Brad D Manion

Washington University in St. Louis, Saint Louis, MO, United States

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Publications (22)109.86 Total impact

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    ABSTRACT: 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. 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. 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. CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.
    Psychopharmacology 04/2014; · 4.06 Impact Factor
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    ABSTRACT: A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: 1) a ketone group at position C-16; 2) an axial 4α-OMe group and 3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents as and drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.
    Journal of Medicinal Chemistry 12/2013; · 5.61 Impact Factor
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    ABSTRACT: Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.
    Molecular pharmacology 05/2012; 82(3):408-19. · 4.53 Impact Factor
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    ABSTRACT: The enantiomer pair androsterone and ent-androsterone are positive allosteric modulators of γ-aminobutyric acid (GABA) type A receptors. Each enantiomer was shown to bind at the same receptor site. Binding orientations of the enantiomers at this site were deduced using enantiomer pairs containing OBn substituents at either C-7 or C-11. 11β-OBn-substituted steroids and 7α-OBn-substituted ent-steroids potently displace [(35)S]-tert-butylbicyclophosphorothionate, augment GABA currents, and anesthetize tadpoles. In contrast, 7β-OBn-substituted steroids and 11α-OBn-substituted ent-steroids have diminished actions. The results suggest that the binding orientations of the active analogues are inverted relative to each other with the 7α- and 11β-substituents similarly located on the edges of the molecules not in contact with the receptor surface. Analogue potentiation of the GABA current was abrogated by an α(1) subunit Q241L mutation, indicating that the active analogues act at the same sites in α(1)β(2)γ(2L) receptors previously associated with positive neurosteroid modulation.
    Journal of Medicinal Chemistry 12/2011; 55(3):1334-45. · 5.61 Impact Factor
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    ABSTRACT: GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. δ-β2 -α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn(2+) . We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors.
    British Journal of Pharmacology 09/2011; 165(7):2228-43. · 5.07 Impact Factor
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    ABSTRACT: This study addresses the hypothesis that the lack of anesthetic activity for (3α,5α)-3-hydroxypregn-16-ene-11,20-dione (Δ(16)-alphaxalone) is explained by the steroid Δ(16) double bond constraining the steroid 20-carbonyl group to a position that prevents it from favorably interacting with γ-aminobutyric acid type A (GABA(A)) receptors. A series of Δ(16) and Δ(17(20)) analogues of Δ(16)-alphaxalone was prepared to evaluate this hypothesis in binding, electrophysiological, and tadpole anesthesia experiments. The results obtained failed to support the hypothesis. Instead, the results indicate that it is the presence of the C-21 methyl group in Δ(16)-alphaxalone, not the location of the constrained C-20 carbonyl group, that prevents Δ(16)-alphaxalone from interacting strongly with the GABA(A) receptor and having anesthetic activity. Consistent with this conclusion, a Δ(17(20)) analogue of Δ(16)-alphaxalone without a C-21 methyl group was found to be very similar to the anesthetic steroid (3α,5α)-3-hydroxypregnane-11,20-dione (alphaxalone) with regard to time of onset and rate of recovery from anesthesia when administered to mice by tail vein injection.
    Journal of Medicinal Chemistry 06/2011; 54(11):3926-34. · 5.61 Impact Factor
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    ABSTRACT: Alphaxalone, a neuroactive steroid containing a 17β-acetyl group, has potent anesthetic activity in humans. This pharmacological activity is attributed to this steroid's enhancement of γ-amino butyric acid-mediated chloride currents at γ-amino butyric acid type A receptors. The conversion of alphaxalone into Δ(16)-alphaxalone produces an analogue that lacks anesthetic activity in humans and that has greatly diminished receptor actions. By contrast, the corresponding 17β-carbonitrile analogue of alphaxalone and the Δ(16)-17-carbonitrile analogue both have potent anesthetic and receptor actions. The differential effect of the Δ(16)-double bond on the actions of alphaxalone and the 17β-carbonitrile analogue is accounted for by a differential effect on the orientation of the 17-acetyl and 17-carbonitrile substituents.
    Bioorganic & medicinal chemistry letters 09/2010; 20(22):6680-4. · 2.65 Impact Factor
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    ABSTRACT: In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.
    Journal of Pharmacology and Experimental Therapeutics 05/2010; 333(2):404-13. · 3.89 Impact Factor
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    ABSTRACT: We have shown that fluorescent, 7-nitro-2,1,3-benzoxadiazol-4-yl amino (NBD)-conjugated neurosteroid analogs photopotentiate GABA(A) receptor function. These compounds seem to photosensitize a modification of receptor function, resulting in long-lived increases in responses to exogenous or synaptic GABA. Here we extend this work to examine the effectiveness of different fluorophore positions, conjugations, steroid structures, and fluorophores. Our results are generally in agreement with the idea that steroids with activity at GABA(A) receptors are the most potent photopotentiators. In particular, we find that an unnatural enantiomer of an effective photopotentiating steroid is relatively weak, excluding the idea that membrane solubility alone, which is identical for enantiomer pairs, is solely responsible for potent photopotentiation. Furthermore, there is a significant correlation between baseline GABA(A) receptor activity and photopotentiation. Curiously, both sulfated steroids, which bind a presumed external neurosteroid antagonist site, and hydroxysteroids, which bind an independent site, are effective. We also find that a rhodamine dye conjugated to a 5beta-reduced 3alpha-hydroxy steroid is a particularly potent and effective photopotentiator, with minimal baseline receptor activity up to 10 muM. Steroid conjugated fluorescein and Alexa Fluor 546 also supported photopotentiation, although the Alexa Fluor conjugate was weaker and required 10-fold higher concentration to achieve similar potentiation to the best NBD and rhodamine conjugates. Filling cells with steroid-conjugated or free fluorophores via whole-cell patch pipette did not support photopotentiation. FM1-43, another membrane-targeted, structurally unrelated fluorophore, also produced photopotentiation at micromolar concentrations. We conclude that further optimization of fluorophore and carrier could produce an effective, selective, light-sensitive GABA(A) receptor modulator.
    Molecular pharmacology 08/2009; 76(4):754-65. · 4.53 Impact Factor
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    ABSTRACT: Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).
    Journal of Medicinal Chemistry 04/2008; 51(5):1309-18. · 5.61 Impact Factor
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    ABSTRACT: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.
    British Journal of Pharmacology 03/2008; 153(3):598-608. · 5.07 Impact Factor
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    ABSTRACT: Allopregnanolone (1) and pregnanolone (2), steroids containing a 17beta-acetyl group, are potent enhancers of GABA (gamma-aminobutyric acid) action at GABAA receptors. Their effects are enantioselective with the non-naturally occurring enantiomers (ent-1 and ent-2) being less potent. Androsterone (3) and etiocholanolone (4), steroids with a C-17 carbonyl group, are weak enhancers of GABA action at GABAA receptors. Unexpectedly, their enantiomers (ent-3 and ent-4) have been found to have enhanced, not diminished, activity at GABAA receptors. Furthermore, the C-17 spiro-epoxide analogues (ent-5 and ent-6) of ent-3 and ent-4, respectively, have activities comparable to those of steroids 1 and 2. The results indicate that some ent-steroids are potent modulators of GABAA receptors and might have clinical potential as GABAergic drugs of the future.
    European Journal of Medicinal Chemistry 02/2008; 43(1):107-13. · 3.50 Impact Factor
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    ABSTRACT: Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the beta configuration at the C2 position of (3alpha,5alpha)-3-hydroxypregnan-20-one (allopregnanolone, 3alpha5alphaP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.
    Nature Neuroscience 05/2007; 10(4):523-30. · 15.25 Impact Factor
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    ABSTRACT: We have examined alpha1beta2gamma2L GABAA receptor modulation by the endogenous steroids allopregnanolone (3alpha5alphaP), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3alpha5alphaP but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3alpha5alphaP, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3alpha5alphaP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma2 subunit, which reduces the ability of 3alpha5alphaP to prolong OT3, also reduces the interaction between ethanol and 3alpha5alphaP. And the presence of the competitive steroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3alpha5alphaP on the GABAA receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3alpha5alphaP for the site. Tadpole behavioral assays showed that the presence of 3alpha5alphaP at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3alpha5alphaP.
    Molecular Pharmacology 03/2007; 71(2):461-72. · 4.41 Impact Factor
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    ABSTRACT: Benz[f]indenes are tricyclic compounds with a linear 6-6-5 fused carbocyclic ring system. When properly substituted, benz[f]indenes can satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids that modulate gamma-aminobutyric acidA (GABAA) receptor function. Thus, the benz[f]indene ring system provides an opportunity to extend the previously well-studied GABAA receptor structure-activity relationships (SAR) of neuroactive steroids to a different ring system. Depending on whether the stereochemistry of the 6-6-5 ring fusions are trans-trans or cis-trans, either planar or nonplanar benz[f]indenes are obtained. We found that the planar trans-trans benz[f]indenes are active, but less active than the steroids they were designed to mimic, whereas the nonplanar cis-trans compounds have little, if any, activity. The results provide new insight into the importance of the steroid framework for the actions of neuroactive steroids at GABAA receptors.
    Journal of Medicinal Chemistry 08/2006; 49(15):4595-605. · 5.61 Impact Factor
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    ABSTRACT: The planar 5alpha-reduced steroid (3alpha,5alpha)-3-hydroxypregnan-20-one and the nonplanar 5beta-reduced steroid (3alpha,5beta)-3-hydroxypregnan-20-one act at GABA(A) receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABA(A) receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5alpha- and 5beta-reduced series of compounds were strikingly similar. In both series, a 6beta-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6alpha-, 7beta- or 7alpha-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABA(A) receptor modulation and tadpole anesthesia. Because of the similar effects of methyl group substitution in the two series of compounds and previous results from other studies showing that structural modifications in the steroid D ring/side chain region produce similar effects regardless of the stereochemistry of the A,B-ring fusion, we propose that either the 3alpha-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABA(A) receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor.
    Journal of Medicinal Chemistry 05/2005; 48(8):3051-9. · 5.61 Impact Factor
  • Journal of Medicinal Chemistry - J MED CHEM. 01/2005; 48(8):3051-3059.
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    ABSTRACT: Although neurosteroids have rapid effects on GABA(A) receptors, study of steroid actions at GABA receptors has been hampered by a lack of pharmacological antagonists. In this study, we report the synthesis and characterization of a steroid analog, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA), that selectively antagonized neurosteroid potentiation of GABA responses. We examined 17PA using the alpha1beta2gamma2 subunit combination expressed in Xenopus laevis oocytes. 17PA had little or no effect on baseline GABA responses but antagonized both the response augmentation and the direct gating of GABA receptors by 5alpha-reduced potentiating steroids. The effect was selective for 5alpha-reduced potentiating steroids; 5beta-reduced potentiators were only weakly affected. Likewise, 17PA did not affect barbiturate and benzodiazepine potentiation. 17PA acted primarily by shifting the concentration response for steroid potentiation to the right, suggesting the possibility of a competitive component to the antagonism. 17PA also antagonized 5alpha-reduced steroid potentiation and gating in hippocampal neurons and inhibited anesthetic actions in X. laevis tadpoles. Analogous to benzodiazepine site antagonists, the development of neurosteroid antagonists may help clarify the role of GABA-potentiating neurosteroids in health and disease.
    Molecular Pharmacology 06/2004; 65(5):1191-7. · 4.41 Impact Factor
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    ABSTRACT: Neuroactive steroids modulate the function of gamma-aminobutyric acid type A (GABA(A)) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3alpha,5beta)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA(A) receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA(A) receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA(A) receptor function and anesthesia. Photolabeling of 35-kDa protein with [(3)H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA(A) receptor function as evidenced by radioligand ([(35)S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA(A) receptor function normally in VDAC-2-deficient fibroblasts transfected with alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA(A) receptor modulation or anesthesia.
    Journal of Pharmacology and Experimental Therapeutics 03/2004; 308(2):502-11. · 3.89 Impact Factor
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    ABSTRACT: The hydrogen-bond-acceptor properties of the carbonyl moiety in the 17beta-acetyl group on the D-ring of the anesthetic steroids (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregan-20-one form an important part of the anesthetic steroid pharmacophore. 13,24-Cyclo-18,21-dinorcholanes containing a ketone or conjugated ketone group at C-20, C-22, C-23, or C-24 were prepared as conformationally constrained analogues of these anesthetic steroids and were used to probe for alternate locations for the D-ring hydrogen-bond-accepting carbonyl group. The analogues were evaluated (1). in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2). in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3). as tadpole anesthetics. In the binding assay, the relative order of potencies for the analogues in the 5alpha- and 5beta-series is identical. For the ketones, the order is 24-one >or= 23-one > 20-one > 22-one. Likewise, for the enones, the order is delta(22)-24-one > delta(20(22))-23-one > delta(22)-20-one > delta(23)-22-one. Similar relative orders of potencies are also found in the other two bioassays. The activities of the 24-one and delta(22)-24-one compounds were expected to be very low, because the carbonyl group in these compounds is located over the steroid C-ring and oriented toward C-8. Instead, these compounds have the highest activities in their respective series, with the delta(22)-24-one compounds having activities comparable to those of the reference anesthetic steroids. The electrophysiology results obtained with the 24-oxo-cyclosteroids suggest that rat alpha(1)beta(2)gamma(2L) GABA(A) receptors contain more than one donor for the hydrogen-bond-acceptor group of anesthetic steroids. The family of cyclosteroids should be useful for future structure-activity relationship studies of steroid modulation of other GABA(A) receptor subtypes.
    Journal of Medicinal Chemistry 12/2003; 46(25):5334-48. · 5.61 Impact Factor

Publication Stats

201 Citations
109.86 Total Impact Points

Institutions

  • 2010–2012
    • Washington University in St. Louis
      • • Department of Anesthesiology
      • • Department of Developmental Biology
      • • Department of Medicine
      Saint Louis, MO, United States
  • 2003–2011
    • University of Washington Seattle
      • Department of Neurology
      Seattle, WA, United States