Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABA_A Receptors

Abstract

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/β- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/β3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the β3- subunit, resulting in a quadruple mutant β3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β- sites.

GABAA Receptor Ligands Often Interact with Binding Sites in the Transmembrane Domain and in the Extracellular Domain—Can the Promiscuity Code Be Cracked?

“…It seems surprising at first sight that the high pharmacophore similarity is not reflected in similar binding properties. However, we have previously observed that interactions with site 1 do not follow pharmacophore features [16], and recently published structures confirm that even ligands of the benzodiazepine chemotype do not display binding modes with The high pharmacophoric overlap of the indole derivatives in comparison with the PQs (Figure 2c,d) suggests similar binding behavior. We therefore considered the three possible binding sites as object of our studies: site 1 (ECD-α+/γ−) site 2 (ECD-α+/β−), and site 3 (TMD-β+/α−).…”

“…It seems surprising at first sight that the high pharmacophore similarity is not reflected in similar binding properties. However, we have previously observed that interactions with site 1 do not follow pharmacophore features [16], and recently published structures confirm that even ligands of the benzodiazepine chemotype do not display binding modes with aligned pharmacophore features. Specifically, flumazenil (PDB structure 6D6T [17]) and alprazolam (PDB structure 6HUO [4]) bind at the high affinity site with completely different binding modes and no overlap of pharmacophore features (Figure 3c,d).…”

“…Among the best known site 1 ligands are the benzodiazepines, for which many pharmacophore models have been developed in the past-all of which rested on the assumption that most or all benzodiazepines share a common binding mode in which common pharmacophore features overlap. Based on a mutational analysis combined with computational docking, we have recently challenged this notion [16], and more recent structures in fact confirmed that benzodiazepines do not generally share a common binding mode [4,17]. Thus, there is no single consensus pharmacophore applicable to the design of site 1 ligands.…”

“…Since all the experimental findings strongly suggest that MTI163 exerts the modulatory effect via the "etomidate site" at the TMD β+/α− site involving β2N265S, we performed computational docking to investigate the possible binding of MTI163 to the equivalent site of 6HUP which harbors diazepam. A redocking of diazepam was performed first to test whether the protocol we use recovers the experimental structure [16]. Poses with very high overlap to the one observed in the cryo-EM structure (6HUP [4]) were indeed found within the top 10 ranked docking results, evaluated with two scoring functions as well in the absence or presence of flexible sidechains (see Methods).…”

“…The recently published cryo-EM structure 6HUP was used to take advantage of the ligand bound conformation in site 3. Standard protein preparation was used, where all hydrogen atoms were added to the protein [16]. The binding site within the respective pocket was defined according to the coordinates of the diazepam -N1 of the amide group with a sphere size of 12A.…”