title: Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators.
abstract: 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 gamma-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 alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, Bern CH-3012, Switzerland.
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Tan
authors.forename: Anne
authors.initials: A
authors.lastname: Gonthier
authors.forename: Roland
authors.initials: R
authors.lastname: Baur
authors.forename: Margot
authors.initials: M
authors.lastname: Ernst
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
authors.forename: Erwin
authors.initials: E
authors.lastname: Sigel
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution; D001569:Benzodiazepines / Q000737:chemistry*; D002491:Central Nervous System Agents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D020125:Mutation, Missense; D020033:Protein Isoforms / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D002491:Central Nervous System Agents; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D008024:Ligands; D020033:Protein Isoforms; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D003545:Cysteine