CORRELATION OF NEUROACTIVE STEROID MODULATION OF [S-35] T-BUTYLBICYCLOPHOSPHOROTHIONATE AND [H-3] FLUNITRAZEPAM BINDING AND GAMMA-AMINOBUTYRIC-ACID, RECEPTOR FUNCTION

Neuroactive steroids, including endogenously occurring metabolites of progesterone and deoxycorticosterone as well as their synthetic deriva tives, are positive allosteric modulators of the gamma-aminobutyric ac id (GABA)(A) receptor complex. They inhibit the binding of [S-35]t-but ylbicyclophosphorothionate ([S-35]TBPS), enhance the binding of [H-3]f lunitrazepam, and potentiate GABA-evoked chloride currents and agonist -stimulated Cl-36- uptake. The structure-activity relationship for 31 neuroactive steroids and related compounds was explored by examining t heir relative ability to inhibit [S-35]TBPS binding in rat brain corti cal membranes. A free 3 alpha-hydroxy group is necessary for high pote ncy inhibition. Whereas hydroxylation in the 21-position and subsequen t esterification maintain activity, 11 alpha- or 12 alpha-hydroxylatio n greatly reduces activity. The rank order of potency for 17-position substitutions in the 5 alpha-reduced series is 17 beta-acetyl > 17 bet a-cyano > 17 beta-methoxycarbonyl > 17 alpha-acetyl > 17-one greater t han or equal to 17-oxime greater than or equal to 17 alpha-cyano. Intr oduction of a double bond between the 9- and 11-positions reduces pote ncy, whereas a double bond in the 4-position reduces the maximal exten t of inhibition. Comparing the activities of these neuroactive steroid s and related compounds in the [S-35]TBPS and [H-3]flunitrazepam assay s, there is a strong correlation between potency (r = 0.90, n = 17) an d magnitude of modulation (r = 0.95, n = 31), indicating that the neur oactive steroid binding site is similarly coupled to the TBPS and benz odiazepine sites in rat cortex. However, there is a weaker correlation (r = 0.74-0.78, n = 31) between the degree of modulation in either bi nding assay and potentiation of muscimol-stimulated Cl-36- uptake in r at cortical synaptoneurosomes. Using an electrophysiological approach, stronger correlations (r = 0.89-0.94, n = 15) were observed between t he magnitude of modulation in the binding assays and potentiation of G ABA-evoked chloride currents in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2L receptor complexes. Thus, neuroactive steroid modul ation of [S-35]TBPS and [H-3]flunitrazepam binding is predictive of fu nctional activity at the GABA(A) receptor complex.