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J. Biol. Chem., Vol. 282, Issue 36, 26316-26325, September 7, 2007

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Proximity-accelerated Chemical Coupling Reaction in the Benzodiazepine-binding Site of -Aminobutyric Acid Type A Receptors

SUPERPOSITION OF DIFFERENT ALLOSTERIC MODULATORS^*

Kelly R. Tan, Anne Gonthier, Roland Baur, Margot Ernst^¶, Maurice Goeldner, and Erwin Sigel¹

From the Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, Bern CH-3012, Switzerland, the Laboratoire de Chimie Bioorganique, Unité Mixte de Recherche 7175 LC1 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, 74 Route du Rhin, 67401 Illkirch Cedex, France, and the ^¶Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the -aminobutyric acid type A (GABA_A) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA_A receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA_A receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly ₁H101C and also three other mutants (₁G157C, ₁V202C, and ₁V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (–NCS) replaces the azide group (–N₃) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, ₁H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.

Received for publication, March 13, 2007 , and in revised form, June 28, 2007.

^* This work was supported by Swiss National Science Foundation Grant 3100A0-105272/1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 41-31-631-4114; Fax: 41-31-631-3737; E-mail: Erwin.sigel{at}mci.unibe.ch.

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