A photoreactive probe that differentiates the binding sites of noncompetitive GABA receptor antagonists.

γ-Aminobutyric acid (GABA) receptors are postsynaptic membrane protein complexes that are important not only in the regulation of the nervous system but also as targets of drugs and insecticides. We synthesized a photoreactive straight-chain noncompetitive antagonist (NCA), 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl 4-(4-methoxycarbonyl-1-butynyl)benzoate (NMB), to probe the NCA binding site. Our data show that this probe labels the NCA site and demonstrate that the NCA insecticide fipronil binds at a site distinct from that of other NCAs, such as picrotoxinin and 4'-ethynyl-4-n-propylbicycloorthobenzoate. The unique molecule NMB will be useful in identifying the cross-linking site of straight-chain NCAs in GABA receptors and mapping allosteric binding sites. Such studies should provide invaluable information in designing novel NCAs.

Synthesis and structure-activity relationship analysis of bicyclophosphorothionate blockers with selectivity for housefly gamma-aminobutyric acid receptor channels.

BACKGROUND: Bicyclophosphorothionates (2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-sulfides) are blockers (or non-competitive antagonists) of gamma-aminobutyric acid (GABA) receptor channels. Twenty-two bicyclophosphorothionates with different 3- and 4-substituents were synthesised, and [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays were performed to evaluate their affinities for housefly and rat GABA receptors. RESULTS: Introduction of an isopropyl group at the 3-position enhanced the affinity of bicyclophosphorothionates for housefly GABA receptors and reduced the affinity towards rat GABA receptors. The 4-isopentyl-3-isopropylbicyclophosphorothionate showed the highest affinity for housefly GABA receptors (IC(50) = 103 nM) among the analogues tested, while the 4-cyclohexylbicyclophosphorothionate showed the highest affinity for rat GABA receptors (IC(50) = 125 nM). Among the bicyclophosphorothionates synthesised to date, the former analogue exhibited the highest selectivity for housefly GABA receptors, with an IC(50)(rat)/IC(50)(fly) ratio of approximately 97. Three-dimensional GABA receptor models successfully explained the structure-activity relationships of the bicyclophosphorothionates. CONCLUSION: The results indicate that minor structural modifications of blockers can change their selectivity for insect versus mammalian GABA receptors. The substituent at the 3-position of the bicyclophosphorothionates dictates selectivity for housefly versus rat GABA receptors. This information should prove useful for the design of safer insecticides and parasiticides.