Affinity probes for the GABA-gated chloride channel: 5 e- tert-Butyl-2 e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with photoactivatable, fluorescent, biotin, agarose and protein substituents

Abstract

Affinity probes for the noncompetitive blocker or picrotoxinin site of the γ-aminobutyric acid (GABA)-gated chloride channel were designed for four types of applications: photoaffinity reagents to covalently label the binding site; fluorescent probes for receptor analysis; biotinylated compounds and agarose/sepharose conjugates for affinity chromatography; ligand-protein/enzyme conjugates for immunoassay. These 5 e- tert-butyl-2 e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes were optimized by structure-activity studies for potency as inhibitors of 3H ethynylbicycloorthobenzoate binding to bovine brain membranes, measured as the concentration for 50% inhibition (IC 50). Preferred compounds are 5 e-(CH 3) 3CCH(CH 2S) 2CH-2 e-C 6H 4-4-CCCH 2OCH 2C(O)R, wherein R confers the following properties and 1C 50 values: R = SCH 2CH 2SCH 2C(O)C 6H 4-4-N 3, photo-affinity, 9 nM; R = NHCH 2CH 2NHC(O)C 6H 2-2-OH,5-1,4-N 3, photoaffinity, 105 nM; R = SCH 2CH 2S-4-benzofurazan-7-NO 2, fluorescent, 13 nM; R = SCH 2CH 2SCH 2-5-fluorescein, fluorescent, 27 nM; R = NHCH 2CH 2NH[C(O)(CH 2) 5NH] 2-biotin, affinity chromatography, 190 nM. The most potent photoaffinity ligand (IC 50 9 nM) was labeled at 7 Ci mmol −1 by reacting the appropriate thiol with 3H 4-azidophenacyl bromide (obtained by alumina-catalyzed tritium exchange of its enolizable hydrogens). The first steps have been taken in using the NCB site for affinity chromatography of the GABA A receptor in CHAPS-solubilized bovine brain membranes with the dithiane-biotin probe and an avidin-acrylic bead system or with an analogous dithiane-agarose/sepharose column eluting with GABA or dithiane as above (R = OH). A protein conjugate of a related dithiane-monosulfone elicited production of specific antisera in rabbits. These findings illustrate the diversity and utility of new affinity probes prepared in the alkynylphenyldithiane series.