Characterization of novel ligands for wild-type and natural mutant diazepam-insensitive benzodiazepine receptors

Abstract

A series of benzodiazepine receptor ligands with different chemical structures were evaluated for their affinities at diazepam-sensitive and diazepam-insensitive binding sites for [ 3H]Ro 15-4513 (ethyl-8-azido-5,60dihydro-5-methyl-6-oxo-4H-imidazo-[1,5a][1,4]benzodiazepine-3-carboxylate) in cerebellar GABA A receptors. Rats of Wistar strain and of alcohol-sensitive (ANT) and alcohol-insensitive (AT) lines were used. The ANT rats possess a single point mutation in their GABA A receptor α6 subunit, which makes their diazepam-insensitive sites sensitive to benzodiazepine agonists, unlike those of AT and Wistar rats. All compounds evaluated displayed high-affinity binding to diazepam-sensitive sites ( K i < 50 nM). In contrast, a wider range of affinities were observed at diazepam-insensitive sites which depended upon the basic structure and substitutions. The 7- and 8-halogen substituted imidazobenzodiazepines and 12-halogen substituted diimidazoquinazolines displayed the highest affinities ( K i < 15 nM), while intermediate to low affinities (100 < K i < 4000 nM) were displayed by imidazoquinazolines, thienopyrimidines, one oxoimidazoquinoxaline, and some cyclopyrrolones. The imidazoquinoxalines evaluated displayed the lowest affinity ( K i > 10000 nM). The oxoimidazoquinoxaline, 6-chloro-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5-dihydro-5-isopropyl-4-oxo-imidazol[1,5-a]quinoxaline (NNC 14-0578) and suriclone represent the first benzodiazepine receptor full agonists to bind with relatively high affinity ( K i ∼ 100 nM) to diazepam-insensitive sites. The 5 position substituted methoxybenzyl, dimethylallyl, and 4-fluorobenzyl oxoimidazoquinoxaline analogs demonstrated a 58–336-fold higher affinity for ANT than AT diazepam-insensitive sites. Classical benzodiazepines having a 5-phenyl substituent have demonstrated a similar preference for ANT sites, suggesting that all these structures bind to diazepam-insensitive sites in the same orientation. The other compounds evaluated demonstrated only a more modest selectivity (1–12-fold), indicating different structural requirements for binding to mutant ANT and wild-type AT and Wistar receptors. These results expand the range of ligands which display high affinity for diazepam-insensitive sites. Such compounds shoudl be helpful in determining intrinsic actions of high-affinity ligands at these sites and in assessing the contribution of these sites in enhanced sedative sensitivity of cerebellar function in the ANT rats.