Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.

m-sulphonate benzene diazonium chloride: a novel GABAA receptor antagonist.

A previously identified irreversible affinity label for the gamma-aminobutyric acid (GABA) binding site in rat brain membranes, m-sulphonate benzene diazonium chloride (MSBD), was characterized in functional studies using patch clamp and two-electrode voltage clamp recording techniques. MSBD did not exhibit any agonist activity on native GABAA receptors in cultured sympathetic ganglionic neurones but acted as an antagonist of GABA-induced membrane currents. Recombinant GABAA receptors composed of alpha 1, beta 1 and gamma 2S subunits were expressed in Xenopus oocytes following microinjection with cDNAs. Equilibrium dose-response curve analyses established that MSBD was a partially reversible, apparently non-competitive GABAA receptor antagonist. The IC50 for MSBD was estimated from an inhibition curve as 87 +/- 3 microM. In addition, the onset and recovery from MSBD-induced inhibition was independent of GABAA receptor activation. The relatively simple structure of this novel GABAA receptor antagonist, MSBD, is compared with known agonists and antagonists at the GABAA receptor. MSBD may be a useful pharmacological tool which could be used to deduce further information about the structure and function of agonist and antagonist binding sites on the GABAA receptor.

m-Sulfonate benzene diazonium chloride: a powerful affinity label for the gamma-aminobutyric acid binding site from rat brain.

m-Sulfonate benzene diazonium chloride (MSBD) was used to affinity-label the gamma-aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter-adsorbed membranes. Inactivation of specific [3H]-muscimol binding sites by MSBD and its prevention by GABA were both time- and concentration-dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.

3-aminopyridazine derivatives with atypical antidepressant, serotonergic, and dopaminergic activities.

Minaprine [3-[(beta-morpholinoethyl)amino]-4-methyl-6-phenylpyridazine dihydrochloride] is active in most animal models of depression and exhibits in vivo a dual dopaminomimetic and serotoninomimetic activity profile. In an attempt to dissociate these two effects and to characterize the responsible structural requirements, a series of 47 diversely substituted analogues of minaprine were synthesized and tested for their potential antidepressant, serotonergic, and dopaminergic activities. The structure-activity relationships show that dopaminergic and serotonergic activities can be dissociated. Serotonergic activity appears to be correlated mainly with the substituent in the 4-position of the pyridazine ring whereas the dopaminergic activity appears to be dependent on the presence, or in the formation, of a para-hydroxylated aryl ring in the 6-position of the pyridazine ring.

Aryl diazo compounds and diazonium salts as potential irreversible probes of the gamma-aminobutyric acid receptor.

The synthesis of different diazonium salts derived from homo- and heterocyclic aromatic amines bearing anionic residues is described. The chemical stabilities of these compounds were established at different pH's, and the compounds were tested accordingly in binding experiments for the rat brain gamma-aminobutyric acid (GABA) receptor, for which they could ultimately be used as irreversible affinity or photoaffinity probes. The aromatic heterocyclic series studied were 2-aminoimidazole, 2-aminothiazole, and 4-aminopyridine N-oxide. The derived diazonium salts are unstable compounds at neutral pH unless they are able to be deprotonated to the corresponding diazo form. As such, the 2-diazoimidazole-4(5)-acetic acid (3b) is stable in neutral medium and recognizes the GABA receptor (IC50 = 70 microM). The homocyclic aromatic diazonium salts showed sufficient stability to be tested in binding experiments. The diazonium salts derived from m-sulfanilic acid and 8-sulfonaphthylamine were the most interesting (10b, IC50 = 10 microM; 15b, IC50 less than 100 microM). In this series, the compounds that deprotonate at neutral pH (hydroxybenzenediazonium derivatives 12b-14b) showed increased chemical stability but decreased affinity for the GABA receptor. This difference between the diazoimidazole and the diazohydroxybenzene series is attributed to a different charge distribution between the two series. The ligands 3b, 10b, and 15b can be used as potential irreversible probes for the GABA receptor.

Synthesis and structure-activity relationships of a series of aminopyridazine derivatives of gamma-aminobutyric acid acting as selective GABA-A antagonists.

We have recently shown that an arylaminopyridazine derivative of GABA, SR 95103 [2-(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride], is a selective and competitive GABA-A receptor antagonist. In order to further explore the structural requirements for GABA receptor affinity, we synthesized a series of 38 compounds by attaching various pyridazinic structures to GABA or GABA-like side chains. Most of the compounds displaced [3H]GABA from rat brain membranes. All the active compounds antagonized the GABA-elicited enhancement of [3H]diazepam binding, strongly suggesting that all these compounds are GABA-A receptor antagonists. None of the compounds that displaced [3H]GABA from rat brain membranes interacted with other GABA recognition sites (GABA-B receptor, GABA uptake binding site, glutamate decarboxylase, GABA-transaminase). They did not interact with the Cl- ionophore associated with the GABA-A receptor and did not interact with the benzodiazepine, strychnine, and glutamate binding sites. Thus, these compounds appear to be specific GABA-A receptor antagonists. In terms of structure-activity, it can be concluded that a GABA moiety bearing a positive charge is necessary for optimal GABA-A receptor recognition. Additional binding sites are tolerated only if they are part of a charge-delocalized amidinic or guanidinic system. If this delocalization is achieved by linking a butyric acid moiety to the N(2) nitrogen of a 3-aminopyridazine, GABA-antagonistic character is produced. The highest potency (approximately equal to 250 times bicuculline) was observed when an aromatic pi system, bearing electron-donating substituents, was present on the 6-position of the pyridazine ring.

Escape behavior produced by the blockade of glutamic acid decarboxylase (GAD) in mesencephalic central gray or medial hypothalamus.

Microinjections into the mesencephalic central gray (CG) or the medial hypothalamus (MH) of three drugs (L-allylglycine, Semicarbazide or 4,5 dihydroxy-isophtalic acid) known to block glutamic acid decarboxylase (GAD) produced a dose-dependent behavioral activation accompanied by jumps. These effects are qualitatively similar to those produced by microinjections of SR 95103 (a GABA-A receptor antagonist) at the same site. These findings suggest that, at both the level of the CG and the MH, gamma-aminobutyric acid (GABA) tonically inhibits a neuronal substrate involved in the generation of flight reactions.