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

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 alpha-, two beta-, and one gamma 2-subunit, whereas the nature of the alpha-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 alpha 5-containing GABA(A) receptors. Beyond the central nervous system, alpha 5-containing GABA(A) receptors in airway smooth muscles are considered as an 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). Although SH53d-ester is only moderately selective for alpha 5-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 human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for alpha 5 beta 3 gamma 2 over other alpha x beta 3 gamma 2 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 alpha-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel alpha 5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for alpha 5-containing GABA(A) receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABA(A) receptor alpha-subunit is the dominant molecular determinant of drug selectivity.

Automated summary

The GABA(A) receptor family is an important drug target group for treating various disorders, and recent drug development research has focused on studying GABA(A) receptors containing the alpha 5 subunit.