Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and γ-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 γ-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 3α-hydroxy group is necessary for high potency inhibition. Whereas hydroxylation in the 21-position and subsequent esterification maintain activity, 11α- or 12α-hydroxylation greatly reduces activity. The rank order of potency for 17-position substitutions in the 5α-reduced series is 17β-acetyl> 17β-cyano > 17β-methoxycarbonyl > 17α-acetyl > 17-one ≥ 17-oxime ≥ 17α-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 α1 β1 γ2L receptor complexes. Thus, neuroactive steroid modulation of [³⁵S]TBPS and [³H]flunitrazepam binding is predictive of functional activity at the GABA_A receptor complex.