Gajanan Vaishnav et al. / Journal of Pharmacy Research 2011,4(9),3162-3165 Research Article Available online through ISSN: 0974-6943 www.jpronline.info Automated in silico modelling and docking of ligands on

benzodiazepine binding site of GABAA receptors Gajanan Vaishnav1*, Kulkarni G. K2and S. Sankar3 1Yash Institute of Pharmacy, South City, Waluj Road, Aurangabad, Maharashtra, Pin 431134, India, 2Dr.Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra Pin 431 003, India, 3Department of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Rocklands, Ooty 643001 Received on: 22-06-2011; Revised on: 08-07-2011; Accepted on:01-08-2011

ABSTRACT Many antianxiety, sedative and antiepileptic ligands such as benzodiazepines (BZDs) act by allosteric modulation of the GABA(A) receptor via the benzodiazepine binding site. This increases the affinity of GABA for the receptors. The exact mode of docking of these ligands in the 1,4-benzodiazepine (BZD) recognition site has been a subject of recent in-silico docking studies. These studies have utilized previous attempts which have been made to superimpose the structures of allosteric modulators to construct a pharmacophore model for the BZD recognition site or manual editing of homology sequences. However, such models are difficult to relate to receptor structure and tedious to produce. The reproducibility of manual editing is also questionable. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnaea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the

BZD binding pocket at the a1 / g2 subunit interface. With fully automated homology modeling pipeline SWISS-MODEL and binding pocket for the homology model was predicted with fuzzy oil drop Gaussian server. Ligands known to interact with BZD binding site were energy minimized, introduced into the binding pocket and molecular simulations were carried out to yield a set of binding poses of the BZD ligands in the binding pocket. A fast, easy and reproducible model providing a basis for the design and selection of GABAA receptor benzodiazepine binding site modulators has been reported in this study. The best docking poses of ligands were found to be in good confirmation with their reported affinities towards receptor binding site. 55GLN and 114 TYR residues were common in all the best ligand docking poses in the binding packet.

Key words: Allosteric Regulation/genetics,Animals,Benzodiazepines/metabolism*,Binding Sites/genetics,Ligands,Models, Molecular,Protein Binding/ genetics,Rats,Receptors, GABA-A/chemistry,Receptors, GABA-A/metabolism*,Structure-Activity Relationship,Fuzzy oil drop model

INTRODUCTION GABA, 4-aminobutyric acid, is the primary inhibitory transmitter in the brain Unfortunately, many of the early benzodiazepines had relative limited subtype and maintains a balance between excitation and inhibition of neurons. Three selectivity resulting in sedation, dependence, cognitive impairment, and ataxia, [5] major classes of GABA receptors have been identified: GABAA, GABAB and potentiating of ethanol effects, tolerance and withdrawal . GABAC. GABAA and GABAC receptors are ligand-gated ion channels (LGIC), while GABA receptors are G-protein coupled receptors. The LGIC receptors Attempts have been made to superimpose the structures of allosteric modula- B [1;6-15] are heteropentamers comprised of a1-6, b 1-3, g1-3, r 1-3, d, e, p and q subunits. Each tors to construct a pharmacophore model for the BZD recognition site . subunit contains four membrane-spanning domains. The N-terminal domain However, such models are difficult to relate to receptor structure. [16] deter- and C-domain are extracellular and the agonist/antagonist binding site is situ- mined affinities for a series of imidazo- and 5-phenyl-1,4-benzodiazepines to ated on the N-terminus. There is an intracellular loop between the 3rd and 4th wild-type and mutant receptors to delineate the orientation of these ligands in membrane spanning regions [1-3]. the recognition site. An extra hydroxyl group of tyrosine introduced by the

g2F77Y mutation interferes with para-substitutions of the C5-phenyl ring, All known GABAA receptors contain a plurality of distinct modulatory sites, suggesting that the phenyl ring is adjacent to g2Phe77 in the binding pocket. one of which is the benzodiazepine (BZD) binding site. Other modulatory sites Kucken (2003) [13] used a series of three substituted imidazobenzodiazepines in include allosteric sites for picrotoxin, barbiturates, neuroactive steroids and combination with amino acid mutations of varying volume at g2Ala79 to infer ethanol. The BZD binding site is the most explored of the GABAA receptor the position of compounds similar to Ro 15-1788 and Ro 15-4513. However, modulatory sites, and is the site through which anxiolytic drugs such as diaz- the methodology for homology model creation included manual deletion/ ad- epam exert their effect. Early radioligand binding studies suggested the exist- dition of amino acid residues, which was tedious and reproducibility was ques- ence of two distinct benzodiazepine-binding sites: BZD1 and BZD2. The BZD1 tionable. subtype has been shown to be GABAA receptor comprising the a1 subunit in combination with a g2 subunit. This is the most abundant GABAA receptor An automatic, simple, fast and reproducible homology model construction subtype. Two other major populations are the a2g2 and a3g2 subtypes. Together method for the BZD binding site located at GABAA receptor a1/ g2 subunits has these constitute approximately 35% of the total GABAA receptor repertoire. been reported in this study. Use of Fuzzy oil drop Gaussian model calculation Pharmacologically, the a2g2 and a3g2 subtypes appear to be equivalent to the for prediction of BZD binding pocket of GABAA receptor has also been re- BZD2 subtype. The physiological role of these subtypes has hitherto been ported. The homology model was constructed based on the crystal structure of unclear because sufficiently selective agonists or antagonists are unknown. acetylcholine binding protein (AChBP) [17]. The binding site was predicted based on a Gaussian Fuzzy oil drop model. The barbiturates and benzodiazepines were among the first clinically useful modulators of the GABA receptors and are among the most widely prescribed MATERIALS AND METHODS medications for anxiety, depression and other psychiatric disorders and as In the present work, all the ligands used were made using Chemdraw 3D Ultra anticonvulsants [4]. Benzodiazepines, with relatively mild side effects, af- 8.0. Before the docking calculation of the ligands, the structures were fully forded an alternative to barbiturates which possess more potent side effects. optimized. As details of the calculations used are available in the literature and therefore, they are not mentioned here. Argus Lab 4.0 was used [18]. The crystal structure used for the present study was found to be acetylcholine binding *Corresponding author. protein (ACHBP) in complex with nicotine, was downloaded from Protein Gajanan A. Vaishnav Data Bank (http://www.rscb.org/) as PDB file to perform all the docking tech- Depatrment of Pharmaceutical Chemistry niques. The file containing the crystal structure of ACHBP complexed with nicotine (PDB entry 1uw6) was downloaded. Yash Institute of Pharmacy, South City, Waluj Road, Aurangabad, Maharashtra, INDIA, PIN 431134 A.Homology modeling by Swiss 3D modeler: Tel: 09049996249 The SWISS-MODEL Repository is a database of annotated three-dimensional Fax: (0240)2551763 comparative protein structure models generated by the fully automated ho- Email: [email protected] mology modeling pipeline SWISS-MODEL, run by the Swiss Institute of

Journal of Pharmacy Research Vol.4.Issue 9. September 2011 3162-3165 Gajanan Vaishnav et al. / Journal of Pharmacy Research 2011,4(9),3162-3165 [19-21] Bioinformatics . The mature protein sequences of the rata1 and g2 subunits where N is the total number of residues in the protein under consideration, H (accession numbers: a , P62813; g , P18508) were automatically aligned with r/ 1 2 j denotes the hydrophobicity of the i-th residue according to the normalized sequences of two adjacent AChBP subunits (A and B, respectively) using scale of hydrophobicity for amino acids, gij denotes the separation of the j-th ClustalW [22] and submitted to the Swiss 3D modeling server for homology grid point and the effective atom of the i-th residue, and c denotes the hydro- modeling and 3D structure generation. The server generated 1 structure for phobic cutoff and has the fixed value of 9.0A0. This means that only residues the sequence. For segment 1 modeled range is from 1 to 209 based on template with r = c influence the j-th point. He is the sum of observed hydrophobic- 2zjuB with sequence identity of 98.565 % and Evalue = 0.00e-1. The 3D ij sum ity for all analyzed grid points. Using1/Hosum2 is a normalizing coefficient the ribbon view of the generated homology model along with projecting amino observed hydrophobicity can be compared to the theoretical hydrophobicity acid residues was obtained (Figure 1A). described previously. The application of this function requires the parameters of hydrophobicity attributed to each amino acid. A new theoretical scale of amino acid hydrophobicity was created.

The PDB file containing 3D co-ordinates of homology model obtained from SWISS MODEL workspace server was submitted to fuzzy oil drop model server at website (http://www.bioinformatics.cm-uj.krakow.pl/activesite/) for determination of ligand binding site. The calculated ligand binding site co- ordinates were saved on hard disk of a computer having Intel Core2Duo TM microprocessor and Windows7 TM operating system as PDB file. The 3D ribbon structure of the binding site is shown in Figure 1B.

1A 1B C. Construction of ligands by molecular builder tool of Argus lab Figure 1: 3D ribbon views of Homology model generated from SWISS – software: MODEL workspace (lA) and binding site generated from fuzzy oil drop The tool provided allows constructing new molecules and modifying existing model server (1B). molecules. Using its molecular formula, the ligands were constructed by Chemdraw4 software. Energy minimization was performed using molecular builder toolkit function of Arguslab 4.0.1[18]. The structures were manually B. Active site identification by Fuzzy oil drop calculation server: checked for inconsistencies and corrected for hybridization states and bond The fuzzy oil drop is a gaussian model oriented on localization of area respon- orders. All the ligands were converted into PDB format for docking purpose. sible for ligand binding or protein-protein complex creation is based on char- acteristics of spatial distribution of hydrophobicity in a protein molecule. It [23] D. Docking and binding evaluation has long been used for recognition of ligand binding site in proteins . It is In the automated Argus Lab 4.0.1 system [18], using a generic algorithm with a assumed that hydrophobicity changes from protein interior (maximal hydro- fast-simplified Potential of Mean Force (PMF) carried docking of flavonoid phobicity) to exterior (close to zero level of hydrophobicity) according to the ligands into 3D active site structure. It was assumed that the protein and the three-dimensional Gauss distribution. It is generally accepted that the core ligand docked non-covalently. The standard PMF implementation used UFF region is not well described by a spheroid of buried residues surrounded by potential for this purpose. The docking was carried with flexible ligand into a surfaces residues due to hydrophobic channels that permeate the molecule. rigid protein active site. The general procedure for the docking process started Therefore the simple comparison of theoretical (idealized according to Gauss with the addition of energy minimized target ligand on the 3D coordinates of function) and empirical spatial distribution of hydrophobicity in protein gives the predicted binding site on homology modeled protein obtained in earlier the opportunity to identify the regions with high deviation versus the ideal step. The predicted active site was defined by amino acid codes obtained from model. Those regions recognized by high hydrophobicity density differences fuzzy oil drop calculations. The ligands were specified in the program. Using seem to reveal functionally important sites in proteins. The model has been 22×22×22 A 0 box located at the centre of the target active site optimized the found to be verified positively for prediction of 3D coordinates of 1NMF, a different starting parameters. The whole procedure of docking was repeated downhill protein [24] and small peptides representing various functional groups [25] [26] until a constant value of docking score was achieved. If a ligand did not dock . have described a method for prediction of ligand binding site based on in ArgusdockTM mode, it was docked with GADdock mode. location of a region of unusual hydrophobicity in a protein structure. Concluding docking results were parameterized in terms of docking score in Theoretical fuzzy-oil-drop [23] Kcal/mol. The docked GABAA receptor benzodiazepine site ligands 1a-l, The fuzzy-oil-drop is described by three-dimensional Gauss function. Gauss complexed with GABAA receptor a1/g2 homology benzodiazepine model was function usually interpreted as a probability distribution is assumed to repre- interpreted by looking at the H-bonding or hydrophobic interactions of the sent the hydrophobicity distribution. If the j-th point described by cartesian ligand with the amino acid residues in the active site. The docking scores coordinates (X Y , Z) belongs to a box with its center at the origin of the j, j j obtained from the docking of these ligands of BZD site of GABA receptor coordinate system (0, 0, 0) the expected hydrophobicity value He for this A j into the predicted target active site pocket are summarized in the Table 1. . As point, is calculated as follows: all the ligands showed difference in their binding energies pointing towards the significant role of various substituents in their binding abilities.

The docked structures of ligands were overlaid and visually compared for characterization of their binding mode and a pharmacophore model was con- structed based on observation of structures of ligands binding in similar con- The hydrophobicity maximum localized in a center of ellipsoid decreases in formation and docking scores. form of distance-dependence according to three-dimensional Gauss function. The parameter - mean value, for which Gauss function reaches its maximum is RESULTS AND DISCUSSION localized in (0, 0,0) point in coordinate system. The second parameter - Validation of potential mean force (PMF) method standard deviation represents the size of drop (the values of three standard To validate the docking model, before docking the test ligands (BZD site deviations determines the size of drop: (s , s , s ), and depends on the length x y z ligands 1a-1l), the docking of diazepam into the active BZD binding site in of polypeptide under consideration. He is the sum of theoretical hydropho- sum homology model of GABA receptor was performed. Diazepam binds into the bicity for all analyzed grid points. Each j-th grid point (X , Y , Z ) is character- A j j j active site cavity with a binding score of -10.6596 kcal/mol and R.M.S.D in ized by the He value, which represents the idealized degree of hydrophobicity j binding scores of two consecutive docking runs of the same ligand was ob- in the fuzzy-oil-drop. served to be 3.12 which was well within acceptance value of NMT 5%. The

[23] docked structure of diazepam in the active site of GABAA receptor homology Observed fuzzy-oil-drop model is shown in Figure 2. The close overlapping of a docked structure with The observed hydrophobicity distribution within the fuzzy-oil-drop is calcu- Zolpidem and SL 651 498 (Figure 3) demonstrates the validity of the model. lated using the simple sigmoid function proposed to quantitavely describe the Docking of GABAA receptor ligands 1a-1l into active site hydrophobic interactions. The j-th point collects hydrophobicity Hoj as fol- lows: Known BZD site ligands 1a-1l (Figure 3) was docked into the active site of the homology model. The ligands were selected on basis of differences their reported subtype selectivity and agonist properties reported in the literature. Diazepam, SL651498 and Flunitrazepam showed highest docking scores (Table 1) which was found to be in good agreement with the fact that the three ligands

Journal of Pharmacy Research Vol.4.Issue 9. September 2011 3162-3165 Gajanan Vaishnav et al. / Journal of Pharmacy Research 2011,4(9),3162-3165 Table 1: Overview of known BZD site ligands with their agonist properties and docking scores on predicted BZD binding pocket of the homology

model of GABAA receptor. S.No. Ligand Structure Description Reference IUPAC Name Docking score (kcal/ mole)

1a Diazepam 1 agonist [27;28] 7-chloro-1,3-dihydro-1-methyl-5-phenyl-1,4-benzodiazepin-2(3H)-one -10.6596

1d SL 651 498 2 Agonist at a2 and a3 subtypes. [5] 6-fluoro-9-methyl-2-phenyl-4-(pyrrolidin-1-yl-carbonyl)-2,9 -10.593 -dihydro-1H-pyrido[3,4-b]indol-1-one 1i Flunitrazepam 3 Agonist, Non subtype selective [29] (E)-5-(2-fluorophenyl)-1-methyl-7-nitro-1H- -10.0461 benzo[e][1,4]diazepin-2(3H)-one 1k FG 7142 4 Non subtype selective [30] N-methyl-9H-pyrido[3,4-b]indole-3-carboxamide -9.59771 1g ELB 139 5 Partial agonist with highest [31] 1-(4-chlorophenyl)-4-(piperidin-1-yl)- -9.402408

potency at a3 subtype. 1H-imidazol-2(5H)-one 1e TPA 023 6 Partial agonist at a2 and a3 subtypes, [32] 6-((2-ethyl-2H-1,2,4-triazol-3-yl)methoxy)-7-tert-butyl- -9.2838 atagonist at a1 and a5 subtypes. 3-(2-fluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine 1f TP003 7 Selective agonist efficacy at a3 subtype [33] 4,2'-Difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl) -9.1692 imidazo[1,2-a]pyridin-3-yl]biphenyl-2-carbonitrile

1h Zolpidem 8 Partial affinity for a1 [34;35] N,N-dimethyl-2-(6-methyl-2-p-tolylH-imidazo[1,2-a]pyridin-3-yl)acetamide -8.36144 1b L-838 417 9 Partial agonist at a2, a3 and a5 [36] 6-((2-methyl-2H-1,2,4-triazol-3-yl)methoxy)-7-tert-butyl-3 -6.99906 antagonist at a1 subtype. -(2,5-difluorophenyl)-[1,2,4]triazolo[4,3-b]pyridazine 1j Flumazenil 10 Antagonist [37] Ethyl-1,2-fluoro- 8-methyl- 9-oxo- 2,4,8- triazatricyclo[8.4.0.02,6 ] -6.39436 tetradeca-1(10),3,5,11,13- pentaene-5-arboxylate

1c Ocinaplon 11 Partial agonist at a2, a3 and a5 subtypes, [38] (pyridin-2-yl)(7-(pyridin-4-yl)pyrazolo -5.44774 nearly full agonist at a1. [1,5-a]pyrimidin-3-yl)methanone 1l DMCM 12 Inverse agonist at benzodiazepine [39] methyl 4-ethyl-6,7-dimethoxy-9H- No suitable

site. a1 selective pyrido[3,4-b]indole-3-carboxylate binding pose found

O O O N N O N N N N N O N Cl N N O F N NH O 2 N N N N N N F F N H C l O N N 4 5 1 6 F 2 3 F N N N O O O F N N N N N N O O O N O N F N N N O O H HO N N F N N N N N N 12 F O 11 N 10 7 8 9

Table 2: Amino acid residues lining the binding site of ligands docked in predicted are known agonists at the GABAA receptor BZD binding BZD binding pocket of GABA a /g subunit homology model. site. Partial agonists such as ELB 139, TPA 023, TP 003 A 1 2 and Zolpidem showed binding scores less than that of the 1a 88ALA, 89ALA, 55GLN, 120GLN, 53PHE, 122PHE, 102GLN, 79ILE, 118ILE, 141ILE, 87LEU, 96PRO, 116PRO, 114TYR 1b 89ALA, 55GLN, 120GLN, 97GLU, 118ILE, 141ILE, 53PHE, 122PHE, 117SER, 100THR, 114TYR 88ALA, 102GLN, 79ILE, agonists but considerably more than the antagonists such 87LEU, 99LEU, 96PRO, 116PRO, 84VAL, as flumazenil (-6.39436 kcal/Mole). 1c 88ALA, 89ALA, 55GLN, 118ILE, 14ILE, 96PRO, 116PRO, 114TYR, 98VAL102GLN, 120 GLN, 139ILE, 34LEU, 87LEU, 140LYS, 53PHE, 122PHE 1d 55GLN, 118ILE, 87LEU, 85PRO, 116PRO, 57THR, 114TYR, 102GLN, 79ILE, 82LEU, 80SER, 84VAL In case of diazepam Figure 2, the negative binding ener- 1e 56GLN, 165TYR, 104ALA, 105ARG, 103LEU, 113LEU, 115MET, 116PRO, 54TRP, 114TYR, 84VAL, 98VAL gies were in agreement with its BZD binding site selectiv- 1f 55GLN, 85PRO, 57THR, 59TRP, 114TYR, 30VAL, 84VAL, 88ALA, 89ALA, 120GLN, 141ILE, 87LEU, 96PRO, ity as reported in literature. During binding of the ligands 1g 89ALA, 55GLN, 120GLN, 118ILE, 141ILE, 53PHE, 122PHE, 88ALA, 102GLN, 79ILE, 87LEU, 96PRO, 116PRO, in the predicted BZD binding pocket of GABA receptor 114TYR, 84VAL A 1h 86ASP, 85PRO, 116PRO, 57THR, 59TRP, 114TYR, 88ALA, 89ALA, 55GLN, 120GLN, 141ILE, 87LEU, 96PRO, a1/ g2 subunit homology model the conformational place- 1i 88ALA, 89ALA, 55GLN, 120GLN, 118ILE, 141ILE, 53PHE, 122PHE, 98VAL102GLN, 87LEU, 96PRO, 116PRO ment of amino acid residues in the active site was ob- 1j 88ALA, 89ALA, 55GLN, 120GLN, 118ILE, 141ILE, 87LEU, 53PHE, 96PRO102GLN, 79ILE, 116PRO, 114TYR served (Table 2). 1k 798ILE, 116PRO, 80SER, 57THR, 84VAL, 55GLN, 120GLN, 118ILE, 96PRO, 98VAL 1l 88ALA, 89ALA, 55GLN, 120GLN, 139ILE, 141ILE, 53PHE, 122PHE, 86ASP, 102GLN, 79ILE, 87LEU, 85PRO, 96PRO, 114TYR, 84VAL Diazepam was found to bind in the active site of GABAA receptor through hydrophobic interactions. The benzo- diazepine ring system was found to be surrounded by 102GLN, 79ILE, 82LEU, 80SER and 84VAL residues, 5- phenyl ring was found surrounded by 88ALA, 89ALA, 55GLN, 118ILE, 14ILE, 96PRO, 116PRO, 114TYR and 98VAL residues. N1 nitrogen of the benzodiazepine ring was surrounded by 88ALA and102GLN residues, while N4 Nitrogen was lined by 55GLN and 114TYR residues.

The binding site was predicted based on a Gaussian Fuzzy oil drop model. The best docking poses of ligands (1a-1l) were found to be in good confirmation with their re- ported affinities towards receptor binding site. 55GLN and 114 TYR residues were common in all the best ligand docking poses in the binding packet.

Figure 2: Docking of diazepam in predicted Figure 3: Structure overlay of Diazepam ACKNOWLEDAGEMENTS (red) and Zolpidem (blue) and SL 651 498 The authors express their gratitude towards DR. S. J. binding site homology model of GABAA a 1/ g 2 receptor subunits. (green) in their best docking poses. Devdhe Patil, Hon.Managing Director, Smt. Taisaheb Journal of Pharmacy Research Vol.4.Issue 9. September 2011 3162-3165 Gajanan Vaishnav et al. / Journal of Pharmacy Research 2011,4(9),3162-3165 Kadam Education and Research Center’s Yash Institute of Pharmacy, Aurangabad 21. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web- and Dr. S.P. Zambre, Professor and Head, Department of Zoology, Dr. Babasaheb based environment for protein structure homology modelling. Bioinformatics 2006; Ambedkar Marathwada University, Aurangabad for providing support and fa- 22(2):195-201. cilities required for this study. 22. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- REFERENCES specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 1. Chebib M, Vandenberg RJ, Johnston GA. Analogues of gamma-aminobutyric acid 22(22):4673-4680. (GABA) and trans-4-aminocrotonic acid (TACA) substituted in the 2 position as 23. Brylinski M, Konieczny L, Roterman I. Ligation site in proteins recognized in silico. GABAC receptor antagonists. Br J Pharmacol 1997; 122(8):1551-1560. Bioinformation 2006; 1(4):127-129. 2. Hirouchi M, Kuriyama K. [Molecular pharmacology of GABAA and GABAB 24. Banach M, Prymula K, Konieczny L, Roterman I. “Fuzzy oil drop” model verified receptors]. Nippon Yakurigaku Zasshi 1994; 104(3):199-206. positively. Bioinformation 2011; 5(9):375-377. 3. Johnston GA, Chebib M, Hanrahan JR, Mewett KN. GABA(C) receptors as drug 25. Prymula K, Salapa K, Roterman I. “Fuzzy oil drop” model applied to individual targets. Curr Drug Targets CNS Neurol Disord 2003; 2(4):260-268. small proteins built of 70 amino acids. J Mol Model 2010; 16(7):1269-1282. 4. Harangozo J, Magyar I, Faludy G. Use of benzodiazepines in psychiatry. Ther Hung 26. Brylinski M, Prymula K, Jurkowski W, Kochanczyk M, Stawowczyk E, Konieczny 1991; 39(3):103-111. L et al. Prediction of functional sites based on the fuzzy oil drop model. PLoS Comput 5. Griebel G, Perrault G, Simiand J, Cohen C, Granger P, Depoortere H et al. SL651498, Biol 2007; 3(5):e94. a GABAA receptor agonist with subtype-selective efficacy, as a potential treatment 27. Huen MS, Leung JW, Ng W, Lui WS, Chan MN, Wong JT et al. 5,7-Dihydroxy-6- for generalized anxiety disorder and muscle spasms. CNS Drug Rev 2003; 9(1):3-20. methoxyflavone, a benzodiazepine site ligand isolated from Scutellaria baicalensis 6. Anzini M, Braile C, Valenti S, Cappelli A, Vomero S, Marinelli L et al. Ethyl 8-fluoro- Georgi, with selective antagonistic properties. Biochem Pharmacol 2003; 66(1):125- 6-(3-nitrophenyl)-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxyl ate as novel, 132. highly potent, and safe antianxiety agent. J Med Chem 2008; 51(15):4730-4743. 28. Huen MS, Hui KM, Leung JW, Sigel E, Baur R, Wong JT et al. Naturally occurring 2'- 7. Berezhnoy D, Gibbs TT, Farb DH. Docking of 1,4-benzodiazepines in the alpha1/ hydroxyl-substituted flavonoids as high-affinity benzodiazepine site ligands. gamma2 GABA(A) receptor modulator site. Mol Pharmacol 2009; 76(2):440-450. Biochem Pharmacol 2003; 66(12):2397-2407. 8. Borea PA, Gilli G, Bertolasi V, Ferretti V. Stereochemical features controlling binding 29. Strakhova MI, Harvey SC, Cook CM, Cook JM, Skolnick P. A single amino acid and intrinsic activity properties of benzodiazepine-receptor ligands. Mol Pharmacol residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at 1987; 31(4):334-344. alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors. Mol Pharmacol 9. Ci S, Ren T, Su Z. Investigating the putative binding-mode of GABA and diazepam 2000; 58(6):1434-1440. within GABA A receptor using molecular modeling. Protein J 2008; 27(2):71-78. 30. Mirza NR, Munro G. The role of GABA(A) receptor subtypes as analgesic targets. 10. Ci SQ, Ren TR, Ma CX, Su ZG. Modeling of alphak/gamma2 (k=1, 2, 3 and 5) interface Drug News Perspect 2010; 23(6):351-360. of GABA A receptor and docking studies with zolpidem: implications for selectivity. 31. Rabe H, Kronbach C, Rundfeldt C, Luddens H. The novel anxiolytic ELB139 displays J Mol Graph Model 2007; 26(2):537-545. selectivity to recombinant GABA(A) receptors different from diazepam. 11. Cid MP, Salvatierra NA, Arce A. Phosphatidylinositol 4,5-bisphosphate induced Neuropharmacology 2007; 52(3):796-801. flunitrazepam sensitive-GABAA receptor increase in synaptosomes from chick 32. Atack JR, Wafford KA, Tye SJ, Cook SM, Sohal B, Pike A et al. TPA023 [7-(1,1- forebrain. Neurochem Res 2007; 32(6):1011-1015. dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluor ophenyl)-1,2,4- 12. Hanson SM, Morlock EV, Satyshur KA, Czajkowski C. Structural requirements for triazolo[4,3-b]pyridazine], an agonist selective for alpha2- and alpha3-containing eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) GABAA receptors, is a nonsedating anxiolytic in rodents and primates. J Pharmacol receptor are different. J Med Chem 2008; 51(22):7243-7252. Exp Ther 2006; 316(1):410-422. 13. Kucken AM, Teissere JA, Seffinga-Clark J, Wagner DA, Czajkowski C. Structural 33. Dias R, Sheppard WF, Fradley RL, Garrett EM, Stanley JL, Tye SJ et al. Evidence for requirements for imidazobenzodiazepine binding to GABA(A) receptors. Mol a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic Pharmacol 2003; 63(2):289-296. effects of benzodiazepines. J Neurosci 2005; 25(46):10682-10688. 14. Sancar F, Ericksen SS, Kucken AM, Teissere JA, Czajkowski C. Structural determinants 34. Crestani F, Martin JR, Mohler H, Rudolph U. Mechanism of action of the hypnotic for high-affinity zolpidem binding to GABA-A receptors. Mol Pharmacol 2007; zolpidem in vivo. Br J Pharmacol 2000; 131(7):1251-1254. 71(1):38-46. 35. Langer SZ, Arbilla S. Imidazopyridines as a tool for the characterization of 15. Villar HO, Uyeno ET, Toll L, Polgar W, Davies MF, Loew GH. Molecular determinants benzodiazepine receptors: a proposal for a pharmacological classification as omega of benzodiazepine receptor affinities and anticonvulsant activities. Mol Pharmacol receptor subtypes. Pharmacol Biochem Behav 1988; 29(4):763-766. 1989; 36(4):589-600. 36. McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR et al. Sedative 16. Sigel E, Schaerer MT, Buhr A, Baur R. The benzodiazepine binding pocket of but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) recombinant alpha1beta2gamma2 gamma-aminobutyric acidA receptors: relative receptor alpha1 subtype. Nat Neurosci 2000; 3(6):587-592. orientation of ligands and amino acid side chains. Mol Pharmacol 1998; 54(6):1097- 37. Eng W, Atack JR, Bergstrom M, Sanabria S, Appel L, Dawson GR et al. Occupancy 1105. of human brain GABA(A) receptors by the novel alpha5 subtype-selective 17. Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der OJ, Smit AB et al. Crystal benzodiazepine site inverse agonist alpha5IA as measured using [(1)(1)C]flumazenil structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic PET imaging. Neuropharmacology 2010; 59(7-8):635-639. receptors. Nature 2001; 411(6835):269-276. 38. Lippa A, Czobor P, Stark J, Beer B, Kostakis E, Gravielle M et al. Selective anxiolysis 18. ArgusLab 4.0.1 [ Planaria Software LLC, Seattle, WA; 2004. produced by ocinaplon, a GABA(A) receptor modulator. Proc Natl Acad Sci U S A 19. Peitsch MC, Wells TN, Stampf DR, Sussman JL. The Swiss-3DImage collection and 2005; 102(20):7380-7385. PDB-Browser on the World-Wide Web. Trends Biochem Sci 1995; 20(2):82-84. 39. Sancar F, Czajkowski C. Allosteric modulators induce distinct movements at the 20. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository GABA-binding site interface of the GABA-A receptor. Neuropharmacology 2011; and associated resources. Nucleic Acids Res 2009; 37(Database issue):D387-D392. 60(2-3):520-528. Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 9. September 2011 3162-3165