Neurosteroid Analogues. 18. Structure–Activity Studies of ent-Steroid Potentiators of γ-Aminobutyric Acid Type A Receptors and Comparison of Their Activities with Those of Alphaxalone and Allopregnanolone

Abstract

A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: 1) a ketone group at position C-16; 2) an axial 4α-OMe group and 3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents as and drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.

Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABAA Receptors

“…Enantiomeric steroids (e18,19dinor3a5a16O, e18nor3a5a16O, e3a5b17O, e3a5a4bOMe16O, e19nor3a5a16O, and e3a5a16) are inverted relative to natural steroids when bound to GABA A receptors. For models of the modes of binding natural steroids and their enantiomers, see (Krishnan et al, 2012;Qian et al, 2014).…”

“…Inorganic salts used in the buffers CNQX, and DL-APV were bought from Sigma-Aldrich (St. Louis, MO). Steroids were bought from Sigma-Aldrich or Steraloids (Newport, RI), or synthesized locally as described previously elsewhere (Hu et al, 1993;Scaglione et al, 2008;Li et al, 2009;Qian et al, 2014). Structures of steroids used are given in Fig.…”

“…Analysis of single-channel currents and simulation of synaptic events was conducted using the QuB Suite (www.qub.buffalo.edu). Previous single-channel data (Li et al, 2009;Qian et al, 2014) were first reanalyzed using Model 1 (Lema and Auerbach, 2006): This model predicts three open states, differing in their mean duration, associated with fully liganded receptors. The model and the fitted rate constants were then used to simulate synaptic events.…”

“…In the presence of 10 mM 3a5b17O or (3b,5b,8a,9b,10a,13a,14b)-3-hydroxygonan-16-one [e18,19dinor3a5a16O], whose sole effect in singlechannel recordings is to increase the relative frequency of long openings (Li et al, 2007a;Qian et al, 2014), the t w was 94 6 2 milliseconds (5 cells) or 53 6 10 milliseconds (four cells), respectively.…”

“…, and (3a,5a,17b)-18-nor-3-hydroxyandrostane-17-carbonitrile [18nor3a5a17bCN], that were known to increase both the duration and prevalence of long openings, but not affect intracluster closed times (Li et al, 2007a(Li et al, , 2009Scaglione et al, 2008;Qian et al, 2014). Exposure to these compounds ranged from no effect on the decay time constant in the presence of e18nor3a5a16O (t w 5 36 6 2 milliseconds, four cells) to a more than 4-fold prolongation in the presence of e3a5b17O (t w 5 143 6 15 milliseconds, five cells).…”