title: A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors.
abstract: In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.
authors.affiliation: Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. eversa@wustl.edu
authors.forename: Alex S
authors.initials: AS
authors.lastname: Evers
authors.forename: Zi-Wei
authors.initials: ZW
authors.lastname: Chen
authors.forename: Brad D
authors.initials: BD
authors.lastname: Manion
authors.forename: Mingcheng
authors.initials: M
authors.lastname: Han
authors.forename: Xin
authors.initials: X
authors.lastname: Jiang
authors.forename: Ramin
authors.initials: R
authors.lastname: Darbandi-Tonkabon
authors.forename: Tristan
authors.initials: T
authors.lastname: Kable
authors.forename: John
authors.initials: J
authors.lastname: Bracamontes
authors.forename: Charles F
authors.initials: CF
authors.lastname: Zorumski
authors.forename: Steven
authors.initials: S
authors.lastname: Mennerick
authors.forename: Joe Henry
authors.initials: JH
authors.lastname: Steinbach
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009570:Nitriles / Q000378:metabolism / Q000494:pharmacology*; D009635:Norandrostanes / Q000378:metabolism / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: C495276:(3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile; D019086:Bridged Bicyclo Compounds, Heterocyclic; D009570:Nitriles; D009635:Norandrostanes; D011963:Receptors, GABA-A; C037476:tert-butylbicyclophosphorothionate