Hawkinson J E, Kimbrough C L, Belelli D, Lambert J J, Purdy R H, Lan N C
CoCensys, Inc., Irvine, California 92718.
Mol Pharmacol. 1994 Nov;46(5):977-85.
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.
神经活性甾体，包括孕酮和脱氧皮质酮的内源性代谢产物及其合成衍生物，是γ-氨基丁酸（GABA）A受体复合物的正变构调节剂。它们抑制[35S]叔丁基双环磷硫代酸盐（[35S]TBPS）的结合，增强[3H]氟硝西泮的结合，并增强GABA诱发的氯离子电流和激动剂刺激的36Cl摄取。通过检测31种神经活性甾体及相关化合物在大鼠脑皮质膜中抑制[35S]TBPS结合的相对能力，探索了它们的构效关系。一个游离的3α-羟基对于高效抑制是必需的。虽然21位羟基化及随后的酯化可维持活性，但11α-或12α-羟基化会大大降低活性。在5α-还原系列中，17位取代的效价顺序为17β-乙酰基＞17β-氰基＞17β-甲氧羰基＞17α-乙酰基＞17-酮≥17-肟≥17α-氰基。在9位和11位之间引入双键会降低效价，而4位的双键会降低最大抑制程度。比较这些神经活性甾体及相关化合物在[35S]TBPS和[3H]氟硝西泮试验中的活性，效价（r = 0.90，n = 17）与调节幅度（r = 0.95，n = 31）之间存在很强的相关性，表明神经活性甾体结合位点在大鼠皮质中与TBPS和苯二氮䓬位点类似地偶联。然而，在任一结合试验中的调节程度与大鼠皮质突触体中蝇蕈醇刺激的36Cl摄取增强之间的相关性较弱（r = 0.74 - 0.78，n = 31）。使用电生理方法，在结合试验中的调节幅度与表达人α1β1γ2L受体复合物的非洲爪蟾卵母细胞中GABA诱发的氯离子电流增强之间观察到更强的相关性（r = 0.89 - 0.94，n = 15）。因此，神经活性甾体对[35S]TBPS和[3H]氟硝西泮结合的调节可预测其在GABAA受体复合物上的功能活性。
