Diastereomeric Selectivity of GABAergic Steroids

Structure-activity studies in several laboratories have established a marked diastereomeric selectivity of 3-hydroxysteroids for GABAa receptors (1-3). Thus,

From: Contemporary Endocrinology: Neurosteroids: A New Regulatory Function in the Nervous System Edited by: E.-E. Baulieu, P. Robel, and M. Schumacher © Humana Press Inc., Totowa, NJ

many 3a-hydroxysteroids in the pregnane (5P) or allopregnane (5a) series are positive allosteric modulators of GABAa receptors, whereas the diastereomeric 3p-hydroxy-steroids are essentially inactive in this respect. (A diasteriomer has the opposite configuration at one of the chiral centers, e.g., at C-3 in this case.) This generalization is not intended to imply that epiallopregnanolone (3p,5a-TH PROG) is devoid of biological activity. For example, Yu and Ticku (4) reported that 5-d treatment of cultured cortical neurons with 3p,5a-TH PROG reversed the effect of 3a,5a-TH PROG at the same concentration (2 ^M).

Esterification and oxidation of the 3a-hydroxyl group greatly reduces activity. There are other notable exceptions, such as 3a-hydroxy derivatives in the 5a-pregn-9(11)-ene series, 11a- and 12a-hydroxylated metabolites, and a C-17 diastereomer with the 17a-acetyl side chain, all of which have negligible activity as allosteric modulators of GABAareceptors (5). In the case of the potent synthetic steroid 3a-hydroxy-5a-androstane-17P-carbonitrile (ACN), its 17a-diastereomer is also inactive (5). However, when there is a ketone group at C-17 rather than the 17P-acetyl group, 3a-hydroxy-5a-androstan-17-one (androsterone) is as active as allopregnanolone or pregnanolone in potentiating GABAa receptors (6). The effect of these structural alterations at C-17 highlight the importance of a hydrogen bond acceptor in the region of the steroid side chain (5-7).

There is also evidence for the presence of multiple steroid-binding sites on GABAa-receptors, initially described by Morrow et al. (8), which limits the interpretation of the results of structure-activity studies. Moreover, because these receptors exist as hetero-oligomers with different subunits, the results of studies employing neuronal membranes from brain tissue provide only an average response from multiple forms of GABAa-receptors from the particular tissue. With these caveats in mind, it is of some interest to examine a few other diastereomeric distinctions.

Hawkinson et al. (9) demonstrated that pregnanolone (but not allopregnanolone) showed two-component allosteric modulation of radioactive flunitrazepam and tert-butylbicyclophosphorothionate (TBPS) binding to neuronal membranes from specific regions of bovine brain. Evidence for both high- and low-affinity binding sites for pregnanolone was also found in membranes from the rat brain. Their results were interpreted to mean that this 5p-reduced steroid, which is a major neuroactive steroid in humans, shows selective binding to different types of GABAa receptors. Furthermore, McCauley et al. (10) have found that 5p-pregnane-3a,20p-diol, which has limited efficacy as an allosteric modulator, is a selective ligand for the high-affinity binding site of pregnanolone. They concluded that partial agonist activity and receptor subtype selectivity are not mutually exclusive, which could explain why certain neuroactive steroids have limited efficacy.

There is accumulating evidence for the ability of other steroids to act as antagonists (or negative allosteric modulators) of GABAa receptors. Among endogenous steroids, epipregnanolone (3 p ,5 P-TH PROG) antagonized the potentiation of flunitrazepam binding produced by 3a,5p-TH PROG, 3a,5a-TH PROG, and alfaxalone (11,12). Through an extensive investigation of the synthesis and in vitro activity of 3p-substituted derivatives of allopregnanolone, Hogenkamp et al. (13) prepared the 3p-trifluoromethyl derivative Co 2-1970, shown in Fig. 1. Hawkinson et al. (14) demonstrated that Co 21970 (10 ^M) produced a concentration-dependent antagonism of GABAa receptors, which resulted in an 11-fold reduction in the apparent binding affinity of allopreg-

Fig. 1. Structures of allopregnanolone (3a,5a-TH PROG) and 3a-hydroxy-3|3-trifluoromethyl-5a-pregnan-20-one (Co 2-1970).

nanolone. In this extensive pharmacological study, the authors also demonstrated the effective partial agonist activity of Co 2-1970.

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