Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the a1b3g2 GABAAR Transmembrane Domain

Abstract

GABAA receptors (GABAARs) 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 ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the a1b3g2 GABAAR transmembrane domain at b1-a2 (b1 sites) and a1-b2/g1-b2 (b2 sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-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 b1, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to b2 sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the a1-b2 and g1-b2 sites. However, we discovered four compounds that bind with different affinities to the two b2 interface sites. Two of these bind with higher affinity to one of the b2 sites than to the b1 sites. We deduce that 4-benzoyl-propofol binds with .100-fold higher affinity to the g1-b2 site than to the a1-b2 or b1-a2 sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the a1-b2 site than to the b1 and g1-b2 sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.