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


OBJECTIVE
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 [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]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.
Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, 63110, USA.
Zi-Wei
ZW
Chen
Cunde
C
Wang
Kathiresan
K
Krishnan
Brad D
BD
Manion
Randy
R
Hastings
John
J
Bracamontes
Amanda
A
Taylor
Megan M
MM
Eaton
Charles F
CF
Zorumski
Joseph H
JH
Steinbach
Gustav
G
Akk
Steven
S
Mennerick
Douglas F
DF
Covey
Alex S
AS
Evers
D018681:Anesthetics, General / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line / Q000187:drug effects; D018682:GABA Agents / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D007202:Indicators and Reagents; D007814:Larva; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D011280:Pregnanolone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects*; D012018:Reflex / Q000187:drug effects; D014982:Xenopus laevis
D018681:Anesthetics, General; D018682:GABA Agents; D007202:Indicators and Reagents; D018377:Neurotransmitter Agents; D011963:Receptors, GABA-A; D011280:Pregnanolone