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Title: Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands

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Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
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Journal Article: Publisher's Accepted Manuscript
Journal Name:
European Journal of Medicinal Chemistry
Additional Journal Information:
Journal Volume: 136; Journal Issue: C; Related Information: CHORUS Timestamp: 2017-10-04 16:26:48; Journal ID: ISSN 0223-5234
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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. France: 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. France. 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. 2017. "Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands". France. doi:10.1016/j.ejmech.2017.04.043.
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 = {},
doi = {10.1016/j.ejmech.2017.04.043},
journal = {European Journal of Medicinal Chemistry},
number = C,
volume = 136,
place = {France},
year = 2017,
month = 8

Journal Article:
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  • 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 anestheticsmore » 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).« less
  • A new heterobifunctional reagent, N-(3-(p-azido-m-iodophenyl)propionyl)-succinimide (AIPPS), was synthesized and chemically characterized. The radiochemical form of the reagent, (/sup 125/I)AIPPS, should be of general use as a photoactive reagent for the derivatization of free amino groups on a large variety of biologically active compounds, including many hormones. Amino-containing ligands can be derivatized with (/sup 125/I)AIPPS in a method which is similar to that used for the /sup 125/I-labeled Bolton-Hunter reagent (N-(3-(p-hydroxyphenyl)propionyl)-succinimide). The added advantage with (/sup 125/I)AIPPS, however, is that the ligand derivative is made both photoactive and radioactive in a single step. As an example of how this reagent canmore » be used, we have prepared carrier-free (/sup 125/I)AIPPS and reacted it with the amino-containing cardiac glycoside, 4-amino-4,6-dideoxyglucosyl digitoxigenin (GluD). The radioiodinated cardiac glycoside, (/sup 125/I)AIPP-GluD, was purified by thin-layer chromatography and was carrier-free with a specific radioactivity of 2175 Ci/mmol. (/sup 125/I)AIPP-GluD was an effective photoaffinity label for Na,K-ATPase as shown by specific photoaffinity labeling of purified canine kidney enzyme and human erythrocyte enzyme.« less
  • Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling wasmore » observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.« less
  • The aims of this study were to investigate whether several histamine receptor agonists and antagonists are subjected to receptor-independent ion trapping into acidic organelles, and whether this sequestration influences their pharmacological or toxicological properties. Vacuolar (V)-ATPase-dependent intracellular sequestration of agonists was recognized as morphological alterations (large fluid-filled vacuoles for betahistine and 1-methylhistamine, granular uptake for fluorescent BODIPY FL histamine) prevented by the specific V-ATPase inhibitor bafilomycin A1 in rabbit vascular smooth muscle cells. Lipophilicity was the major determinant of these cellular effects (order of potency: BODIPY FL histamine > betahistine > 1-methylhistamine > histamine) that occurred at high concentrations. Thismore » ranking was dissociable from the potency order for H{sub 1} receptor-mediated contraction of the rabbit aorta, a response uninfluenced by bafilomycin. Antihistamines are inherently more lipophilic and caused vacuolization of a proportion of cells at 5-500 {mu}M. Agonist or antagonist-induced vacuoles were of macroautophagic nature (labeled with GFP-conjugated LC3, Rab7 and CD63; detection of LC3 II). Further, the 2 most lipophilic antihistamines tested, astemizole and terfenadine, were potentiated by V-ATPase blockade in the aortic contractility assay (13- and 3.6-fold more potent, respectively, pA{sub 2} scale), suggesting that V-ATPase-mediated cation trapping sequesters these antagonists from the vicinity of H{sub 1} receptors in the therapeutic concentration range. This potentiation did not apply to less lipophilic antagonists (pyrilamine, diphenhydramine). While some agonists and all tested antagonists of the histamine H{sub 1} receptors induce the V-ATPase-dependent vacuolar and autophagic cytopathology, sequestration affects the pharmacology of only the most lipophilic antagonists, the ones prone to off-target arrhythmogenic side effects.« less
  • Square-planar palladium complexes containing tridentate ligands with PXP (X = C, N, O, S, and As) donor sets have been prepared. For [Pd(POP-R)(CH[sub 3]CN)[sub 2]](BF[sub 4])[sub 2] complexes (where POP-R is bis((diethylphosphino)ethyl) ether, bis((diphenylphosphino)ethyl) ether, or bis((dicyclohexylphosphino)ethyl) ether), the POP-R ligands function as bidentate ligands. For all other complexes, the PXP ligands function as tridentate ligands. The complex [Pd(PCP)(PEt[sub 3])](BF[sub 4]) (where PCP is 2,6-bis(diphenylphosphino)methyl)phenyl crystallizes in the triclinic space group P[bar 1] with a = 10.276(2) angstroms, b = 10.978(2) angstroms, c = 18.807(3) angstroms, [alpha] = 84.820(10)[degrees], [beta] = 76.910(10)[degrees], [gamma] = 63.870(10)[degrees], V = 1855.2(6) angstroms[sup 3],more » and Z = 2. The structure was refined to R = 0.0335 and R[sub w] = 0.0442 for 7570 independent reflections with F > 4.0[sigma](F). [Pd(PNP)(PEt[sub 3])](BF[sub 4])[sub 2] (where PNP is 2,6-bis(disphenylphosphino)methyl)pyridine crystallizes in the monoclinic space group C2/c with a = 13.246(2) angstroms, b = 14.299(2) angstroms, c = 20.847(4) angstroms, [beta] = 100.96(2)[degrees], V = 3876.5(11)angstroms[sup 3], and Z = 4. 40 refs., 6 figs., 4 tabs.« less