科研之友
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摘要
GABA(A) receptors mediate the action of many clinically important drugs interacting with different binding sites. For some potential binding sites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular alpha 1+beta 3- interface, which is homologous to the benzodiazepine binding site at the alpha 1+gamma 2- interface. On screening of >100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3-c]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at alpha 1 beta 3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at alpha 1 beta 3 gamma 2 and alpha 1 beta 3 receptors. By mutating pocket-forming amino acid residues at the alpha 1+ and the beta 3- side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a general inactivation of GABA(A) receptors, because the GABA-enhancing effect of ROD 188 or the steroid alpha-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the alpha and beta subunit types forming the interface. CGS 9895 thus represents the first prototype of drugs mediating benzodiazepine-like modulatory effects via the alpha+beta-interface of GABA(A) receptors. Since such binding sites are present at alpha beta, alpha beta gamma, and alpha beta delta receptors, such drugs will have a much broaderaction than benzodiazepines and might become clinical important for the treatment of epilepsy.
- 出版日期2011-1-19
