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

Positive allosteric modulation of the GABAA receptor (GABAAR) 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 1/ 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 structureactivity and stereospecificity characteristics of BZDs and the impact of the 1H101R 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 sitedirected 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 GABAAR modulators with enhanced therapeutic potential. GABAA receptors (GABAARs) are pentameric transmembrane proteins that belong to the cysteine-loop superfamily of ligand-gated ion channels and function as GABA-gated Cl selective channels, which mediate most fast inhibitory neurotransmission in the central nervous system (Berezhnoy et al., 2007). There are 20 related GABAAR subunits in mammals, designated 1–6, 1–4, 1–3, , , , , and 1–3, that can assemble in multiple combinations to produce different GABAAR subtypes (Barnard et al., 1998; Bonnert et al., 1999). The regional and cellular distribution of different GABAAR subunits is distinct but overlapping, and individual receptor subtypes exhibit distinct subcellular localizations (Berezhnoy et al., 2007). Most GABAARs in the adult mammalian central nervous system are composed of , , and subunits, with 1 2/3 2 being the most abundant subtype (Sieghart and Sperk, 2002). GABAARs are activated by binding of agonist to recognition sites located at ( )/ ( ) subunit interfaces (Berezhnoy et al., 2007). Agonist-induced receptor activation can be modulated through allosteric binding sites located at the 1( )/ 2( ) subunit interface (the BZD recognition site) (Choh et al., 1977; Chan and Farb, 1985). Residues implicated in the formation of the GABA and BZD binding sites are located at equivalent positions within six loops in the extracellular N-termini of the , , and subunits (Supplemental Fig. 1) (Berezhnoy et al., 2007). Previous attempts have been made to superimpose the structures of allosteric modulators to construct a pharmacophore model for the BZD recognition site (Borea et al., 1987; Villar et al., 1989; Schove et al., 1994; Zhang et al., 1995; Huang et al., 1998, 1999; He et al., 2000; Marder et al., 2001; Verli et al., 2002). However, such models are difficult to relate to receptor structure. Sigel et al. (1998) determined affinities for a series of imidazoand 5-phenyl-1,4-benzodiazThis work was supported by the National Institutes of Health National Institute of Mental Health [Grant R01-MH049469]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.054650. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: GABAAR, GABAA receptor; BZD, benzodiazepine; FNZ, flunitrazepam; DZ, diazepam; RMSD, root-mean-square deviation; DZ-NCS, diazepam carrying a thiol-reactive –N C S group; AChBP, acetylcholine binding protein; Ro 15-4513, 4H-imidazo(1,5a)(1,4)benzodiazepine-3-carboxylic acid, 8-azido-5,6-dihydro-5-methyl-6-oxo-, ethyl ester; Ro 15-1788, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4Himidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester. 0026-895X/09/7602-440–450$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 2 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 54650/3500013 Mol Pharmacol 76:440–450, 2009 Printed in U.S.A. 440 http://molpharm.aspetjournals.org/content/suppl/2009/05/29/mol.109.054650.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 6, 2017 m oharm .aspeurnals.org D ow nladed from epines to wild-type and mutant receptors to delineate the orientation of these ligands in the recognition site. An extra hydroxyl group of tyrosine introduced by the 2F77Y mutation interferes with para-substitutions of the C5-phenyl ring, suggesting that the phenyl ring is adjacent to 2Phe77 in the binding pocket (Sigel et al., 1998). Kucken and colleagues (2003) used a series of three substituted imidazobenzodiazepines in combination with amino acid mutations of varying volume at 2Ala79 to infer the position of compounds similar to Ro 15-1788 and Ro 15-4513 (Kucken et al., 2003). Photoaffinity labeling using [H]flunitrazepam identified the major site that incorporates radioactivity as His101 of loop A (McKernan et al., 1995; Davies et al., 1996; Duncalfe et al., 1996) and a second less abundant site as Pro96 (Smith and Olsen, 2000). Likewise, photoaffinity labeling using the imidazobenzodiazepine [H]Ro 15-4513 identified residue Tyr209 of loop C of the 1 subunit as proximal to the benzodiazepine binding site (Sawyer et al., 2002). However, the docking position with respect to specific contact residues cannot be deduced because of uncertainty of photoaffinity labeling in an environment containing multiple aromatic residues (Kotzyba-Hibert et al., 1995). Using a C7-modified diazepam (DZ) carrying a thiol-reactive –N C S group (DZ-NCS), 1H101C was confirmed to be in or near the binding pocket. At the functional level, the reacted receptor becomes irreversibly locked in a positively modulated state (Berezhnoy et al., 2004, 2005; Tan