This work is supported by the SPARC Grant SPARC/2018- 2019/P435/SL, Early Career Research Grant ECR/2016/
001846, and Core Research Grant CRG/2020/002621 awarded to V.T. by the Ministry of Human Resource &
Development and Science and Engineering Research Board, Government of India, and Russian Foundation for Basic Research Grant 20-515-18008 and the State Assignment AAAA-A18-118012290427-7 awarded to N.N.S.
The authors declare no competing financial interest.
We acknowledge the National Institute of Pharmaceutical Education and ResearchAhmedabad, Gandhinagar, Gujarat, and Department of Pharmaceutical Engineering and Technol- ogy, Indian Institute of Technology, Banaras Hindu University, Varanasi, Uttar Pradesh, India, for providing the necessary facilities and infrastructure.
Fernandes, V., Sharma, D., Vaidya, S., Shantanu, P. A., Guan, Y., Kalia, K., and Tiwari, V. (2018) Cellular and molecular mechanisms driving neuropathic pain: recent advancements and challenges. Expert Opin. Ther. Targets 22 (2), 131-142.
Kamerman, P. R., Wadley, A. L., Davis, K. D., Hietaharju, A., Jain, P., Kopf, A., Meyer, A. C., Raja, S. N., Rice, A. S. C., Smith, B. H., et al. (2015) World Health Organization essential medicines lists: Where are the drugs to treat neuropathic pain? Pain 156 (5), 793.
Cruccu, G., and Truini, A. (2017) A review of neuropathic pain: from guidelines to clinical practice. Pain Ther. 6, 35-42.
Belinskaia, D. A., Belinskaia, M. A., Barygin, O. I., Vanchakova, N. P., and Shestakova, N. N. (2019) Psychotropic drugs for the management of chronic pain and itch. Pharmaceuticals 12 (2), 99.
González-Gil, I., Zian, D., Vázquez-Villa, H., Hernandez-Torres, G., Martínez, R. F., Khiar-Fernandez, N., Rivera, R., Kihara, Y., Devesa, I., Mathivanan, S., del Valle, C. R., et al. (2020) A novel agonist of the type 1 lysophosphatidic acid receptor (LPA1), UCM- 05194, shows efficacy in neuropathic pain amelioration. J. Med. Chem. 63 (5), 2372-90.
Uta, D., Kato, G., Doi, A., Andoh, T., Kume, T., Yoshimura, M., and Koga, K. (2019) Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo. Biochem. Biophys. Res. Commun. 512, 352-359.
Inquimbert, P., Moll, M., Latremoliere, A., Tong, C.-K., Whang, J., Sheehan, G. F., Smith, B. M., Korb, E., Athié, M. C. P., Babaniyi, O., et al. (2018) NMDA receptor activation underlies the loss of spinal dorsal horn neurons and the transition to persistent pain after peripheral nerve injury. Cell Rep. 23, 2678-2689.
Kreutzwiser, D., and Tawfic, Q. A. (2019) Expanding Role of NMDA Receptor Antagonists in the Management of Pain. CNS Drugs 33 (4), 347-74.
Stepanenko, Y. D., Boikov, S. I., Sibarov, D. A., Abushik, P. A., Vanchakova, N. P., Belinskaia, D., Shestakova, N. N., and Antonov, S. M. (2019) Dual action of amitriptyline on NMDA receptors: enhancement of Ca-dependent desensitization and trapping channel block. Sci. Rep. 9, 19454.
Zhang, X.-L., Ding, H.-H., Xu, T., Liu, M., Ma, C., Wu, S.-L., Wei, J.-Y., Liu, C.-C., Zhang, S.-B., and Xin, W.-J. (2018) Palmitoylation of δ-catenin promotes kinesin-mediated membrane trafficking of Nav1.6 in sensory neurons to promote neuropathic pain. Sci. Signaling 11 (523), eaar4394.
Shantanu, P. A., Sharma, D., Sharma, M., Vaidya, S., Sharma, K., Kalia, K., Tao, Y.-X., Shard, A., and Tiwari, V. (2019) Kinesins: Motor Proteins as Novel Target for the Treatment of Chronic Pain. Mol. Neurobiol. 56, 3854-3864.
Xing, B. M., Yang, Y. R., Du, J. X., Chen, H. J., Qi, C., Huang, Z. H., Zhang, Y., and Wang, Y. (2012) Cyclin-dependent kinase 5 controls TRPV1 membrane trafficking and the heat sensitivity of nociceptors through KIF13B. J. Neurosci. 32 (42), 14709-14721.
Freund, R. K., Gibson, E. S., Potter, H., and Dell’Acqua, M. L. (2016) Inhibition of the Motor protein Eg5/Kinesin-5 in amyloid β- mediated impairment of hippocampal long-term potentiation and dendritic spine loss. Mol. Pharmacol. 89, 552-559.
Ari, C., Borysov, S. I., Wu, J., Padmanabhan, J., and Potter, H. (2014) Alzheimer amyloid beta inhibition of Eg5/kinesin 5 reduces neurotrophin and/or transmitter receptor function. Neurobiol. Aging 35 (8), 1839-1849.
Shen, Y., Ding, Z., Ma, S., Zou, Y., Yang, X., Ding, Z., Zhang, Y., Zhu, X., Schäfer, M. K. E., Guo, Q., et al. (2020) Targeting aurora kinase B alleviates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury. J. Neurochem. 152 (1), 72-91.
Meadows, J. C. (2013) Interplay between mitotic kinesins and the Aurora kinase-PP1 (protein phosphatase 1) axis. Biochem. Soc. Trans. 41 (6), 1761-1765.
Harrington, E. A., Bebbington, D., Moore, J., Rasmussen, R. K., Ajose-Adeogun, A. O., Nakayama, T., Graham, J. A., Demur, C., Hercend, T., Diu-Hercend, A., et al. (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med. 10 (3), 262-267.
Martens, S., Goossens, V., Devisscher, L., Hofmans, S., Claeys, P., Vuylsteke, M., Takahashi, N., Augustyns, K., and Vandenabeele, P. (2018) RIPK1-dependent cell death: A novel target of the Aurora kinase inhibitor Tozasertib (VX-680). Cell Death Dis. 9 (2), 211.
El-Nassan, H. B. (2013) Advances in the discovery of kinesin spindle protein (Eg5) inhibitors as antitumor agents. Eur. J. Med. Chem. 62, 614-631.
Hofmans, S., Devisscher, L., Martens, S., Van Rompaey, D., Goossens, K., Divert, T., Nerinckx, W., Takahashi, N., De Winter, H., Van Der Veken, P., et al. (2018) Tozasertib analogues as inhibitors of necroptotic cell death. J. Med. Chem. 61, 1895-1920.
Zhang, L. N., Ji, K., Sun, Y. T., Hou, Y. B., and Chen, J. J. (2020) Aurora kinase inhibitor tozasertib suppresses mast cell activation in vitro and in vivo. Br. J. Pharmacol. 177 (12), 2848-2859.
Yin, C., Huang, G., Sun, X., Guo, Z., and Zhang, J. H. (2016) Tozasertib attenuates neuronal apoptosis via DLK/JIP3/MA2K7/
JNK pathway in early brain injury after SAH in rats. Neuro- pharmacology 108, 316-323.
Williams, R. (2009) Discontinued drugs in 2008: oncology drugs. Expert Opin. Invest. Drugs 18, 1581-1594.
Zhao, B., Smallwood, A., Yang, J., Koretke, K., Nurse, K., Calamari, A., Kirkpatrick, R. B., and Lai, Z. (2008) Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallog- raphy studies on Aurora A interactions with VX-680 and with TPX2. Protein Sci. 17 (10), 1791-1797.
Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kalé, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26 (16), 1781-1802.
Martignoni, M., Groothuis, G. M. M., and de Kanter, R. (2006) Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin. Drug Metab. Toxicol. 2 (6), 875-894.
Blais, E. M., Rawls, K. D., Dougherty, B. V., Li, Z. I., Kolling, G. L., Ye, P., Wallqvist, A., and Papin, J. A. (2017) Reconciled rat and human metabolic networks for comparative toxicogenomics and biomarker predictions. Nat. Commun. 8, 14250.
Cusack, B., Nelson, A., and Richelson, E. (1994) Binding of antidepressants to human brain receptors: focus on newer generation compounds. Psychopharmacology (Berl). 114 (4), 559-565.
Bobylev, I., Peters, D., Vyas, M., Barham, M., Klein, I., von Strandmann, E. P., Neiss, W. F., and Lehmann, H. C. (2017) Kinesin- 5 Blocker Monastrol Protects Against Bortezomib-Induced Peripheral Neurotoxicity. Neurotoxic. Res. 32 (4), 555-562.
Li, L., Wang, H., Kim, J. S., Pihan, G., and Boussiotis, V. A. (2009) The cyclin dependent kinase inhibitor (R)-roscovitine prevents alloreactive T cell clonal expansion and protects against acute GvHD. Cell Cycle 8, 1794-1802.
Liu, J., Du, J., Yang, Y., and Wang, Y. (2015) Phosphorylation of TRPV1 by cyclin-dependent kinase 5 promotes TRPV1 surface localization, leading to inflammatory thermal hyperalgesia. Exp. Neurol. 273, 253-262.
SIMAlignment Tool for protein sequences. https://web. expasy.org/sim/ (accessed on November 2, 2020).
The UniProt Consortium. (2019) UniProt: A worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506-D515.
Guedes, I. A., de Magalhaes, C. S., and Dardenne, L. E. (2014) Receptor-ligand molecular docking. Biophys. Rev. 6 (1), 75-87.
Schrodinger Release 2020-3: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2020. Maestro-Desmond Interoperability Tools, Schrodinger, New York, NY, 2020.
Bowers, K. J., Chow, E., Xu, H., Dror, R. O., Eastwood, M. P., Gregersen, B. A., Klepeis, J. L., Kolossvary, I., Moraes, M. A., Sacerdoti, F. D., et al. (2006) Scalable algorithms for molecular dynamics simulations on commodity clusters. Proceedings of the 2006 ACM/IEEE Conference on Supercomputing, 43-43.
Thakkar, S., Sharma, D., and Misra, M. (2018) Comparative evaluation of electrospraying and lyophilization techniques on solid state properties of Erlotinib nanocrystals: Assessment of In-vitro cytotoxicity. Eur. J. Pharm. Sci. 111, 257-269.
Sharma, K., Sharma, D., Sharma, M., Sharma, N., Bidve, P., Prajapati, N., Kalia, K., and Tiwari, V. (2018) Astaxanthin ameliorates
behavioral and biochemical alterations in in-vitro and in-vivo model of neuropathic pain. Neurosci. Lett. 674, 162-170.
Tiwari, V., He, S. Q., Huang, Q., Liang, L., Yang, F., Chen, Z., Tiwari, V., Fujita, W., Devi, L. A., Dong, X., et al. (2020) Activation of μ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents. Pain 161 (4), 842-855.
Tiwari, V., Anderson, M., Yang, F., Tiwari, V., Zheng, Q., He, S. Q., Zhang, T., Shu, B., Chen, X., Grenald, S. A., et al. (2018) Peripherally Acting μ-Opioid Receptor Agonists Attenuate Ongoing Pain-associated Behavior and Spontaneous Neuronal Activity after Nerve Injury in Rats. Anesthesiology 128 (6), 1220-1236.
Sleigh, J. N., West, S. J., and Schiavo, G. (2020) A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels. BMC Res. Notes 13, 302.
Xu, B., Liu, S.-S., Wei, J., Jiao, Z.-Y., Mo, C., Lv, C.-M., Huang, A.-L., Chen, Q.-B., Ma, L., and Guan, X.-H. (2020) Role of Spinal Cord Akt-mTOR Signaling Pathways in Postoperative Hyperalgesia Induced by Plantar Incision in Mice. Front. Neurosci. 14, 766.
Fernandes, V., Sharma, D., Kalia, K., and Tiwari, V. (2018) Neuroprotective effects of silibinin: An in silico and in vitro study. Int. J. Neurosci. 128 (10), 935-945.
Uniyal, A., Singh, R., Akhtar, A., Bansal, Y., Kuhad, A., and Sah, S. P. (2019) Co-treatment of piracetam with risperidone rescued extinction deficits in experimental paradigms of post-traumatic stress disorder by restoring the physiological alterations in cortex and hippocampus. Pharmacol., Biochem. Behav. 185, 172763.
Rahman, I., Kode, A., and Biswas, S. K. (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1 (6), 3159.
Sharma, D., Singh, J. N., and Sharma, S. S. (2016) Effects of 4- phenyl butyric acid on high glucose-induced alterations in dorsal root ganglion neurons. Neurosci. Lett. 635, 83-89.
Anderson, M. E., Powrie, F., Puri, R. N., and Meister, A. (1985) Glutathione monoethyl ester: Preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239 (2), 538-548.
Ishola, I. O., Akinleye, M. O., Oduola, M. D., and Adeyemi, O. O. (2016) Roles of monoaminergic, antioxidant defense and neuroendocrine systems in antidepressant-like effect of Cnestis ferruginea Vahl ex DC (Connaraceae) in rats. Biomed. Pharmacother. 83, 340-348.
Wu, D., and Yotnda, P. (2011) Production and detection of reactive oxygen species (ROS) in cancers. J. Visualized Exp. No. 57, e3357.
Ishola, I. O., Chaturvedi, J. P., Rai, S., Rajasekar, N., Adeyemi, O. O., Shukla, R., and Narender, T. (2013) Evaluation of amentoflavone isolated from Cnestis ferruginea Vahl ex DC (Connaraceae) on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells. J. Ethnopharmacol. 146 (2), 440-448.