2 research outputs found
Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABA<sub>A</sub> Receptors
The
anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic
effects of benzodiazepine site ligands are mainly elicited by allosteric
modulation of GABA<sub>A</sub> receptors via their extracellular αx+/γ2–
(<i>x</i> = 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 <i>in silico</i> 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
Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ‑Aminobutyric Acid Type A Receptor (GABA<sub>A</sub>R) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability
Recent
reports indicate that α6β2/3γ2 GABA<sub>A</sub>R
selective ligands may be important for the treatment of
trigeminal activation-related pain and neuropsychiatric disorders
with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2
GABA<sub>A</sub>R selective pyrazoloquinolinones, 42 novel analogs
were synthesized, and their in vitro metabolic stability and cytotoxicity
as well as their in vivo pharmacokinetics, basic behavioral pharmacology,
and effects on locomotion were investigated. Incorporation of deuterium
into the methoxy substituents of the ligands increased their duration
of action via improved metabolic stability and bioavailability, while
their selectivity for the GABA<sub>A</sub>R α6 subtype was retained. <b>8b</b> was identified as the lead compound with a substantially
improved pharmacokinetic profile. The ligands allosterically modulated
diazepam insensitive α6β2/3γ2 GABA<sub>A</sub>Rs
and were functionally silent at diazepam sensitive α1β2/3γ2
GABA<sub>A</sub>Rs, thus no sedation was detected. In addition, these
analogs were not cytotoxic, which render them interesting candidates
for treatment of CNS disorders mediated by GABA<sub>A</sub>R α6β2/3γ2
subtypes