Docking of 1,4-Benzodiazepines in the 1/ 2 GABAA Receptor Modulator Site

Laboratory of Molecular Neurobiology, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA.
Molecular pharmacology (Impact Factor: 4.13). 06/2009; 76(2):440-50. DOI: 10.1124/mol.109.054650
Source: PubMed


Positive allosteric modulation of the GABA(A) receptor (GABA(A)R) via the benzodiazepine recognition site is the mechanism whereby diverse chemical classes of therapeutic agents act to reduce anxiety, induce and maintain sleep, reduce seizures, and induce conscious sedation. The binding of such therapeutic agents to this allosteric modulatory site increases the affinity of GABA for the agonist recognition site. A major unanswered question, however, relates to how positive allosteric modulators dock in the 1,4-benzodiazepine (BZD) recognition site. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the BZD binding pocket at the alpha(1)/gamma(2) subunit interface. A tethered BZD was introduced into the binding pocket, and molecular simulations were carried out to yield a set of candidate orientations of the BZD ligand in the binding pocket. Candidate orientations were refined based on known structure-activity and stereospecificity characteristics of BZDs and the impact of the alpha(1)H101R mutation. Results favor a model in which the BZD molecule is oriented such that the C5-phenyl substituent extends approximately parallel to the plane of the membrane rather than parallel to the ion channel. Application of this computational modeling strategy, which integrates site-directed affinity labeling with structure-activity knowledge to create a molecular model of the docking of active ligands in the binding pocket, may provide a basis for the design of more selective GABA(A)R modulators with enhanced therapeutic potential.

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Available from: David H Farb, Apr 17, 2014
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    • "So far, drug discovery efforts have relied mainly on indirect structural insight from focused [8]–[12] or unified pharmacophore models recapitulating the structure-activity relationships (SAR) of compounds synthesized during more than fifty years of active medicinal chemistry research in the field [13], [14]. Homology models, on the other hand, have had little practical impact on the design process despite a number of models reported in the literature [15]–[25]. The models were mainly built using the homologous acetylcholine binding proteins (AChBPs) as templates. "
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    ABSTRACT: We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1.
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    ABSTRACT: GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of alpha(1)S205C and alpha(1)T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (-NCS). We also provide new data that identify alpha(1)H101C and alpha(1)N102C as exclusive sites of the reaction of a diazepam derivative where the -Cl atom is replaced by a -NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of alpha(1)beta(2)gamma(2) receptors.
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    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.
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