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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.

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