11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors

Rationale

While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABA_A) 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 GABA_A 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 GABA_A receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α₁β₂γ_2L 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 GABA_A 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 GABA_A receptor. Photolabeling of rat brain GABA_A 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 GABA_A receptors.