Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands

Neuroactive steroids are potent positive allosteric modulators of GABA_A receptors (GABA_AR), but the locations of their GABA_AR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABA_AR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABA_AR responses, left-shifting the GABA concentration - response curve for human α1β3γ2 GABA_ARs expressed in Xenopus oocytes, and enhancing [³H]muscimol binding to α1β3γ2 GABA_ARs expressed in HEK293 cells. With EC₅₀ of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [³H]F4N3Bzoxy-AP and [³H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABA_ARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by ³H-azietomidate and ³H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology2009, 34S1, S59-S66).