A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C

Abstract

The family of GABA A receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures and many others. The most frequent GABA A receptor subtype is composed of two α, two β and one γ2-subunit, while the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABA A receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABA A receptors. Beyond the CNS, α5-containing GABA A receptors in airway smooth muscles are considered as emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2′F-R-CH3 (SH53d-ester). While SH53d-ester is only moderately selective for α5-subunit containing GABA A receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity, and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with HEK 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5β3γ2 over other αxβ3γ2 GABA A receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABA A receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate.

Electroacupuncture-induced activation of GABAergic system alleviates airway inflammation in asthma model by suppressing TLR4/MyD88/NF-κB signaling pathway

“…In our study, the administration of GABAAR antagonist diminished the therapeutic effects of EA in asthma, which further confirmed that the EA-induced alleviation of airway inflammation in asthma may be associated with activation of the GABAergic system. GABAARs are expressed in ASM, airway epithelium, and inflammatory cells, and GABA is associated with goblet cells; therefore, the GABAergic system is considered as an emerging target for asthma [37,38] . Activation of these receptors directly leads to ASM relaxation, and modulation of these receptors has been shown to affect immune cell function [5] .…”

“…GABAARs are expressed in ASM, airway epithelium, and inflammatory cells, and GABA is associated with goblet cells; therefore, the GABAergic system is considered as an emerging target for asthma. [ 37 , 38 ] Activation of these receptors directly leads to ASM relaxation, and modulation of these receptors has been shown to affect immune cell function. [ 5 ] It is reasonable to believe that the GABAergic system plays a critical role in mediating the therapeutic effect of EA in asthma; this is in accordance with a previous study in which EA up-regulated the expression of GABA, and in turn suppressed inflammation.…”

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors

“…9h ), explaining its lack of α5 selectivity 44 , 47 . However, expanding the contact zone of a diazepam analog by adding an extra acidified triazole group within 3 Å of T208 does lead to α5 selectivity, presumably by recruiting a T208 methyl-specific interaction 48 . From the radioligand binding data α5 I215 also impacts selectivity, albeit to a lesser extent, with an α1 Val substitution reducing affinity 9-, 3- and 4-fold for basmisanil, RO7015738 and RO7172670, respectively (Fig.…”

A Comparison of Epileptogenic Effect of Status Epilepticus Treated With Diazepam, Midazolam, and Pentobarbital in the Mouse Pilocarpine Model of Epilepsy

“…However, the exact mechanisms of modulation of GABA A receptors by benzodiazepines are complicated, as there are many types of GABA A receptors with different binding affinity for GABA and benzodiazepine. This is in that each GABA A receptor comprises 5 subunits and there are up to 19 different subunits of the GABA A receptor ( 23 , 24 ). Therefore, even for the same type of GABA A receptor, benzodiazepines (e.g., diazepam) may produce biphasic actions via separable subunit-dependent mechanisms ( 25 ).…”