title: 3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor.
abstract: Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors.
authors.affiliation: CoCensys, Inc., Irvine, California 92718, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Hawkinson
authors.forename: J A
authors.initials: JA
authors.lastname: Drewe
authors.forename: C L
authors.initials: CL
authors.lastname: Kimbrough
authors.forename: J S
authors.initials: JS
authors.lastname: Chen
authors.forename: D J
authors.initials: DJ
authors.lastname: Hogenkamp
authors.forename: N C
authors.initials: NC
authors.lastname: Lan
authors.forename: K W
authors.initials: KW
authors.lastname: Gee
authors.forename: K Z
authors.initials: KZ
authors.lastname: Shen
authors.forename: E R
authors.initials: ER
authors.lastname: Whittemore
authors.forename: R M
authors.initials: RM
authors.lastname: Woodward
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D003292:Convulsants / Q000378:metabolism; D018755:GABA Agonists; D006801:Humans; D015640:Ion Channel Gating; D011280:Pregnanolone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects*; D011994:Recombinant Proteins; D014982:Xenopus laevis
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; C099326:Co 2-1970; D003292:Convulsants; D018755:GABA Agonists; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C037476:tert-butylbicyclophosphorothionate; D011280:Pregnanolone