Characterization of the Interaction of Zopiclone with γ-Aminobutyric Acid Type A Receptors

Abstract

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

Molecular Actions of (S)-Desmethylzopiclone (SEP-174559), an Anxiolytic Metabolite of Zopiclone

“…Racemic zopiclone [(R,S)-zopiclone] has been shown to enhance GABA A receptor currents via the benzodiazepine site (Im et al, 1993;Davies et al, 2000). The molecular actions of its principal metabolite, SEP-174559, have not been described.…”

“…Indeed, (R,S)-zopiclone modulation has been demonstrated for the ␣1␤2␥2 subtype but also for ␤2␥2 absent an ␣ subunit (Im et al, 1993), suggesting the ␣ subunit is not important for (R,S)-zopiclone modulation. Yet, paradoxically, (R,S)-zopiclone binding has been shown to involve the ␣-subunit His101 residue, which is a necessary component of the benzodiazepine binding site (Davies et al, 2000). Likewise, our studies also indicate at least some involvement of the ␣ subunit in (R,S)-zopiclone binding because K d estimates from kinetic analyses of ␣1-, ␣2-, and ␣3-bearing subtypes differed by as much as 7-fold between ␣1-bearing (highest affinity) and ␣3-bearing (lowest affinity) subtypes.…”

“…Racemic zopiclone, which consists of (R)-and (S)-enantiomers, acts at the benzodiazepine site to enhance GABA A receptor binding and function (Im et al, 1993;Davies et al, 2000). Although the selectivity of zopiclone is not fully characterized, it is known to act at ␥2-bearing GABA A receptors, including ␣1␤2␥2 (Im et al, 1993;Reynolds and Maitra, 1996;Davies et al, 2000).…”

“…Racemic zopiclone, which consists of (R)-and (S)-enantiomers, acts at the benzodiazepine site to enhance GABA A receptor binding and function (Im et al, 1993;Davies et al, 2000). Although the selectivity of zopiclone is not fully characterized, it is known to act at ␥2-bearing GABA A receptors, including ␣1␤2␥2 (Im et al, 1993;Reynolds and Maitra, 1996;Davies et al, 2000). Recent behavioral studies in rodents suggest that the sedative and anxiolytic activities of (R,S)-zopiclone are produced mainly by the (S)-enantiomer (Carlson et al, 2001), which is metabolized in vivo to (S)-desmethylzopiclone (SEP-174559).…”

Zopiclone Overdose Responsive to Flumazenil

“…Zopiclone's binding site appears to be distinct from that of the benzodiazepines but photoaffinity labeling and analysis of the allosteric modification of GABA suggest that the sites are adjacent and may overlap (1). A MEDLINE literature search (key words: zopiclone, cyclopyrrolones) revealed no cases that involved successful reversal of the drug effects with an antidote.…”