Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α 1 β 3 γ 2 GABA A R Transmembrane Domain.

GABA _A receptors (GABA _A Rs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([ ³ H]azietomidate) and mephobarbital [[ ³ H]1-methyl-5-allyl-5-( m -trifluoromethyl-diazirynylphenyl)barbituric acid ([ ³ H] R - m TFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α 1 β 3 γ 2 GABA _A R transmembrane domain at β ⁺ - α ^- ( β ⁺ sites) and α ⁺ - β ^- / γ ⁺ - β ^- ( β ^- sites) subunit interfaces. We now use competition photolabeling with [ ³ H]azietomidate and [ ³ H] R-m TFD-MPAB to identify para -substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to β ⁺ , while derivatives with bulkier lipophilic substitutions [4-( tert -butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β ^- sites. Similar to R-m TFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α ⁺ - β ^- and γ ⁺ - β ^- sites. However, we discovered four compounds that bind with different affinities to the two β ^- interface sites. Two of these bind with higher affinity to one of the β ^- sites than to the β ⁺ sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ ⁺ - β ^- site than to the α ⁺ - β ^- or β ⁺ - α ^- sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α ⁺ - β ^- site than to the β ⁺ and γ ⁺ - β ^- sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABA _A R transmembrane domain, a property that may facilitate the development of subtype selective GABA _A R PAMs.
(Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)