Correlation of neuroactive steroid modulation of [35S]t- butylbicyclophosphorothionate and [3H]flunitrazepam binding and gamma- aminobutyric acidA receptor function

JE Hawkinson, CL Kimbrough, D Belelli, JJ Lambert, RH Purdy and NC Lan

CoCensys, Inc., Irvine, California 92718.

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.

Volume 46, Issue 5, pp. 977-985, 11/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

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