Characterization of the enantiomeric 3-substituted-1,4-benzodiazepin-2-ones binding to the GABA—BDZ—chloride ionophore receptor complex

Abstract

Gamma aminobutyric acid (GABA) is an important and abundant inhibitory neurotransmitter in the mammalian central nervous system (CNS). Its interaction with the GABAA receptor subtypes opens anion channels in the membrane through which chloride ions pass down their electrochemical gradient, thus stabilizing the resting level of membrane potential. GABA* receptors, associated with the anion channel, are part of a protein complex containing distinct but interacting recognition sites for convulsants [such as picrotoxin and t-butylbicyclo-phosphorothionate (TBPS)] and depressants [such as benzodiazepines (BDZs) and barbiturates] [ 11. Therefore, the macromolecular make-up and interactions of the BDZ binding sites with the GABA receptor and chloride channel have to be accounted for in models of BDZ receptor function [2-41. Several papers report the structure activity relationships as well as the influence of the stereochemistry of BDZs on their in vitro binding to the CNS receptor [5-111. However, to the best of our knowledge, nothing is known about BDZ receptor function depending on the enantioselective binding of chiral BDZs to their receptor. In the present paper the interaction to the CNS receptor of a series of chiral3-substituted1,4-benzodiazepin-2-ones, resolved as pure enantiomers by preparative chiral HPLC, was studied. The study of the interaction of the BDZ receptor with the GABA receptor and chloride channel was utilized to profile the in vitro differences between enantiomeric BDZs as agonists, antagonists, and inverse agonists. Quantifiable differences in the coupling of the BDZ receptors to GABA receptors can be determined by measuring the GABA-shift in the affinities of BDZs to displace 3H-flunitrazepam [2, 11-131. Thus, the influence of the stereochemistry on the pharmacological profile of the BDZ receptor ligands has been investigated as GABA-ratio determination. Further, the study of possible interaction between BDZ receptor and the chloride channel has been performed by the use of 35S-TBPS (a selective label of chloride anion recognition site). Thus the effect exerted by chiral BDZs on the chloride channel regulation was evaluated by the modulation of 35S-TBPS binding.