A Synthetic 18-Norsteroid Distinguishes between Two Neuroactive Steroid Binding Sites on GABAA Receptors
A Synthetic 18-Norsteroid Distinguishes between Two Neuroactive Steroid Binding Sites on GABAA Receptors
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DOI：
10.1124/jpet.109.164079
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发表时间：
2010-05-01
影响因子：
3.5
通讯作者：
Covey, Douglas F.
中科院分区：
文献类型：
--
作者：
Evers, Alex S.;Chen, Zi-Wei;Covey, Douglas F.
在不存在GABA的情况下，增强GABA介导电流的神经活性类固醇以双相方式调节[S-35]叔丁基双环硫代磷酸酯的结合，在低浓度（位点NS 1）下增强结合，在高浓度（位点NS 2）下抑制结合。在当前的研究中，化合物（3 α，5 β，17 β）-3-羟基-18-去甲雄甾烷-17-腈（3 α 5 β-18-去甲ACN），一种18-去甲类固醇，在大鼠脑细胞膜和异源表达的GABA（A）受体中均显示为NS 1位点的完全激动剂和NS 2位点的弱部分激动剂。3 α 5 β-18-norACN也抑制完全神经类固醇激动剂（3 α，5 α，17 β）-3-羟基-17-腈（3 α 5 β ACN）在NS 2位点的作用。结构-活性研究表明，C18甲基基团的缺失和5 β-还原构型都有助于在NS 2位点的弱激动剂作用。使用异源表达的GABA（A）受体进行的电生理学研究表明，3 α 5 β-18-norACN可有效地增强低浓度GABA引起的GABA电流，但作为GABA（A）受体的直接激活剂的效力较低。3 α 5 β-18-norACN也抑制3 α 5 α ACN对GABA（A）受体的直接激活。3 α 5 β-18-norACN也在蝌蚪和小鼠中产生翻正反射的丧失，表明NS 1的作用足以介导神经类固醇的镇静作用。这些数据提供了对NS 2位点神经类固醇功效所需药效团的深入了解，并可能证明可用于开发GABA（A）受体上神经类固醇位点的选择性激动剂和拮抗剂。
In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [S-35] t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3 alpha,5 beta,17 beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3 alpha 5 beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABA(A) receptors. 3 alpha 5 beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3 alpha,5 alpha,17 beta)-3-hydroxy-17-carbonitrile (3 alpha 5 beta ACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5 beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABA(A) receptors show that 3 alpha 5 beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABA(A) receptors. 3 alpha 5 beta-18-norACN also inhibits direct activation of GABA(A) receptors by 3 alpha 5 alpha ACN. 3 alpha 5 beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABA(A) receptor.