Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and gamma-aminobutyric acidA receptor function.
Neuroactive steroids, including endogenously occurring metabolites of progesterone and deoxycorticosterone as well as their synthetic derivatives, are positive allosteric modulators of the gamma-aminobutyric acid (GABA)A receptor complex. They inhibit the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), enhance the binding of [3H]flunitrazepam, and potentiate GABA-evoked chloride currents and agonist-stimulated 36Cl- uptake. The structure-activity relationship for 31 neuroactive steroids and related compounds was explored by examining their relative ability to inhibit [35S]TBPS binding in rat brain cortical membranes. A free 3 alpha-hydroxy group is necessary for high potency inhibition. Whereas hydroxylation in the 21-position and subsequent esterification maintain activity, 11 alpha- or 12 alpha-hydroxylation greatly reduces activity. The rank order of potency for 17-position substitutions in the 5 alpha-reduced series is 17 beta-acetyl > 17 beta-cyano > 17 beta-methoxycarbonyl > 17 alpha-acetyl > 17-one > or = 17-oxime > or = 17 alpha-cyano. Introduction of a double bond between the 9- and 11-positions reduces potency, whereas a double bond in the 4-position reduces the maximal extent of inhibition. Comparing the activities of these neuroactive steroids and related compounds in the [35S]TBPS and [3H]flunitrazepam assays, there is a strong correlation between potency (r = 0.90, n = 17) and magnitude of modulation (r = 0.95, n = 31), indicating that the neuroactive steroid binding site is similarly coupled to the TBPS and benzodiazepine sites in rat cortex. However, there is a weaker correlation (r = 0.74-0.78, n = 31) between the degree of modulation in either binding assay and potentiation of muscimol-stimulated 36Cl- uptake in rat cortical synaptoneurosomes. Using an electrophysiological approach, stronger correlations (r = 0.89-0.94, n = 15) were observed between the magnitude of modulation in the binding assays and potentiation of GABA-evoked chloride currents in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2L receptor complexes. Thus, neuroactive steroid modulation of [35S]TBPS and [3H]flunitrazepam binding is predictive of functional activity at the GABAA receptor complex.
CoCensys, Inc., Irvine, California 92718.
J E
JE
Hawkinson
C L
CL
Kimbrough
D
D
Belelli
J J
JJ
Lambert
R H
RH
Purdy
N C
NC
Lan
D000494:Allosteric Regulation; D000731:Androstanes / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005445:Flunitrazepam / Q000378:metabolism*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D011278:Pregnanes / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D000731:Androstanes; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011278:Pregnanes; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005445:Flunitrazepam; C037476:tert-butylbicyclophosphorothionate