title: Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.
abstract: Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 +/- 14% and 109 +/- 14% at beta3 and beta2, respectively, compared with 62 +/- 6% and 56 +/- 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which betasubunits are an important determinant of efficacy and potency.
authors.affiliation: Sharp and Dohme Research Laboratories, Merck, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK. alison_smith@merck.com
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Smith
authors.forename: Beth
authors.initials: B
authors.lastname: Oxley
authors.forename: Sallie
authors.initials: S
authors.lastname: Malpas
authors.forename: Gopalan V
authors.initials: GV
authors.lastname: Pillai
authors.forename: Peter B
authors.initials: PB
authors.lastname: Simpson
mesh_terms: D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology*; D058787:GABA-A Receptor Antagonists; D006801:Humans; D051379:Mice; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000777:Anesthetics; D000893:Anti-Inflammatory Agents; D018756:GABA Antagonists; D058787:GABA-A Receptor Antagonists; D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid