Enantiomeric Selectivity Of Gabaergic Steroids

Studies of the binding and electrophysiological activity of neuroactive steroids have demonstrated that the effects of these steroids do not result from steroid binding at the benzodiazepine, barbiturate, picrotoxin or GABA recognition sites associated with GABAa receptor complexes. These results have led to the concept of unique binding site(s) for a group of neuroactive steroids on GABA receptors (1-3, 5-7). Although there has been no satisfactory demonstration of the specific binding of radioactive steroid ligands to such sites, a significant correlation has been obtained between physiological and behavioral effects and in vitro structure-activity relationships of GABAergic neu-roactive steroids (7).

Nevertheless, because of the lipophilic nature of these steroids, it was important to determine whether their effects on ion channels (such as the GABA-gated chloride ion channel) were simply due to nonspecific perturbation of lipid bilayers (15). In a series of elegant studies, Covey and colleagues, have synthesized a series of steroid and benz[e]indene enantiomers, in order to provide a test of the hypothesis that these compounds exert their effects on chiral region(s) of protein recognition sites in the ion channels (16-18). The six compounds initially chosen for study are shown in Fig. 2.

As isolated in pure form from women's pregnancy urine by Marker et al. in 1937 (19), an ethanol solution of 3 a,5 a-TH PROG rotated plane-polarized light in a positive direction, indicated as (+). This steroid has eight chiral centers, six at the ring fusions of the four rings, and two more at C-3 and C-17. This leads to a possible 28 =256 different stereoisomers. One of these stereoisomers is (-)3a,5a-TH PROG, shown on the upper right of Fig. 2, where all eight of the eight chiral centers of natural (+)3a,5a-TH PROG have been inverted in the rigorous total chemical synthesis of the (-) enantiomer. It is an absolute mirror image of the natural (+) enantiomer, as seen in the two-dimensional drawing where solid lines (P configuration) are used for bonds above the plane of the drawing in Fig. 2, and dotted lines (a configuration) for bonds below the plane. These enantiomers (as opposed to diastereomers) have identical physical properties except for their rotation of plane-polarized light and would be expected to perturb the lipid bilayer in a similar manner, as contrasted with their expected dissimilar binding to protein

Fig. 2. Structures of the enantiomeric pairs of compounds prepared by Hu et al. (20).

receptor site(s). Similarly, the total synthesis of the benz[e]indene enantiomer (-)-BI-1 was performed (20), as well as the multi-step preparation of the enantiomer (-)-ACN of the synthetic GABAa potentiator (+)-ACN (3a-hydroxy-5a-androstane-17P-cya-nonitrile).

The two pairs of steroidal enantiomers (Fig. 2) were compared for their ability to potentiate GABA-mediated chloride ion currents in rat hippocampal neurons (16). The unnatural synthetics (-)3a,5a-TH PROG and (-)-ACN, were essentially inactive, compared with (+)3a,5a-TH PROG and (+)-ACN, respectively. This was also the case for direct activation of GABAA receptors seen at concentrations up to 100 ^M in these hippocampal neurons. The (+) enantiomers of 3a,5a-TH PROG and ACN also produced anesthesia in tadpoles at considerably lower concentrations than the respective (-) enantiomers (16). This was also the case for (+)-BI-1 compared to (-)-BI-1 (20).

Surprisingly, the (-) enantiomers of 3a,5a-TH PROG and ACN did have significant anesthetic activity in tadpoles even though their maximal electrophysiological effect was a doubling of the GABA current. Conversely, the (+) enantiomers of 3a,5a-TH PROG and ACN caused anesthesia in tadpoles at concentrations much lower than those that produced maximal electrophysiological effects. Additionally, these enantiomers showed synergistic effects both electrophysiologically and as anesthetics, where threshold doses of the enantiomers gave an augmented effect when they were administered together. The authors concluded from these results that (1) neuroactive steroid-induced anesthesia probably results from potentiation of GABA currents, rather than from direct activation of chloride currents; and (2) the (+) and (-) enantiomers probably bind to different sites on GABA receptors (16).

In a further report, Zorumski et al. (17) demonstrated that the three (+) enantiomers shown in Fig. 2 were more potent and effective than their respective unnatural (-) enantiomers in the enhancement of GABAA-receptor-mediated evoked synaptic currents in microcultures of rat hippocampal neurons. Together, these results strongly support the action of these steroids and benz[e]indenes at unique chiral binding sites on protein regions of GABAa receptors, rather than through perturbation of membrane lipids.

In a further extension of this approach, the unnatural enantiomers of pregnanolone sulfate (3a,5p-TH PROGS), pregnenolone sulfate (PREGS), and dehydroepian-drosterone sulfate (DHEAS) were prepared by total synthesis (21). The natural (+) enantiomers of these sulfate esters have been established by electrophysiological methods to be negative allosteric modulators of GABAA-receptors in cultured rat hip-pocampal neurons (6). The inhibitory effect of natural (+)-DHEAS was about sevenfold greater than that of the synthetic (-)-DHEAS (21). However, negligible enantioselectivity was found for 3a,5^-TH PROGS or PREGS compared to their unnatural enantiomers. These results suggest that different mechanisms and/or sites of action may be operating for the effect of DHEAS, compared with the pregnane sulfate esters, on GABA-mediated chloride ion currents (21). The data also provide novel evidence for a direct binding interaction of DHEAS with a chiral recognition site of these receptor(s). The extension of this approach, exploring the enantioselectivity of neuroactive steroids, to other neu-rotransmitter systems will be invaluable.

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