Terry Clayton

Are GABAA Receptors Containing α5 Subunits Contributing to the Sedative Properties of Benzodiazepine Site Agonists

Classical benzodiazepines (BZs) exert anxiolytic, sedative, hypnotic, muscle relaxant, anticonvulsive, and amnesic effects through potentiation of neurotransmission at GABAA receptors containing α1, α2, α3 or α5 subunits. Genetic studies suggest that modulation at the α1 subunit contributes to much of the adverse effects of BZs, most notably sedation, ataxia, and amnesia. Hence, BZ site ligands functionally inactive at GABAA receptors containing the α1 subunit are considered to be promising leads for novel, anxioselective anxiolytics devoid of sedative properties. In pursuing this approach, we used two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABAA receptor subtypes to investigate functional selectivity of three newly synthesized BZ site ligands and also compared their in vivo behavioral profiles. The compounds were functionally selective for α2-, α3-, and α5-containing subtypes of GABAA receptors (SH-053-S-CH3 and SH-053-S-CH3-2′F) or essentially selective for α5 subtypes (SH-053-R-CH3). Possible influences on behavioral measures were tested in the elevated plus maze, spontaneous locomotor activity, and rotarod test, which are considered primarily predictive of the anxiolytic, sedative, and ataxic influence of BZs, respectively. The results confirmed the substantially diminished ataxic potential of BZ site agonists devoid of α1 subunit-mediated effects, with preserved anti-anxiety effects at 30 mg/kg of SH-053-S-CH3 and SH-053-S-CH3-2′F. However, all three ligands, dosed at 30 mg/kg, decreased spontaneous locomotor activity, suggesting that sedation may be partly dependent on activity mediated by α5-containing GABAA receptors. Hence, it could be of importance to avoid substantial agonist activity at α5 receptors by candidate anxioselective anxiolytics, if clinical sedation is to be avoided.

The differential role of α1- and α5-containing GABAA receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats

The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that alpha1- and alpha5-containing GABA(A) receptors mediate and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands: the alpha1-subunit affinity-selective antagonist beta-CCt, and the alpha5-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by beta-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABA(A) receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg beta-CCt and 10 mg/kg XLi093. Combining a BZ with beta-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects.

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors.

Allosteric Modulation of GABAA Receptor Subtypes: Effects on Visual Recognition and Visuospatial Working Memory in Rhesus Monkeys

Non-selective positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) are known to impair anterograde memory. The role of the various GABAAR subtypes in the memory-impairing effects of non-selective GABAAR PAMs has not been fully elucidated. The current study assessed, in rhesus monkeys, effects of modulation of α1, α2/3, and α5GABAARs on visual recognition and spatial working memory using delayed matching-to-sample (DMTS) and self-ordered spatial search (SOSS) procedures, respectively. The DMTS procedure (n=8) involved selecting a previously presented ‘sample’ image from a set of multiple images presented after a delay. The SOSS procedure (n=6) involved touching a number of boxes without repeats. The non-selective GABAAR PAM triazolam and the α1GABAA preferential PAMS zolpidem and zaleplon reduced accuracy in both procedures, whereas the α5GABAA preferential PAMs SH-053-2′F-R-CH3 and SH-053-2′F-S-CH3, and the α2/3GABAA preferential PAM TPA023B were without effects on accuracy or trial completion. The low-efficacy α5GABAAR negative allosteric modulator (NAM) PWZ-029 slightly increased only DMTS accuracy, whereas the high-efficacy α5GABAAR NAMs RY-23 and RY-24 did not affect accuracy under either procedure. Finally, the slopes of the accuracy dose-effect curves for triazolam, zolpidem, and zaleplon increased with box number in the SOSS procedure, but were equivalent across DMTS delays. The present results suggest that (1) α1GABAARs, compared with α2/3 and α5GABAARs, are primarily involved in the impairment, by non-selective GABAAR PAMs, of visual recognition and visuospatial working memory in nonhuman primates; and (2) relative cognitive impairment produced by positive modulation of GABAARs increases with number of locations to be remembered, but not with the delay for remembering.

Synthesis, pharmacological studies and molecular modeling of some tetracyclic 1,3-diazepinium chlorides.

Seven new 1,3-diazepinium chlorides exhibiting some structural similarities to the 1,4-benzodiazepines were synthesized. In a Hippocratic screen using mice, three of these salts, 3-methoxy-6-oxo-7,13-dihydro-6H-benzofuro[2,3-e]pyrido[1,2-a][1,3]diazepin-12-ium chloride (8a), 3-methoxy-9-methyl-6-oxo-7,13-dihydro-6H-benzofuro[2,3-e]pyrido[1,2-a][1,3]diazepin-12-ium chloride (8c) and 3-methoxy-11-methyl-6-oxo-7,13-dihydro-6H-benzofuro[2,3-e]pyrido[1,2-a][1,3]diazepin-12-ium chloride (8e) were examined for their effect on the central nervous system, and their activities compared to that of diazepam. On their own, salts 8a, 8c and 8e solicited no sedative effects on the behaviour of the animals. However, they elicited significant effects in combination with diazepam on diazepam-induced activities such as decreased motor activity, ataxia and loss of righting reflex. Compounds 8a and 8c were fitted into the pharmacophore/receptor model developed by Cook et al. with interaction at the L(1), H(1) and A(2) sites indicating that they are potential inverse agonists of the Bz receptor. The compounds displayed some affinity for the alpha1 isoform of the GABA(A)/BzR (L(Di) interaction) but are non-selective for alpha5 (no L(2) interaction). Results of binding affinity studies showed that compound 8a is mildly selective for the alpha1 receptor although not very potent (K(i)=746.5nM). The significant potentiation of diazepam-induced ataxia and decreased motor activity by compounds 8a and 8c in the Hippocratic screen may be associated with alpha1 selectivity.

A study of the structure-activity relationship of GABAA-benzodiazepine receptor bivalent ligands by conformational analysis with low temperature NMR and X-ray analysis

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in the solid state. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for alpha5 subtype selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both in solution and the solid state. Dimers which are folded do not bind to Bz receptors.

Erratum: Allosteric modulation of GAB aa receptor subtypes: Effects on visual recognition and visuospatial working memory in rhesus monkeys (Neuropsychopharmacology (2013) 38 (2553) DOI: 10.1038/npp.2013.162)

Correction to: Neuropsychopharmacology advance online publication, 26 June 2013; doi:10.1038/npp.2013.137 In this article, there is an error in the title. The correct title is ‘Allosteric Modulation of GABAA Receptor Subtypes: Effects on Visual Recognition and Visuospatial Working Memory in Rhesus Monkeys.’ Neuropsychopharmacology (2013) 38, 2553 & 2013 American College of Neuropsychopharmacology. All rights reserved 0893-133X/13