Synthesis of GABA_A receptor agonists and evaluation of their α-subunit selectivity and orientation in the GABA binding site

Drugs used to treat various disorders target GABA_A receptors. To develop α subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [³H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA_A α_iβ ₃γ₂ receptors (i = 1-6). The effects of 5-aminomethyl-3H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all α subunit isoforms. 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at α₂-, α₃-, and α₅-containing receptors. When coapplied with GABA, they were antagonistic in α₂-, α₄-, and α₆-containing receptors and potentiated α₃-containing receptors. 6a protected GABA binding site cysteine-substitution mutants α₁F64C and α₁S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the α₁R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing α subtype selective GABA mimetic drugs.