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Title: Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands

Abstract

Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 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 GABAAR responses, left-shifting the GABA concentration – response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs 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 GABAARs, 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 frommore » 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. (Psychoneuroendocrinology 2009, 34S1, S59-S66).« less

Authors:
; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1373772
Resource Type:
Journal Article
Resource Relation:
Journal Name: European Journal of Medicinal Chemistry; Journal Volume: 136; Journal Issue: C
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 60 APPLIED LIFE SCIENCES

Citation Formats

Savechenkov, Pavel Y., Chiara, David C., Desai, Rooma, Stern, Alexander T., Zhou, Xiaojuan, Ziemba, Alexis M., Szabo, Andrea L., Zhang, Yinghui, Cohen, Jonathan B., Forman, Stuart A., Miller, Keith W., and Bruzik, Karol S. Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands. United States: N. p., 2017. Web. doi:10.1016/j.ejmech.2017.04.043.
Savechenkov, Pavel Y., Chiara, David C., Desai, Rooma, Stern, Alexander T., Zhou, Xiaojuan, Ziemba, Alexis M., Szabo, Andrea L., Zhang, Yinghui, Cohen, Jonathan B., Forman, Stuart A., Miller, Keith W., & Bruzik, Karol S. Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands. United States. doi:10.1016/j.ejmech.2017.04.043.
Savechenkov, Pavel Y., Chiara, David C., Desai, Rooma, Stern, Alexander T., Zhou, Xiaojuan, Ziemba, Alexis M., Szabo, Andrea L., Zhang, Yinghui, Cohen, Jonathan B., Forman, Stuart A., Miller, Keith W., and Bruzik, Karol S. Tue . "Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands". United States. doi:10.1016/j.ejmech.2017.04.043.
@article{osti_1373772,
title = {Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands},
author = {Savechenkov, Pavel Y. and Chiara, David C. and Desai, Rooma and Stern, Alexander T. and Zhou, Xiaojuan and Ziemba, Alexis M. and Szabo, Andrea L. and Zhang, Yinghui and Cohen, Jonathan B. and Forman, Stuart A. and Miller, Keith W. and Bruzik, Karol S.},
abstractNote = {Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 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 GABAAR responses, left-shifting the GABA concentration – response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs 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 GABAARs, 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. (Psychoneuroendocrinology 2009, 34S1, S59-S66).},
doi = {10.1016/j.ejmech.2017.04.043},
journal = {European Journal of Medicinal Chemistry},
number = C,
volume = 136,
place = {United States},
year = {Tue Aug 01 00:00:00 EDT 2017},
month = {Tue Aug 01 00:00:00 EDT 2017}
}