Abstract
Rationale
While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified.
Objectives
This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors.
Results
Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C–H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [35S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [3H]CW12 and [3H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific.
Conclusions
CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.
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Acknowledgments
This work was supported by grant PO1-GM47969 from the National Institute of General Medical Sciences to A.S.E, D.F.C., J.H.S and C.F.Z and by grant MH099658 to S.M. from the National Institute of Mental Health. Washington University receives income and equity based on a license of related technology to Sage Therapeutics. D.F.C., C.F.Z. and A.S.E. have equity holdings in Sage Therapeutics, Inc. Sage Therapeutics, Inc. did not support this research or have any other role in the research or its conclusions.
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Chen, ZW., Wang, C., Krishnan, K. et al. 11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors. Psychopharmacology 231, 3479–3491 (2014). https://doi.org/10.1007/s00213-014-3568-4
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DOI: https://doi.org/10.1007/s00213-014-3568-4