An advantage of photolabeling with

An advantage of photolabeling with tritiated photoprobes followed by microsequencing with Edman degradation is the ability to both identify the photolabeled salubrinal and to quantify photoincorporation which enables assessment of pharmacological specificity and allosteric interactions with other ligands. For example, tritium-labeled photoreactive analogs of etomidate, mephobarbital, and propofol have identified multiple binding sites in purified α1β3 and α1β3γ2L GABAARs (Fig. 1) [[18], [19], [20]], and were useful in photolabeling protection studies that compared other drug's relative affinity for these sites. Multiple steroid photolabels 5–11, incorporating photoreactive groups at various positions on the steroid backbone, have been tested in GABAARs [17,21]. However, to date no tritiated steroid photolabels have proven useful in such protection studies at steroid binding sites. Previously, we developed tritiated allopregnanolone analogs 12 and 13 containing photoreactive substituents at the 11-position, which were potent anesthetics and GABAAR modulators, but their photoincorporation in GABAARs was not inhibited by other steroid modulators [22]. Our goal in this study was to develop additional anesthetic steroid photolabels suitable for photolabeling the modulatory binding sites in typical heteropentameric receptors. We hypothesized that the upper edge of the steroid framework, where our C-11 photolabels were located, projected out of the binding site, perhaps facing towards the lipids [22]. Consequently, in this work, we placed the photoreactive residues on the opposite edge of the steroid (C-6), where previous studies on the β3 homomer had been successful [17,21], and on the steroid side-chain (C1-7 of the framework). Here, we report the synthesis of three neurosteroid analogs equipped with diazirines located at C-6 or C-21 (14–16) that act as positive allosteric modulators of heteropentameric α1β3 and α1β3γ2L receptors. We found that tritiated compound 16 (21-pTFDBzox-AP) photoincorporated with pharmacological specificity into full-length human α1β3 and α1β3γ2L GABAARs. In addition, photolabeling was inhibited by other neurosteroid enhancers at concentrations close to their modulatory EC50 values, but not by the inhibitory neurosteroid pregnenolone sulfate, nor by etomidate, or a potent barbiturate anesthetic.
Results
Discussion In our previous work we have described two allopregnanolone derivatives, 11-F4N3Bzox-AP (12) and 11-azi-AP (13), that displayed potent positive modulatory properties and efficiently photolabeled GABAARs [22]. However, the photolabeling by these reagents was not inhibited by other neurosteroids, and hence was not site-specific. The most important result of this report is the successful design of a photoreactive neurosteroid analog 21-pTFDBzox-AP (16) that meets the following criteria: (i) It is a potent, albeit low-efficacy, allosteric modulator of human heteromeric GABAA receptors. (ii) It photolabels GABAARs in a pharmacologically specific manner, as evidenced by the observed inhibition of photolabeling at the subunit level by other positive steroid modulators, but not by steroid antagonists nor by ligands such as etomidate and R-mTFD-MPAB, which act at separate sites. (iii) Its photoincorporation is not strongly dependent on the presence or absence of GABA, which is consistent with the low efficacy with which it positively modulates currents induced by low concentrations of GABA. (iv) Lastly, the modulation of GABA currents by 21-pTFDBzox-AP is completely eliminated by the α1Q242W mutation suggesting that this ligand binds in the same site as that of other neurosteroids [4,28,30]. We compared the potency of common steroids to inhibit the photoincorporation of [3H]21-pTFDBzox-AP into its site (IC50) with their potency for enhancing the binding of the agonist [3H]muscimol (EC50), a measure of desensitization (Table 1). These experiments were carried out with heteromeric, human full-length, glycosylated GABAARs with subunit composition α1β3 and α1β3γ2L. In neither case, did the presence of the γ2-subunit exert a major influence on the results, so we pursued more detailed studies with α1β3 receptors. For 3α,5α-steroids: THDOC (3), alphaxalone (4), 6-Azi-OAP (6) and 21-pTFDBzox-AP itself (16), 3α,5β-steroid (pregnanolone, 1) and 6-azi-OP (5), the agreement between IC50 and EC50 was very good. Furthermore, pregnenolone sulfate, a 3β-steroid that inhibits GABA currents [31], neither bound to the 21-pTFDBzox-AP site nor enhanced desensitization. Considering that the binding studies were carried out in much more dilute suspensions than the photolabeling studies, we regard the pharmacological evidence establishing that photoincorporation was occurring in the site that was responsible for enhancing desensitization to be strong.