Shengming Huang

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.

Antiseizure activity of novel γ-Aminobutyric acid (A) receptor subtype-selective benzodiazepine analogues in mice and rat models

The antiseizure activity of benzodiazepines (BDZs) 1-5 in mice and rats as animal models is described. These BDZs have selective efficacy for alpha2beta3gamma2 and alpha3beta3gamma2 GABA(A)-receptors. Significant anticonvulsant activity with little or no motor impairment and therapeutic indexes (TI) of 2.8-44 (mice, ip) were observed for compounds 2-4 in the subcutaneous metrazole seizure (scMET) test. In rats, orally (po) the TI was >5 to 105. These compounds represent novel leads in the search for anticonvulsants devoid of sedative, ataxic, and amnestic side effects.

Subtype selectivity of α+β− site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2‐(4‐methoxyphenyl)‐2H‐pyrazolo[4,3‐c]quinolin‐3(5H)‐one) elicits a strong and subtype‐dependent enhancement of GABA‐induced currents via a novel drug‐binding site at extracellular αx+βy− (x = 1–6, y = 1–3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA‐induced currents of various GABAA receptor subtypes.

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β- interface.

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2‐(4‐methoxyphenyl)‐2H‐pyrazolo[4,3‐c]quinolin‐3(5H)‐one) acts as a null modulator (antagonist) at the high affinity benzodiazepine binding site, but in addition elicits a strong enhancement of GABA‐induced currents via a novel drug binding site at the extracellular α+β− interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+β3− interface of GABAA receptors.

Different Benzodiazepines Bind with Distinct Binding Modes to GABAA Receptors

Benzodiazepines are clinically relevant drugs that bind to GABAA neurotransmitter receptors at the α+/γ2– interfaces and thereby enhance GABA-induced chloride ion flux leading to neuronal hyperpolarization. However, the structural basis of benzodiazepine interactions with their high-affinity site at GABAA receptors is controversially debated in the literature, and in silico studies led to discrepant binding mode hypotheses. In this study, computational docking of diazepam into α+/γ2– homology models suggested that a chiral methyl group, which is known to promote preferred binding to α5-containing GABAA receptors (position 3 of the seven-membered diazepine ring), could possibly provide experimental evidence that supports or contradicts the proposed binding modes. Thus, we investigated three pairs of R and S isomers of structurally different chemotypes, namely, diazepam, imidazobenzodiazepine, and triazolam derivatives. We used radioligand displacement studies as well as two-electrode voltage clamp electrophysiology in α1β3γ2-, α2β3γ2-, α3β3γ2-, and α5β3γ2-containing GABAA receptors to determine the ligand binding and functional activity of the three chemotypes. Interestingly, both imidazobenzodiazepine isomers displayed comparable binding affinities, while for the other two chemotypes, a discrepancy in binding affinities of the different isomers was observed. Specifically, the R isomers displayed a loss of binding, whereas the S isomers remained active. These findings are in accordance with the results of our in silico studies suggesting the usage of a different binding mode of imidazobenzodiazepines compared to those of the other two tested chemotypes. Hence, we conclude that different chemically related benzodiazepine ligands interact via distinct binding modes rather than by using a common binding mode.