Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Mol Pharmacol
2021 Jan 01;991:39-48. doi: 10.1124/molpharm.120.000067.
Show Gene links
Show Anatomy links
A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.
Simeone X
,
Koniuszewski F
,
Müllegger M
,
Smetka A
,
Steudle F
,
Puthenkalam R
,
Ernst M
,
Scholze P
.
???displayArticle.abstract???
The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two β-, and one γ2-subunit, whereas 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 GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA 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 α5-subunit-containing GABAA 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 α5β3γ2 over other αxβ3γ2 GABAA 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 GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-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 α5-containing GABAA 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 GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.
Fig. 2. Affinity and efficacy data in the four diazepam-sensitive GABAA receptor subtypes. To directly compare the properties of SH53d-acid and SH53d-ester, both were measured at identical conditions side by side (A and B): Inhibition of binding of [3H]flunitrazepam to recombinant αxβ3γ2 GABAA receptors. Membranes from HEK 293 cells transfected with the GABAA receptor subunit combinations were incubated with 2 nM [3H]flunitrazepam in the presence of various concentrations of SH53d-ester (A) or SH53d-acid (B). Values are given as mean ± S.D. of three experiments performed in duplicates each. (C and D) Concentration-response curves of the compounds SH53d-ester (C) or SH53d-acid (D) in αxβ3γ2 GABAA receptors expressed in X. laevis oocytes using GABA EC3–5. Values are given as mean ± S.D., n = 3 or higher from at least two batches of oocytes.
Fig. 3. The benzodiazepine pocket of the cryo-EM structure (6D6T) and the human α5 subunit in 6A96. Color coding: Amino acids differing between α5 and α1 are marked in cyan. γ2-Subunit is light gray. α5- and α1-subunits are in pale yellow. Concecutive numbers are used in the images and the partial alignment to identify the depicted sidechains. (A) The benzodiazepine pocket of the α1 (pale yellow)/γ2 (gray) GABAA receptor with the pocket-forming amino acids in stick rendering. (B) The principal subunit of the benzodiazepine pocket of the α5-subunit. (C) Partial alignment of the α1- and α5-subunits, with the mutated amino acids marked by cyan boxes, pocket-forming amino acids from the γ2-subunit. For comparison with the rat amino acid numbering, see Supplemental Table 2.
Fig. 4. Comparison of alprazolam and flumazenil binding modes with representative results from the top-20 SH53d-acid poses. (A) 6HUO with alprazolam; (B) representative alprazolam-like (BMII) pose; (C) 6D6T with flumazenil; (D) representative flumazenil-like (BMI) pose. Color coding: pale yellow ribbons: α1/α5-subunits; gray ribbons: γ2-subunit; stick rendering: O is red, N is blue. The Ser/Thr position in which α5 sequence is uniquely featuring Thr is also shown in stick representation.
Fig. 5. 3H]flunitrazepam equilibrium binding assays. Membranes from transfected HEK 293 cells were incubated with 1–20 nM (A and B) and 5–150 nM (C) [3H]flunitrazepam in absence or presence of 5 µM diazepam (to determine nonspecific binding). Radioactivity bound to the membranes was determined after rapid filtration. Inserts show the Scatchard transformation of the results. Data represent a single experiment performed in duplicates each. Experiments were repeated three to four times with similar results.
Fig. 6. Inhibition constants (Ki) of SH53d-acid competition for [3H]Ro 15-4513 binding and TEV functional data in α5T239S-β3γ2– and α5P197T-β3γ2–injected oocytes. (A–C) Membranes from transfected HEK 293 cells were incubated with 5 nM [3H]Ro 15-4513 in the presence of various concentrations of SH53d-acid. The concentrations resulting in half-maximal inhibition of radioligand binding were converted into Ki values by using the Cheng-Prusoff relationship, and the respective KD values were given in Table 4. For detailed statistical analysis, see Supplemental Fig. 3. ( D and E ) Concentration-response curves and respective fitting parameters of SH53d-acid in mutated αxβ3γ2 GABAA receptors expressed in X. laevis oocytes. Values are given as mean ± S.D., n = 4 to 5 for at least two batches of oocytes.
