Exploring the affinity and recognition between VPA , major neurotransmitters and their natural substrate for anticonvulsant activity.

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Keywords: α-ketoglutarate dehydrogenase, Docking, GABA-transaminase, Glutamate Decarboxylase, Molecular modeling, Valproic acid, Succinate Semialdheyde dehydrogenase

BACKGROUND

Epilepsy is a deliberate state of brain in which normal electrical activity gets disturbed causing temporary communication problems between nerve cells, leading to repetitive seizures [1,2] (McNamara, Jefferys, J.G.). Nearly 1% of world population is affected with epileptic disorders. Several anti-epileptic drugs (AEDs) are being used for the treatment of epilepsy but Valproic acid (VPA, 2-propylpentanoic acid) has been used with choice, for more than a decade for epilepsy therapy as it enjoys the uniqueness of being effective in generalized seizures, including primary tonic-clonic and myoclonic seizures and partial seizures, as well [3,4] (Llyod, K.A, Vajda, F.J.E). To the darker side, VPA suffers from the major application disadvantage of having teratogenicity, reproductive dysfunction alongwith hepatic and pancreatic dysfunction [5](Nau H,Hauck RS). Therefore, there has been a constant interest and efforts till date, to search for a VPA like broad spectrum anti-epileptic agent without these major side effects. The search for newer VPA like drug candidate suffers from a limitation of lack of equivocal pharmacological basis of mechanism of action of VPA [6,7](Johannesen , C.U, Nau H,Löscher W)

Broadly, VPA has been reported to have pharmacological basis of anti-epileptic effects due to its reductive ability of neuronal excitability [8,9] (BARBARA, Löscher, W). This reductive effect has been attributed to three unequivocal hypotheses (Figure 1) that includes – a) increase in GABAergic activity; b) lowering of glutamatergic excitatory activity and c) lowering of action potential by negatively regulating voltage gated sodium channel (Chapman A) [10]

VPA reportedly increases the GABAergic activities by virtue of numerous enzymatic activities of GABA transaminase (GABAt), α-keto glutarate dehydrogenase (ALDH), Succinate Semialdehyde dehydrogenase (SSADH) and Glutamate Decarboxylase (GAD) [11,12][Bialer, M.; Yagen, B, Löscher, W.]. VPA elevates the GABA level by increasing the availability of α-ketoglutarate precursor or by inactivation of α-ketoglutarate dehydrogenase and by increasing the GAD activity, the enzyme responsible for GABA synthesis. VPA also contribute in GABA mediated inhibition by blocking GABA catabolism, declining its deterioration that is regulated by GABAt and SSADH. [13-17]( Löscher, W, Löscher, W, Whittle, S.R.;Turner, Luder, A.S, Löscher, W. Biochem Pharmacol.,)

To obtain a clear insight into the pharmacological effect of VPA, the present study was performed to analyze, with in-silico tools, the role of VPA in enzymatic control and coordination responsible for uncontrolled synchronization of GABA level thus having anticonvulsant effect [18-20](Löscher, W, Battistin, L.;Varotto, M.;Berlese, G.;Roman, G). The patterns and energetic of natural ligands binding to their respective enzymes changes if effected by any external ligand, VPA in our study, giving us the qualitative insight while the degree of these changes provide us with quantitative view. Therefore, molecular docking-simulation and binding energetic analyses were carried out for the four major enzymes involved in GABAergic and glutamergic control of GABA level, i.e., GABAt, ALDH, SSADH and GAD. These enzymes were docked against their respective natural ligands and VPA for the study. Also, these enzymes bound with VPA were docked again against the same natural ligands to observe and analyze the docking profile difference generated due to VPA.

METHODS

Human-ALDH (PDB ID-1DTW), Human-SSADH (PDB ID-2W8O) and Human-GAD (PDB ID-2OKK) were retrieved from RCSB Protein Data Bank . VPA, and all the natural substrate α-Ketoglutarate, Succinate Semialdehyde, Glutamate were downloaded from chemical database NCBI Pubchem. Since the solved crystal structure of human GABAt was not available, its 3D structure was generated by homology modelling using Modeller v9.11. For homology modelling of human GABAt, the target sequence was retrieved from NCBI protein sequence database with accession number NP_001120920.1, having residue count of 500 amino acid. PSI-BLAST was performed for the identification of template for modelling human GABAt against PDB structure database. Sus scrofa GABAt (PDB ID- 1OHV) with 96% similarity was identified as a template sequence. The generated models were evaluated for Dope score and GA341 score. The model with the best score was refined for loops using loop refining script of Modeller [21](Fiser and Sali) . The refined model was subjected to structure validation using Swissmodel server [22] (Arnold, K), using various estimation parameters i.e. Z-score, QMean Score [23] (Benkert), Anolea [24] (Melo), D-fire Energy [25] (Zhou and Zhou). The online tools - ERRAT[26](Colovos), Verify3D [27](Luthy R), PROCHECK [28] (Laskowski,) at Structure Analysis and Verification Server (SAVES) (nihserver.mbi.ucla.edu/SAVES/) were further used for further verification and assessment. The 3D-structure of human GABAt was energy minimized using SPDBV. GROMOS96 force field was used for the running the energy minimization program. Then all these enzyme models (h-ALDH, h-SSADH, h-GAD and hGABAt) were subjected to docking against their natural substrates (α-Ketoglutarate, Succinate Semialdehyde, Glutamate and GABA) and with VPA using AutoDock 4.2.5.1 [29](Morris). The docked complex of VPA and these 4 proteins were saved as .pdb file and were further docked individually with their substrate. All the docking simulations were performed using the grid and docking parameters as detailed in Table 1 and the docked conformation of lowest binding energy or maximum cluster (if the lowest binding energy and maximum cluster binding energy difference was more than 2.5kcal/mol) were saved. The analysis of docked complexes thus obtained were done using Analyze module of Auto Dock Tools (ADT).

RESULTS AND DISCUSSION

In this study, we used docking study to explore the affinity and recognition between VPA , major neurotransmitters and their natural substrate for anticonvulsant activity. The first and major step was to retrieve all neurotransmitters from PDB. The GABAt structure is still under X-ray crystallographic investigation but not yet available and thus modelled using Modeller9v8. The target sequence Sus scrofa (PDB ID 1OHV) was used as a template to generate the 3D human GABAT structure with accession number NP_001120920.1 with 500 amino acid residue count. The generated structure was assessed using Protein Structure and Model Assessments Tools at Swiss Model Server. The Predicted Z-Score was -2.243, QMean6 score was 0.579, and DFire energy was 701.11kj/mol (Table 1). 3D human GABAT structure was also evaluated with SAVES that includes PROCHECK, ERRAT and Verify3D. The Ramachandran Plot in PROCHECK shows 89.9% in core region. ERRAT depicts the statistical analysis of non-bonded interactions between different atom types. The overall quality factor for ERRAT is 86.850. VERIFY 3D analyze the Compatibility of atomic model with its own amino acid sequence and predicted 81.04% of residue had average 3D-1D score>0.2% (Table 2). Further analysis with Pro-Q [30](Wallner B, Elofsson A), Pro-SA [31] (Wiederstein. M.), and RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) revealed the model is of good quality and can be used in our investigation. The geometry of modelled structured and elimination of unfavourably non-bonded contacts were energy minimized in SwissPDBviewer. The energy computation was done with GROMOS96 force field and minimised to -23203.176kJ/mol-1. Molecular docking study was carried out between all the above said neurotransmitters enzymes with their substrate and with Anti-epileptic drug VPA. The docked complexes of all the four neurotransmitters enzymes with VPA were saved (Figure) and they were finally docked independently with their substrates.

The hydrophilic pocket of human GABAt was predicted to interact through amino acid residues SER356, ARG220, GLY164, CYS163, LYS357, HIS218, VAL328 (Fig1a ). The free energy of binding and estimated inhibition constant were determined to be -4.99Kcal/mol and 218.28uM. Seven carbon atoms of VPA were involved in hydrophobic interactions with amino acid residue ARG220, GLY219, ASP326, HIS218, VAL328, SER356 of human GABAt and 4 residues were involved in Hydrogen bonds are tabulated in (Table 4). The docked complex of GABAt and VPA were saved and were used for further docking analysis. The free energy and Ki value from the docked complex of VPA with Gabat and with their natural substrate GABA is -3.55kCal/mol and 2.52mM. The binding pocket was predicted to interact with VPA and GABA through amino acid residues SER149, LYS400, VAL150, GLU341, PHE338 (Fig 1b). The carbon atom of GABA C1 and C2, oxygen atom O1 and O2 and atom N are involved in hydrophobic interaction. Atoms O1 and N are involved in Hydrogen bonds with amino acid residues LYS400, SER149, GLU342 (Table 4). The polar and hydrophilic pocket of GABAt was predicted to interact with GABA through amino acid residues SER149, LYS400, PHE338, GLU342, VAL150 (Fig 1c). The free energy of binding and inhibition constant for GABAt and GABA interaction were -3.83 and 1.56mM, respectively. Atoms C1, C2, C3, C4, N,O1 and O2 are involved in hydrophobic interactions through amino acid residues SER149, LYS400, GLU342, VAL150, PHE338.The oxygen and Nitrogen atom of GABA are involved in Hydrogen bonding with amino acid residue SER149, LYS400, GLU342. The carboxylate group of GABA interacts with K400 and S149, determining the specificity of monocarboxylic amino acids GABA. Atom NZ of K400 is donating a proton to GABA and atom O of GABA accepting the proton maintaining the acid base catalyst reaction. The resultant binding energy difference between the interaction of docked complex and GABAt with GABA is -0.28Kcal/Mol (Table 3). The interaction study analysis revealed that atom O1 and N orienting the atom NZ of K400 donating proton and OE2 of GLU342 accepting proton and changes the bond length. The docking position and interacting amino acid residues showing that VPA is not altering the actual site of GABA and thus showing non-competitive inhibition. The higher binding affinity because of lower binding energy and difference in binding energy of docked complex and GABAt with GABA attribute the Valproate inhibitory effect on GABAt.

The docking study of VPA with SSADH reveals that the binding energy and inhibition constant were -5.33kcal/mol and 124.44uM (Table 3). Further the interacting residues study reveals the binding pocket of VPA is ASN385, TRP204, ALA388, LYS391, ILE384, PHE440 (Fig 1g). Oxygen atom (O2) of VPA was observed to make hydrogen contact with amino acid residue F440 with bond length 2.77 (Table 4). In addition, carbon atom C1-C8 and Oxygen atom O1 and O2 were predicted to be involved with hydrophobic interactions with all the residues present in active site. The docked complex of SSADH and VPA were saved and further docked with SSA and reveals the estimated binding energy and Ki -4.28kcal/mol and 801.38uM (Table 3). The docked complex oh VPA and SSADH was determined to interact through residues LYS192, ILE521, GLU170, ARG173, TYR175, VAL174. Atom NZ of amino acid residue LYS192 is donating proton to atom O2 of carboxyl group of SSA and forms hydrogen bond of length 2.69. Carbon atom C1,C2,C3,C4 and Oxygen atom O1,O2 and O3 were involved in hydrophobic interactions with all the active site residues (Fig 1h). Further, the docking study of Human SSADH with SSA releases the binding energy and Ki of -4.5Kcal/mol and 503.27uM (Table 3). The active site of human brain SSADH was determined to interact with SSA through residues LYS391, TRP204, PHE440, ALA388, THR439 (Fig 1i). The oxygen atom O1 forms hydrogen bond with atom N of amino acid residue of F440, O2 forms hydrogen contact with atom NZ of K391 with bond length of 2.76, Atom O3 is involve in hydrogen contact with NG W204 (Table 4). Carbon atom C1,C2,C3,C4 and Oxygen atom O1, O2, O3 of SSA were involved in hydrophobic interaction with all the active site residues of binding pocket. VPA . The binding energy difference of docked complex and SSADH with SSA was -0.22kcal/mol (Table 3), showed the inhibitory action of VPA towards SSADH. The docking position and interacting amino acid residues , indicate the competitive inhibition system.

VPA was found to interact with human ALDH with 5 amino acid residues , namely, VAL367, TYR119, PRO158, LYS116, ALA115, ASP366 (Fig 1d). The binding energy and inhibition constant of ALDH and VPA interaction were determined to be -1.99kcal/mol and 34.68mM (Table 3). Carbon atom of VPA, C2-C8 and Oxygen atom O1 and O2 were predicted to be involved in hydrophobic interactions with amino acid residues present in active site. The Oxygen atom (O2) of VPA was involved in hydrogen bond interaction with NZ atom of LYS116 (Table 4). The complex were saved and used for further analysis. The docked complex of ALDH and VPA were further docked with AKG and predicted free energy of binding and ki were -3.5kcal/mol and 2.7mM (Table 3). Further analysis revealed the active site residues are HIS51, LEU52, LYS56, LYS266, TYR262, PRO53 (Fig 1e). The carbon atom C1-C8 and Oxygen atom are involved in Hydrophobic interactions with all the amino acid residues present in active site but only 4 amino acid residues are involved in hydrogen bond formation (Table 4). The O2 atom of AKG forming hydrogen bond with atom N of LEU52. similarly O1 atom was involved in hydrogen bond formation with OH atom of TYR262 and NZ atom of LYS56 donating the proton. The O3 atom with a bond length of 3.07 make hydrogen contact with H51. Further the free energy of binding and Ki of human ALDH and AKG interaction were determined to be -3.66kCal/mol and 2.06mM respectively(Table3). The active site residues involved in this interaction are LYS66, LYS179, TYR176, PHE70, ASP67, ARG61, LEU211 (Fig 1f). The O1 atom of AKG forms H-bond with atom O of LYS66 with bond length of 2.86, whereas atom O1 and O2 of AKG forming hydrogen contact with atom NH1 and NH2 of ARG61, similarly atom O3 and O5forms hydrogen bond with OH of TYR176 (Table). Carbon atom C1,C4 and C5 and Oxygen atom O1, O3 and O5 of AKG were making hydrophobic contact with all the amino acid residues involved in active site. In this case, it is relevant to intricate this interaction as VPA is altering the site of action. The docked complex after interacting with natural substrate AKG altering the site and the difference in binding energy is -0.16kcal/mol, showing competitive inhibition (Table 3 ). Lowest binding energy and higher affinity of VPA shows the inhibitory action towards ALDH. VPA showed inhibitory action towards GABAt, ALDH and SSADH.

The docking simulation of GAD with glutamate releases the free energy of binding and inhibition constant of -6.21kcal/mol and 28.29uM (Table3). The polar and hydrophobic domain of human GAD with VPA interact through HIS377, GLU476, GLU479, LYS498, PRO499, ALA478, GLN500, HIS501, LEU475, LYS380(Fig1j). The 8 carbon atom of VPA C1-C8 and oxygen atom O1 and O2 were involved in hydrophobic interactions and 2 amino acid residues LYS380 and HIS501 were forming Hydrogen bonds (Table4). The free energy of binding and ki of docked complex of VPA and GAD with glutamate were -4.91kcal/mol and 252.64uM(Table3). The binding pocket of docked complex and Glutamate was determined to interact through residues SER382, PHE344, GLN500, ASP345, LEU347, LYS309, LEU348, PRO346(Fig 1k). The oxygen atom (O1) of glutamate was involved in hydrogen bond interaction with NZ of LYS380 and atom N of HIS501 (Table4). The carbon atom C1-C8 and oxygen atom O1 and O2 were involved in hydrophobic contact with all the active binding site residues. The free energy of binding and Ki of GAD with glutamate were -4.87kcal/mol and 271.15uM, respectively(Table3). The amino acid residue of binding pockets are LYS309, PRO344, LEU348, SER382, ASP345, LEU347, GLN500 (Fig1l). The atom OE1 and OE2 forms hydrogen bond with atom N of LEU347 and LEU348. The OXT atom of glutamate involves hydrogen bonding with atom NZ of LYS309. Atom C, CA, CB, CG,CD, O, OE1 and OE2 were involved in hydrophobic interaction with all the active amino acid residue in the present binding pocket.

Conclusion

Abbreviations

GABAt- Gaba Transaminase

SSADH- Succinic semialdehyde dehydrogenase

ALDH- alpha ketoglutarate dehydrogenase

GAD- glutamate decarboxylase

VPA- Valproic acid

DFIRE- distance scaled finite ideal gas reference

Conflict of interest

The authors confirm that this article content has no conflict of interest.

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