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Back to Journal »Journal of Inflammation Research» Volume 14

Studies on 1, 3, 4 Oxadiazole Derivatives in PTZ-induced Neurodegeneration: Simulation and Molecular Methods

Authors Faheem M, Althobaiti YS, Khan AW, Ullah A, Ali SH, Ilyas U

Published on November 1, 2021, the 2021 volume: 14 pages 5659-5679

DOI https://doi.org/10.2147/JIR.S328609

Single anonymous peer review

Editor who approved for publication: Dr. Monika Sharma

Muhammad Faheem,1 Yusuf S Althobaiti,2,3 Abdul Waheed Khan,4 Aman Ullah,1 Syed Hussain Ali,1 Umair Ilyas1 1 Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan; 2 Pharmacology and Toxicology, Faculty of Pharmacy, Taif University Department of Science, Taif, 21944, Saudi Arabia; 3 Addiction and Neuroscience Research Laboratory, Taif University, Taif, 21944, Saudi Arabia; 4 Department of Pharmacy, Lahore University, Islamabad, Pakistan Corresponding author: Muhammad Faheem; Yusuf S Althobaiti Email [email protected]; [email protected] Purpose: This study investigated 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3) in acute epileptic shock Role in animal models. Method: Check the pharmacokinetic characteristics of B3 by SwissADME software. The binding affinity of B3, Diazepam and Flumazenil (FLZ) is obtained through Auto Dock and PyRx. The analysis and hydrogen bond interpretation after docking were performed through Discovery Studio Visualizer 2016. The molecular dynamics simulations of the three complexes were carried out by the Desmond software package. Then perform B3 in the PTZ-induced acute epilepsy model. Flumazenil is used in animal studies to elucidate the possible mechanism of B3. After the behavioral study, the animals were sacrificed, and the brain samples were separated and stored in 4% formalin for molecular studies, including H and E staining, IHC staining, and Elisa. Results: The results showed that B3 of 20 and 40 mg/kg prolonged the onset time of generalized seizures. B3 significantly increased the expression of protective glutathione S-transferase and glutathione reductase, and reduced lipid peroxidation and inducible nitric oxide synthase in the cortex (P <0.001). B3 significantly inhibited (P <0.01) the overexpression of inflammatory mediator tumor necrosis factor-α, which was reported to be up-regulated in acute epileptic shock. Conclusion: Therefore, it can be concluded from the above results that B3 provides neuroprotection in PTZ-induced acute epilepsy models. FLZ pretreatment resulted in the suppression of the anticonvulsant effect of B3. B3 has anticonvulsant effects, which may be mediated through GABAA-mediated antiepileptic pathways. Keywords: docking, molecular dynamics simulation, γ-aminobutyric acid A

Epilepsy is a serious and heterogeneous neurological disease that will have a negative impact on the quality of life and the standard of living of human beings. Seizures are electrical discharges in the brain caused by excessive excitement or too little inhibition in the cerebral cortex. The excitation and inhibition of neurons may be mediated by many unique neurotransmitters. 1 Glutamate is related to the pathophysiology of epilepsy and has been established. 2 It directly affects more than 4 lac people in the UK and as many as 60 million people worldwide. 3 Approximately 30% of patients do not receive adequate medication due to tolerance and aggressive treatment options. Most patients with epilepsy have behavioral comorbidities, including depression, anxiety, psychosis, and cognitive impairment. Risk factors for epilepsy include intracranial hemorrhage or stroke, tumors, central nervous system infections, prolonged febrile seizures, and other status epilepticus. 4

In addition to glutamate, which is excitatory in nature, an inhibitory neurotransmitter called Gamma Aminobutyric Acid A (GABAA) is also provided in the cortex to prevent or balance excitatory neurotransmitters. Excessive stimulation. 5 Currently, benzodiazepines are considered for management to reduce seizures by increasing chloride ion conductance and causing hyperpolarization, while reducing hyperexcitability and minimizing seizures. 6

Since the long-term use of benzodiazepines is related to dependence and tolerance, it was decided to use the synthetic compound 1,3,4-oxadiazole derivative (B3) in phenyltetrazole (PTZ)-induced acute and chronic epilepsy The pharmacological study in the onset is studied. PTZ, a benzodiazepine receptor antagonist, is a widely used animal model during drug discovery and development. A single high-dose intraperitoneal injection can cause acute convulsions, while a continuous medium dose can cause seizures. 7 Another antidote for benzodiazepine (BDZ) receptors is flumazenil, which is also used to reverse BDZ overdose. 8,9 Since PTZ is involved in causing neuroinflammation, the selected compounds used in the study must have potential anti-inflammatory properties to deal with acute epileptic shock caused by PTZ. In this context, the selected compounds have been reported to have antioxidant, analgesic, anti-inflammatory, toxicity assessment, tumor suppressive activity, and effective relief of pain symptoms caused by chronic contractions. 10,11 Two-dimensional (2D) and three-dimensional B3 (3D) structures are shown in Figures 1A and 2A, respectively. This study uses computational techniques, behavioral methods, and molecular studies to clarify the effect of B3 in improving PTZ-induced seizures. Figure 1 (A) shows the structure and protein data band of 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiazole-2thiol (B3) and (B) the target protein γ-aminobutyric acid (GABA) (PDB ID = 6D6T). Figure 2 (A-C) shows the ligands 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3), diazepam (DZM) and flumazenil (FLZ) The three-dimensional structure), respectively, using Discovery Studio Visualizer 2016 to draw.

Figure 1 (A) shows the structure and protein data band of 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiazole-2thiol (B3) and (B) the target protein γ-aminobutyric acid (GABA) (PDB ID = 6D6T).

Figure 2 (A-C) shows the ligands 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3), diazepam (DZM) and flumazenil (FLZ) The three-dimensional structure), respectively, using Discovery Studio Visualizer 2016 to draw.

Proteinase K and PBS tablets were purchased from MP-Biomedicals, USA. N-(1-naphthyl) ethylenediamine dihydrochloride, trichloroacetic acid (TCA), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 1-chloro-2 ,4-Dinitrobenzene (CDNB) and reduced glutathione (GSH) were purchased from Sigma Aldrich, USA. Mouse monoclonal anti-TNF-α (SC-52B83722), 3,3'-diaminobenzidine (DAB, SC-216567) and avidin-biotin complex (ABC kit, SC-516216) were purchased from the United States Santa Cruz Biotechnology Company. Mounting medium (ab-10431) and horseradish peroxidase conjugated secondary antibodies (ab-6789, ab-6721) were purchased from Abcam, UK. Flumazenil (FLZ) and Diazepam (DZM) were purchased from Shifa International Hospital in Islamabad, Pakistan. Phenyltetrazole (PTZ) was purchased from Sigma Aldrich, USA. TNF-α enzyme-linked immunosorbent assay (ELISA) kit (product number SU-B3098) was purchased from Shanghai Yuchun Biotechnology Co., Ltd. Compound 1, 3, 4-oxadiazole derivative (B3) was obtained as a gift from the Graduate Laboratory of Medicinal Chemistry, Riphah International University, Islamabad, Pakistan.

In this study, Swiss albino mice weighing 25-30 grams were used. The mice are allowed to drink water and eat freely, each type of light and shade is supplied for 12 hours, and they are kept in a standard environment of humidity and temperature (25-30°C). The study was conducted in accordance with the guidelines established by the ethics committee of the Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad (REC/RIPS/2021/01).

The mice were divided into the following groups (6 rats in each group).

PTZ NS: Group I as a sham operation (saline 10 mL/Kg) PTZ (90 mg/Kg)

PTZ B3: Group II uses B3 (20 mg/Kg) and PTZ

PTZ B3: Group III uses B3 (40 mg/Kg) and PTZ

PTZ DZM: The IV group provides PTZ DZM (1 mg/Kg) for the standard group

PTZ NS: Group I as a sham operation (saline 10 mL/Kg) PTZ (90 mg/Kg)

PTZ DZM: Group II uses DZM (1 mg/Kg) PTZ.

PTZ FLZ: FLZ (2mg/Kg) PTZ was given to the group III inhibitor group

PTZ B3: Group IV uses B3 (40 mg/Kg) and PTZ

PTZ B3 FLZ: Group V was given B3 (40 mg/Kg) FLZ (2 mg/Kg) and DZM

PTZ DZM FLZ: Group VI was given B3 (30 mg/Kg) FLZ (2 mg/Kg) and DZM

The ligands 1, 3, and 4 oxadiazole as shown in Figure 1A are prepared by ChemSketch, and then converted to 3D structure by Discovery Studio Visualizer 2016 (DSV 2016) and saved in PDB (Protein Data Band) format, as shown in the figure Shown in 2A. The structures of diazepam and flumazenil were retrieved from PubChem structure search in mol format, converted to PDB format via Open Bebel, and saved in PDB format via DSV 2016, as shown in Figure 2B and C.

The PDB ID of the target protein γ-aminobutyric acid A (GABAA): 6D6T was retrieved from the protein database and purified by DSV 2016 for further processing, as shown in Figure 1B.

The active pocket of the target protein shown in Figure 3 is identified by Dog site scorer (an online tool for active site search). 13 Dog site scorer can give a simple score of volume, surface area, drug score, active pocket. The first pocket is considered to be the most important part to check whether the ligand can be a drug. Tables 1A-F show some important descriptions of the target protein. Table 1 Dog Site Scorer value of target protein GABAa (GABAa), Protein Data Band (PDB) ID: 6D6T. 1A represents the first three pockets P_0, P_1 and P_2, and then randomly selects three pockets for docking P_11, P_15 and P_17. 1B represents the size and shape of each pocket (P_0, P_1 and P_2, P_11, P_15), including volume , Surface area, depth, ellipsoid principal axis ratio c/a, ellipsoid principal axis ratio b/a and shell. 1C represents the elements of each pocket, including pocket atoms, carbon, nitrogen, oxygen, and sulfur. 1D represents the functional group of each pocket that participates in the bonding as a hydrogen bond donor, acceptor, metal, hydrophobic interaction, and hydrophobic ratio. 1E expresses the amino acid composition of each pocket involved in bonding in terms of non-polar amino acid ratio, polar amino acid ratio, positive amino acid ratio, and negative amino acid ratio. 1F represents that the specific amino acid in the specific target pocket participates in the bonding into the polar amino acid ratio, polar amino acid ratio, positive amino acid ratio and negative amino acid ratio. Figure 3 Active site of γ-aminobutyric acid (GABA) (P0, P1, P2 P11, P15 and p17) are obtained through Dog site scorer.

Table 1 Dog Site Scorer value of target protein GABAa (GABAa), Protein Data Band (PDB) ID: 6D6T. 1A represents the first three pockets P_0, P_1 and P_2, and then randomly selects three pockets for docking P_11, P_15 and P_17. 1B represents the size and shape of each pocket (P_0, P_1 and P_2, P_11, P_15), including volume , Surface area, depth, ellipsoid principal axis ratio c/a, ellipsoid principal axis ratio b/a and shell. 1C represents the elements of each pocket, including pocket atoms, carbon, nitrogen, oxygen, and sulfur. 1D represents the functional group of each pocket that participates in the bonding as a hydrogen bond donor, acceptor, metal, hydrophobic interaction, and hydrophobic ratio. 1E expresses the amino acid composition of each pocket involved in bonding in terms of non-polar amino acid ratio, polar amino acid ratio, positive amino acid ratio, and negative amino acid ratio. 1F represents that the specific amino acid in the given target pocket participates in the bonding into the ratio of polar amino acids, the ratio of polar amino acids, the ratio of positive amino acids and the ratio of negative amino acids

Figure 3 Gamma-aminobutyric acid (GABA) active sites (P0, P1, P2, P11, P15, and p17) obtained by Dog site scorer.

After selecting the active site, an important step is to use the PyRx software to select the Grid box. The grid center of the grid box is X = 133.02, Y = 147.50, and Z = 133.19, and X = 50, Y = 50, and Z = 50. The dimensions in angstroms are X = 98.17, Y = 121.63 and Z = 100.16 and the spacing in angstroms is 0.375. The grid frame is set in such a way that all proteins are covered in the grid frame.

SwissADME is an online technology used to check various parameters before processing drugs into animal models, including pharmacokinetic properties, drug possibilities, physical and chemical properties, lipophilicity and medicinal chemical ligands.

Auto Dock vina and PyRx are used to dock the ligand to the target. Ligand and target are first converted to PDBQT by AutoDock, and then loaded into PyRx. Docking is done by selecting the amino acid at the active site of the target protein. After running the ligand-receptor complex through PyRx, the binding energy expressed in kcal/mol is obtained. The best posture obtained by PyRx is then subjected to post-docking analysis, and hydrogen bonding (classical and non-classical), hydrophobic interaction and amino acid residues related to the best docking posture of the ligand protein complex are performed by DSV 2016. 14

After the molecular docking study, the Desmond software package was used to perform molecular dynamics simulation studies on the three complexes B3-GABA, DZM-GABA and FLZ-GABA to track molecular interactions. 16 In the MD simulation, the compound was immersed in an automatically calculated orthorhombic crystal box using a single-point charge water model to add 68444, 68446, and 68441 to the compounds B3-GABA, DZM-GABA, and FLZ-GABA, respectively. The optimization of the model is further completed by the optimized liquid simulation potential (OPLS5). Neutralize the complex of the system and make the system isotonic by adding NaCl (Na = 50.73 and Cl = 60.30 mM). In order to make the system electrically stable, a counter ion is added. The nasal Hoover thermostat is used to provide a temperature of 300 K. Martyna-Tobias-Klienbarostate is used to maintain a pressure of 1.01325 bar. The total time of MD simulation is 50 ns. The electrostatic interaction is calculated by the grid Ewald method. 17 The interaction behavior between ligand and protein is analyzed by the simulation interaction diagram tool in the Desmond package. The density functional theory method was applied to the structural optimization of all three ligands. The Desmond software package can obtain the RMSD of the protein and the RMSF of the residue, which can be used for further analysis. 17

The target information in the form of total residues, protein chains, atoms, heavy atoms, and charged atoms is shown in Figure 4A. The root mean square fluctuation (RMSF) of the target protein and the average RMSF of the protein need not exceed 2.5 angstroms, as shown in Figure 4B. Figure 4C shows the protein secondary structure elements (SSE), the alpha helix in red, and the beta chain in blue. Figure 4 (A) shows the total residues, protein chains, atoms, heavy atoms and charged atoms. Part (B) shows the root mean square fluctuation (RMSF) of the target protein in Angstroms (Å). The part (C) represents the protein secondary structure element (SSE), the alpha helix shown in red and the beta chain shown in blue, monitored by simulation. This figure summarizes the SSE composition of each trajectory frame during the simulation.

Figure 4 (A) shows the total residues, protein chains, atoms, heavy atoms and charged atoms. Part (B) shows the root mean square fluctuation (RMSF) of the target protein in Angstroms (Å). The part (C) represents the protein secondary structure element (SSE), the alpha helix shown in red and the beta chain shown in blue, monitored by simulation. This figure summarizes the SSE composition of each trajectory frame during the simulation.

The information of the ligands (B3, DZM and FLZ) is in the form of RMSD value, radius of return (RG) value, intramolecular hydrogen bond, molecular surface area (MSA), solvent accessible surface area (SASA) and polar surface area (PSA) as shown in Figure 5. Shown. Throughout the simulation process, the torsion area of ​​each ligand B3, DZM and FLZ is shown in different colors in Figure 6A-C. The color-coded rotatable bond of the ligand is also shown in Figures 6A-C. Each rotatable key twist is supplemented by the dial graphs 6A1-C1 and the bar graphs of the same color. In addition, Figures 7A-C show the number of ion-like atoms, atomic mass, charge, molecular formula, number of fragments, and number of rotatable bonds of the ligand. Figure 5 Parts (1), (2) and (3) represent the ligand (5-[(naphth-2-yloxy)methyl]-1,3,4-oxadiazole 2-thiol [B3] , Diazepam [DZM] and Flumazenil [FLZ]) Root Mean Square Deviation (RMSD) Value, Recirculation Radius (RG) Value, Intramolecular Hydrogen Bond, Molecular Surface Area (MSA), Solvent Accessible Surface Area (SASA) And polar surface area (PSA), respectively. The part (AC) represents the torsion area of ​​each ligand B3, DZM, and FLZ throughout the simulation, and is displayed in different colors. (A1-AC) stands for rotatable key. Each rotatable key twist is supplemented by a dial graph (A1–C1) and a bar graph of the same color. Figure 6 (A–C) shows the ligands (5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol [B3], diazepam [DZM] and flumazenil [FLZ ]) Information about the number of atoms, atomic mass, charge, molecular formula, number of fragments, and the number of rotatable bonds. Figure 7 The best posture of B3 (A), the interaction of B3 (B) and the two-dimensional representation of the hydrogen bond of B3 (C) drawn using Discovery Studio Visualizer 2016.

Figure 5 Parts (1), (2) and (3) represent the ligand (5-[(naphth-2-yloxy)methyl]-1,3,4-oxadiazole 2-thiol [B3] , Diazepam [DZM] and Flumazenil [FLZ]) Root Mean Square Deviation (RMSD) Value, Recirculation Radius (RG) Value, Intramolecular Hydrogen Bond, Molecular Surface Area (MSA), Solvent Accessible Surface Area (SASA) And polar surface area (PSA), respectively. The part (AC) represents the torsion area of ​​each ligand B3, DZM, and FLZ throughout the simulation, and is displayed in different colors. (A1-AC) stands for rotatable key. Each rotatable key twist is supplemented by a dial graph (A1–C1) and a bar graph of the same color.

Figure 6 (A–C) shows the ligands (5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol [B3], diazepam [DZM] and flumazenil [FLZ ]) Information about the number of atoms, atomic mass, charge, molecular formula, number of fragments, and the number of rotatable bonds.

Figure 7 The best posture of B3 (A), the interaction of B3 (B) and the two-dimensional representation of the hydrogen bond of B3 (C) drawn using Discovery Studio Visualizer 2016.

The mice were randomly divided into four groups (6 animals in each group). After 30 minutes of treatment with NS, B3, DZM and FLZ, all groups received chemical convulsion PTZ, and the animals' seizures, seizure duration and protection percentage were observed for 30 minutes. Percent mortality was also observed 24 hours after PTZ administration. 18,19

In order to explore the possible mechanism of the anticonvulsant effect of B3, FLZ, a BDZ receptor antagonist, was injected 5 minutes before B3 and DZM administration. After induction of convulsions in mice by intraperitoneal injection of PTZ 35 minutes later, the animals' seizures, seizure duration, and 30-minute protection percentage were observed. PTZ also noticed the mortality percentage after 24 hours. 20

Lipid hydroperoxide determination method for the determination of thiobarbituric acid reactive substances (TBARS) by colorimetry. 21 According to this assay, 200 uL of the supernatant of the homogenized sample was mixed with 200 uL of ascorbic acid, 20 uL of ferric chloride, and 580 mL of phosphate buffer. The mixture was incubated at 37°C for 1 hour. After this step, add each 1000 uL 0.66% thiobarbituric acid and 10% trichloroacetic acid to stop the reaction. The sample tube was then passed through water at 25°C, then cold water and centrifuged at 3000×g for 10 minutes. Calculate the concentration of TBARS – Nm/min/mg protein by collecting and measuring the supernatant at 535 nm.

NO detection is carried out according to established methods. 22 According to this protocol, mix the supernatant (50uL) and Griess reagent (50uL) together. The reagent is composed of 5% phosphoric acid, 0.1% naphthalene ethylene diamine dihydrochloride and 1% sulfonamide in distilled water. The mixture was incubated at 37°C for 30 minutes, and then read on a microplate reader (Bio-ELx 808). Calibrate the absorbance coefficient with sodium nitrate solution.

The damage caused by PTZ is determined by the measurement of oxidative stress markers. The sample (cortex) was homogenized in a phosphate buffer containing phenylmethylsulfonyl fluoride (PMSF) as a protease blocker, and centrifuged at 4000×g for 10 minutes at 4°C, and the supernatant was collected. 23 To determine the GST level, the supernatant (0.2mL) was mixed with 2mL of sodium phosphate (0.2M) and DTNB (0.6Mm) solution. Add about 3 mL of phosphate buffer to mark the volume, and then incubate at room temperature for 10 minutes. Spectrophotometer is used for absorbance measurement at 412nm. A solution of phosphate buffer and DTNB was used as a control. The value obtained by subtracting the control absorbance from the tissue-containing sample is considered the actual absorbance, which represents the GSH level in umol/mg protein. Determine the GST level based on the previously used protocol. 24 Pour the supernatant (60uL) into 1.2 mL of a freshly prepared solution (5mM GSH, 1mM CDNB in ​​0.1M phosphate buffer) through a glass vial to make a triplet. Put 210uL from the mixture into the microplate, and find the reaction rate at 340nm with the help of ELIZA microplate reader (Bio-Tek ELx-808, Winooski, VT, USA). Instead of tissue, 60uL of water was added as a control. GST is expressed as umol CDNB conjugate/minute/mg protein.

The H and E staining is performed according to an established and clearly explained protocol. 25

The immunohistochemistry research is carried out according to the explained enlightenment program. 26

ELISA kit is used to detect the expression of inflammation marker TNF-α in cerebral cortex. In a 96-well plate, treat the cerebral cortex supernatant with the designated antibody, and then use an ELISA microplate reader to detect the expression level of the inflammation marker TNF-α. These values ​​are expressed as pictograms per milliliter (pg/mL). This procedure was repeated three times. 27

Data are shown as mean±SEM. H and E staining, IHC staining, behavioral data, oxidative stress data, and ELISA were analyzed using one-way analysis of variance, and then a post-hoc Tukey test was performed in Graph Pad Prism. The morphological data was analyzed using Image J software. P value is calculated by Graph Prad prism symbol, *** and *** represent significant difference value P <0.05, 0.01 and 0.001, respectively.

The pharmacokinetic characteristics of B3 are shown in Table 2. Table 2 shows the pharmacokinetic characteristics of the ligand 5-[(naphthalene-2-yloxy)methyl]-1,3,4-oxadiazole 2-thiol (B3)

Table 2 shows the pharmacokinetic characteristics of the ligand 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3)

The optimal positions of the ligand molecules B3, DZM and FLZ against the target protein GABA are shown in Figures 5A, 8A and 9A. The interaction of the ligands is shown in Figures 5B, 8B, and 9B, and the hydrogen bonding is shown in Figures 5C, 8C, and 9C. B3 (6.3 kcal/mol), which has the highest binding affinity, forms four hydrogen bonds. DZM and FLZ form 1 and 2 hydrogen bonds, and the binding affinity is 5.7kcal/mol and 5.3kcal/mol, respectively. Table 3 summarizes the number of hydrogen bonds, binding energy (kcal/mol) and residues of amino acids involved in the interaction of B3, DZM and FLZ. Table 3 Hydrogen bond and amino acid binding affinity (Kcal/mol) 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3), Diazepam (DZM) and Flumazenil (FLZ) docking The remaining Pi alkyl bond, van der Waals force to Gamma Aminobutyric acid (GABA) Figure 8 Diazepam (DZM) (A), DZM interaction (B) and DZM (C) drawn using Discovery Studio Visualizer 2016 Two-dimensional representation of hydrogen bonds. Figure 9 The interaction of the optimal posture of Flumazenil (FLZ) (A) and FLZ (B) and the two-dimensional representation of the FLZ (C) hydrogen bond drawn using Discovery Studio Visualizer 2016.

Table 3 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol after docking hydrogen bond, amino acid residue, Pi alkyl bond, van der Waals force binding affinity (Kcal/mol) (B3 ), diazepam (DZM) and flumazenil (FLZ) against gamma aminobutyric acid (GABA)

Figure 8 The best posture of the interaction of diazepam (DZM) (A), DZM (B) and the two-dimensional representation of the hydrogen bond of DZM (C) drawn using Discovery Studio Visualizer 2016.

Figure 9 The two-dimensional representation of flumazenil (FLZ) (A), FLZ (B) and FLZ (C) hydrogen bonds drawn using Discovery Studio Visualizer 2016.

Use Desmond software package for MD simulation. After providing a physiological environment for the complexes, all complexes were run for 50 ns to obtain results. The root mean square fluctuations (RMSF) of B3, DZM and FLZ are shown in Figure 10A-C. The stability of the ligand-receptor complex can be predicted by the RMSD value shown in Figure 11. Small changes in RMSD showed a more stable combination. B3, DZM and FLZ and the RMSD of the target are shown in 11 (a, b, and c). Figures 12A1-A3 show the interactions in the form of H bonds, hydrophobicity, ionicity, and water bridges. The top panel shows the total number of specific contacts between the ligand and the protein, shown in blue. The bottom panel (orange) represents the interaction of the ligand with the exact residue of the target. Monitor protein-ligand interactions throughout the simulation process. These interactions are shown in Figures 12B1-B3. The detailed schematic diagram of ligand-atom interaction with protein residues is shown in Figure 12C1-C3. Quantitative analysis of MD simulations specifically targeting ligands B3 and DZM (GABA receptor agonists) showed that B3 forms hydrogen with aspartic acid (ASP) 56 (95%) and threonine (THR) 133 (70%) key. It is found that DZM interacts at similar points and forms hydrogen bonds in ASN, THR, SER and ILE. The data provided is consistent with previously published data. 16,17 Figure 10 (AC) shows the root mean square fluctuation (RMSF) of the atomic position of the ligand 5-[(naphthalen-2-yloxy)methyl] -1,3,4-oxadiaszole2-thiol (B3), diazepam (DZM) and Flumazenil (FLZ) use Desmond packaging. Figure 11 (A–C) shows the root mean square deviation (RMSD) of the protein GABA (6D6T) and the RMSD of the ligand shown in blue, including 5-[(naphthalen-2-yloxy)methyl]-1,3, 4-oxadiaszole2-thiol (B3), diazepam (DZM) and flumazenil (FLZ), as shown in red, use the Desmond software package. Figure 12 The timeline representation of interactions and contacts (hydrogen bonds, hydrophobicity, ions and water bridges) is shown in (A1-A3). The top panel shows the total number of specific contacts of the protein with the ligand. The bottom panel shows the residues that interact with the ligand. According to the ratio on the right side of the figure, some residues form more than one specific contact with the ligand, which is indicated by a darker orange shading. The interaction between protein and ligand is shown in (B1-B3). These interactions are classified as hydrogen bonding, hydrophobicity, ionicity, and water bridges. Schematic diagram of ligand-atom interaction with protein residues. (C1-C3) shows interactions that occurred over 30% of the simulation time.

Figure 10 (AC) shows the root mean square fluctuation (RMSF) of the atomic position of the ligand 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3), diazepam (DZM) ) And flumazenil (FLZ) in the Desmond package.

Figure 11 (AC) shows the root mean square deviation (RMSD) of the protein GABA (6D6T) and the RMSD of the ligand shown in blue, including 5-[(naphthalen-2-yloxy)methyl]-1,3,4- Oxadiaszole2-thiol (B3), diazepam (DZM) and flumazenil (FLZ), as shown in red, use the Desmond software package.

Figure 12 The timeline representation of interactions and contacts (hydrogen bonds, hydrophobicity, ions and water bridges) is shown in (A1-A3). The top panel shows the total number of specific contacts of the protein with the ligand. The bottom panel shows the residues that interact with the ligand. According to the ratio on the right side of the figure, some residues form more than one specific contact with the ligand, which is indicated by a darker orange shading. The interaction between protein and ligand is shown in (B1-B3). These interactions are classified as hydrogen bonding, hydrophobicity, ionicity, and water bridges. Schematic diagram of ligand-atom interaction with protein residues. (C1-C3) shows interactions that occurred over 30% of the simulation time.

Intraperitoneal (ip) injection of B3 at doses of 20 and 40 mg/kg resulted in 50.0% and 83.33% of animals being protected from PTZ-induced mortality, respectively. In addition, B3 also significantly increased the duration of seizures, P <0.01, and reduced the duration of seizures, P <0.01 in PTZ-induced seizures. Compared with the 100% mortality and 0% protection in the PTZ group shown in Table 4, B3 also reduced the mortality to 16.66% and increased protection (83.33%). The results of the B3 anticonvulsant study and the recently published data of Van and colleagues, who interpreted PTZ-induced epilepsy in mice as an animal model. 28 Table 4 Effects of 1,3,4 oxadiazole derivatives (B3) on epileptic seizures induced by phenyltetrazole (PTZ) in mice. Data are shown as mean ± standard error of mean (SEM) (n = 6). **P <0.01 with PTZ group, one-way analysis of variance (ANOVA) and Post Hoc Tukey test

Table 4 Effects of 1,3,4 oxadiazole derivatives (B3) on seizures in mice induced by phenyltetrazole (PTZ). Data are shown as mean ± standard error of mean (SEM) (n = 6). **P <0.01 with PTZ group, one-way analysis of variance (ANOVA) and Post Hoc Tukey test

The ip injection of 2 mg/kg FLZ 5 minutes before B3 and 35 minutes before PTZ resulted in a reduction in the percentage of protection from 83.33% of PTZ-induced mortality to 66.6%. FLZ also significantly reduced (P <0.01) the duration of seizures and increased the duration of seizures (P <0.01). As shown in Table 5, the administration of flumazenil also increased the mortality rate to 33.3% compared with saline (100%). Table 5 Effect of flumazenil on the anticonvulsant effect of 1,3,4-oxadiazole derivatives (B3) induced by phenyltetrazole (PTZ) in mice with flumazenil. Data are shown as mean ± standard error of mean (SEM) (N = 6). **P <0.01 and ***P <0.001 compared with the PTZ group, one-way analysis of variance (ANOVA) and post-hoc Tukey test

Table 5 The effect of flumazenil on the anticonvulsant effect of 1,3,4 oxadiazole derivatives (B3) in phenyltetrazole (PTZ)-induced seizures in mice. Data are shown as mean ± standard error of mean (SEM) (N = 6). **P <0.01 and ***P <0.001 compared with the PTZ group, one-way analysis of variance (ANOVA) and post-hoc Tukey test

The effects of B3 on lipid peroxidation (LPO) and inducible nitric oxide synthase (iNOS) in the cerebral cortex were evaluated. Oxidative stress markers are the main molecules responsible for promoting inflammation and neurodegeneration. 29 Several studies have explained the up-regulation of these destructive factors, which are involved in increasing the possibility of harmful consequences. Compared with the normal saline group, iNOS (70.22 ± 1.2) and LPO (253.13 ± 3.1), B3 significantly reduced the levels of iNOS (46.41 ± 2.6) and LPO (187.16 ± 3.1), P <0.01 and P <0.001, As shown in Table 6 below. Table 6 The effect of 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3) on the expression of cerebral cortex oxidase in an acute epilepsy model. Data are shown as mean ± standard error of mean (SEM) (n = 6). . *P <0.05, **P <0.01 and ***P <0.001 vs PTZ group, analysis of variance (analysis of variance)) and Post Hoc Tukey test

Table 6 The effect of 5-[(naphthalen-2-yloxy)methyl]-1,3,4-oxadiaszole2-thiol (B3) on the expression of cerebral cortex oxidase in an acute epilepsy model. Data are shown as mean ± standard error of mean (SEM) (n = 6). . *P <0.05, **P <0.01 and ***P <0.001 vs PTZ group, analysis of variance (analysis of variance)) and Post Hoc Tukey test

The effects of B3 on certain oxidative stress-related enzymes such as GSH and GST in the cerebral cortex were studied, as shown in Table 6. The levels of GSH and GSH in the PTZ-induced convulsion group were 7.22±4.2 and 5.88±2.3, respectively. Treatment of B3 significantly increased (P <0.01) the levels of GSH and GST to 47.26 ± 1.2 and 69.78 ± 2.6, respectively. The results of B3 show the effect of regulating protective biomarkers and reversing signs of epileptic shock and are well-studied markers for their role in neurodegenerative diseases. 29

The morphological changes of brain tissue after PTZ administration is a well-known report parameter that can explain the damage to the affected area. The administration of PTZ resulted in increased infiltration, disorder of cell structure, formation of edema, increased intracellular space and disorder pattern, as shown in Figure 13. B3 (40mg/kg) treatment reversed the pathological changes induced by PTZ and resulted in well-organized cell structure, no infiltration, and intact intracellular space without edema. The results of the current study are consistent with the results of the published literature. 30 Figure 13 Representative immunohistochemical images of hematoxylin and eosin (H and E), and quantitative histograms of the reactivity and integral density of surviving neurons in the cortex.

Figure 13 Representative immunohistochemical images of hematoxylin and eosin (H and E) and quantified histograms of responsiveness and integral density of surviving neurons in the cortex.

The results of immunohistochemistry are shown in Figure 14A and B. The results showed a significant increase/expression of TNF-α in the PTZ-induced group. B3 administration attenuated the level of overexpressed TNF-α in the cortex, significantly (P <0.001). The results are consistent with the previous literature. 30 Figure 14 Immunohistochemical results of tumor necrosis factor (TNF)-α in mouse cortex. The bar is 20µm, and the magnification is 40×. The histogram shows that the expression of TNF-α is relatively higher in the PTZ-induced seizure group*** P <0.001 There is a significant difference compared with the phenyltetrazole (PTZ) group. The data is expressed as the mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey test in Graph Pad Prism. Use Graph Pad Instate to calculate the p-value.

Figure 14 Mouse cortical tumor necrosis factor (TNF)-α immunohistochemical results. The bar is 20µm, and the magnification is 40×. The histogram shows that the expression of TNF-α is relatively higher in the PTZ-induced seizure group*** P <0.001 There is a significant difference compared with the phenyltetrazole (PTZ) group. The data is expressed as the mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey test in Graph Pad Prism. Use Graph Pad Instate to calculate the p-value.

We studied the effect of B3 on the expression of TNF-α in the cortex, as shown in Figure 15. The overexpression level of TNF-α in the PTZ group was observed in the cortex. B3 administration significantly (P <0.001) down-regulated TNF-α and reduced signs of convulsions, and inhibited PTZ-induced inflammation. The results obtained can be correlated with published data. 30 Figure 15 TNF-α protein expression, quantified by using enzyme-linked immunosorbent assay. (ELISA). Data are shown as mean ± standard error of mean (SEM). ***P<0.001 indicates a significant difference compared with the treatment group, ###P <0.001 indicates a significant difference with PTZ. Analyze the data by using one-way analysis of variance (ANOVA), and then perform a post-hoc Tukey test in Graph Pad Prism. The P value is calculated using Graph Pad Instate.

Figure 15 Protein expression of TNF-α, quantified using enzyme-linked immunosorbent assay. (ELISA). The data are shown as mean±standard error of mean (SEM). ***P<0.001 indicates a significant difference compared with the treatment group, ###P <0.001 indicates a significant difference with PTZ. Analyze the data by using one-way analysis of variance (ANOVA), and then perform a post-hoc Tukey test in Graph Pad Prism. The P value is calculated using Graph Pad Instate.

In order to improve the quality of life (QOL) of patients and reduce the burden of disease, there are some commonly used drugs on the market, including carbamazepine, phenobarbital, phenytoin, diazepam, etc., which are used to control epileptic seizures. However, the use of these drugs It is also associated with gastrointestinal problems, dizziness, drowsiness and the possibility of addiction. In addition, these drugs were found to be ineffective for 30% of the population. Therefore, the health system needs to develop safe and effective epilepsy drugs to improve the quality of life of patients. 31 Epilepsy is a neurological disease characterized by frequent and unpredictable interference discharges, affecting 50 million people worldwide, regardless of age. The mechanism of epilepsy is not clear, but it is speculated that infection, neuroinflammation, stroke, or GABA and glutamate pathway disorders may cause epileptic shock. GABA has always been a well-known epilepsy target, and has been the focus of attention and discussion by many researchers for decades. Recently, newly synthesized pyrimidine-triazine compounds have been studied because of their potential to attenuate seizures through GABA mimicry. 32 This research also aims to develop and add new parts with anti-epileptic activity mediated by GABA receptors. GABA: 6D6T with PDB-ID is also used in the previous computational research of many researchers. 33 Based on literature research and evidence about the involvement of GABA in epileptic seizures, we also selected it in our study. In order to evaluate the role of B3 in epileptic shock, the ligands B3 and GABA were first screened through molecular docking studies. Do molecular docking to check the binding affinity of the ligand to GABA. Nowadays, docking is the first step to confirm the interaction of ligands with their respective targets. 10,11 The advantageous result of docking in the form of binding affinity and hydrogen bonding is to perform molecular dynamics (MD) simulations to further verify the cause of the interaction. B3 opposes GABA. MD simulation is attracting the attention of researchers involved in drug discovery and development. 34 This tool can be used to check conformational changes, the positioning of various atomic structures, detect mutations, protonation, and phosphorylation. What's more interesting is that any atom, target or ligand and 35 MD simulation were first used in 1970, and later attracted many Biologists screen their newly developed/discovered compounds through MD simulations. Many researchers use it to compare and correlate results obtained from animal studies. 36,37 It is also used in neuroscience to examine neuronal signaling pathways, their work, and how these pathways change to develop disease conditions and how to target it to reverse pathological processes. 38-40 We also performed 50ns MD simulations for the three complexes. The stability of the composite was verified and the Desmond software package was used for 50 ns MD simulation. It is found that the complex is stable and present within the range of RMSF and RMSD values. The favorable interaction determines the affinity of the ligand to its target. The results obtained by docking and MD simulation are consistent with the published data, so we conducted animal studies. 15-17 In animal research, various types of chemical convulsants are used in animal models to explore synthetic or natural sources. 18,41 We chose the acute epilepsy model induced by PTZ to study the role of B3 in acute epileptic shock. PTZ is a BDZ receptor antagonist, a chemical convulsant, and it is related to the up-regulation of a variety of inflammatory mediators, such as interleukin 6 (IL-6), TNF-α, interleukin 1β (IL-1β), Interleukin 10 (IL-10) and interferon gamma (INF-γ) in the hippocampus and cerebral cortex. 42,43 Pro-inflammatory cytokines including IL-6, interleukin 17 (IL-17) and IL-17 receptor (IL-17R) and TNF-α also proved their role in destroying the BBB and inducing seizures. The main cells that produce these IFMs are called brain glial cells and are involved in the pathogenesis of many neurological diseases. 44-46 TNF-α activates TNF receptors-1 and 2 (TNFR1 and TNFR2). TNFR1 and TNFR2 are receptors with overexcitatory/teratogenic and anti-teratogenic properties. PTZ injection, bacterial or viral infection can cause the activation of TNFR1, which leads to neuronal cell excitement and epilepsy. 47 The relationship between IFM and neuroinflammation and neurodegenerative diseases has been extensively studied in the past. 48 These two important areas of the IFM brain (such as the cortex and hippocampus) play a crucial role in disease development and pathogenicity, and these areas are susceptible to this neuronal crisis. 49,50 In order to deal with the worse situation, anti-inflammatory drugs are the most commonly used drugs to reverse the pathogenic state and limit the release of IFM, thereby correcting behavioral defects. 51 Among the various categories effective for neurodegeneration and neuroinflammation, oxadiazole is an important and promising synthetic source that can alleviate various neuroinflammation and neurodegenerative diseases. Oxadiazole is a biologically active moiety with a five-membered heterocyclic structure. Such drugs have good anti-inflammatory, analgesic and antioxidant potential. 10,11 The potential of this type of active drug to attenuate stroke caused by middle cerebral artery occlusion has also recently been studied. 52 also studied the anti-Alzheimer's disease potential, anti-cancer, antibacterial, anti-inflammatory, anti-allodynia and anti-insomnia properties of oxadiazole. 53-55 In animal studies, it was found that taking B3 can correct behavioral defects by increasing the incidence, as shown in the results section, seizure time and reducing seizure duration. In molecular studies, literature studies have confirmed that PTZ is involved in increasing the expression of nitric oxide, lipid peroxidase and TNF-α, which are the reasons for mediating epileptic shock. B3 down-regulates the overexpression levels of nitric oxide, lipid peroxidase and TNF-α, which indicates that it has anti-neuro-inflammation potential.

The results show that B3 is a promising candidate, and it has good binding affinity with GABA, and the formation of a stable complex has been confirmed by molecular docking and molecular dynamics simulation studies. In addition to correcting the behavioral defects caused by PTZ, it also increases the incubation period of seizures, increases the levels of GSH and GST, and reduces the levels of LPO and iNOS. It can reverse the pathological changes induced by PTZ established by H & E and IHC staining of the brain. Therefore, the neuroprotective properties of B3 may be related to the down-regulation of TNF-α, which was found to be up-regulated in PTZ-induced seizures. Therefore, the available results explain the potential of B3 to reduce neurodegeneration and neuroinflammation caused by PTZ.

This work was approved by the ethics committee of the Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad.

All data is included in the manuscript.

Thanks to the Rifah International University of Islamabad for providing laboratory space and chemicals to conduct experiments and successfully complete this research work. Yusuf S. Althobaiti was supported by the researcher support project number (TURSP-2020/78) of Taif University, Taif University, Saudi Arabia.

All authors have made significant contributions to the work in the manuscript, including concept, research design, execution, data acquisition, analysis and interpretation. Participate in drafting and critically review the article, finally approve the version to be published, and agree to be responsible for all aspects of the work.

No external funding was obtained for this research.

The author declares that there is no conflict of interest for this work.

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