Activity of B-Nor Analogues of Neurosteroids on the GABA_A Receptor in Primary Neuronal Cultures

Abstract

A GABA(A) receptor study of several B-nor analogues of allopregnanolone and pregnanolone has been carried out. B-norallopregnanolone (i.e., 3alpha-hydroxy-7-nor-5alpha-pregnan-20-one) was found comparable to allopregnanolone when measured with labeled TBPS. Analogous results were obtained from their effect on neurons in culture: this time, both 3alpha-hydroxy-7-nor-5xi-pregnan-20-ones (5 and 6) were found to stimulate [3H]flunitrazepam binding and GABA-induced 36Cl- influx. These effects were inhibited by GABA(A) receptor antagonists. Other analogues carrying electronegative substituents (epoxides 9 and 10 and ketone 12) in the B ring were inactive. Similarly, B-normal ketones 17, and 18 and 6-azasteroids 20 and 21 were also inactive. B-Nor analogues 5 and 6 did not induce neurotoxicity at relevant concentrations. A computational analysis of active and inactive neurosteroid analogues allowed the proposal of a 3D pharmacophoric hypothesis of their interaction with the GABA(A) receptor.

GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor

“…Chloride influx was determined as 36 Cl -uptake in intact cell cultures (Vale et al, 2003;García et al, 2006 …”

“…In particular, this compound specifically interacts with synaptic neural functions on neuronal Na + and GABA-operated Cl -channels (Mohammadi et al, 2001;Haeseler et al, 2002;Priestley et al, 2003;García et al, 2006) as well as with nociception-sensitive receptor channels (Lee et al, 2008). Its capacity to increase [ 3 H]flunitrazepam binding to its specific site in the GABA A receptor by using synaptosomal membranes or cultured cortical neurons has been described (Sánchez et al, 2004;García et al, 2006). We also demonstrated the ability of thymol to both enhance GABA-induced chloride influx and open the chloride channel in the GABA A receptor in the absence of GABA.…”

Allopregnanolone and Pregnanolone Analogues Modified in the C Ring: Synthesis and Activity

“…Previously, 11 Table 1). The binding was measured in the presence of the varying concentration of these compounds.…”

“…These analogues either involve compounds with additional substituents (eg., alphaxalone, 3), or have their skeleton modified by increasing 9 or decreasing [10][11][12] the flexibility of the molecule or even reversing its chirality. 13 Various changes were also made to the steroid side chain.…”

“…In our previous paper we reported structure modification of the steroidal B ring. 11 Some anomalies in the structure of the analogues were tolerated by the GABA A receptor, others not; the biological consequences of the structure modification were rationalized using methods of molecular modelling. A similar study of the effect of C ring substituents upon the activity of neurosteroid analogues is the subject of this paper, which also takes into account earlier results published by other authors.…”