0022-3565/00/2933-1009$03.

00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 2000 by The American Society for Pharmacology and Experimental Therapeutics JPET 293:10091116, 2000 /2306/824792

Vol. 293, No. 3 Printed in U.S.A.

Enantioselectivity of Pregnanolone-Induced -Aminobutyric AcidA Receptor Modulation and Anesthesia1

DOUGLAS F. COVEY, DEVI NATHAN, MELISSA KALKBRENNER, KENT R. NILSSON, YUEFEI HU, CHARLES F. ZORUMSKI, and ALEX S. EVERS
Departments of Molecular Biology and Pharmacology (D.F.C., Y.H., K.R.N., A.S.E.), Anesthesiology (D.N., M.K., A.S.E.), and Psychiatry (C.F.Z.), Washington University School of Medicine, St. Louis, Missouri Accepted for publication February 22, 2000 This paper is available online at http://www.jpet.org

ABSTRACT This study reports the actions of enantiomer pairs of anesthetic steroids 3 5 P/ent-3 5 P and 3 5 P/ent-3 5 P as modulators of -aminobutyric acid (GABA)A receptors and as anesthetics. The enantiomers of structurally related 17-carbonitrile analogs also are examined. These studies were aimed at 1) determining whether the steroid recognition site could distinguish between molecules differing in shape, but not other physical properties (enantioselectivity); 2) providing further insight into the structure-activity relationships of anesthetic steroids; and 3) determining whether modulation of GABAA receptor function correlates with anesthetic potency for anesthetic steroid enantiomers. Stereoselective actions of the compounds were evaluated in four different bioassays: 1) noncompetitive displacement of [35S]t-butylbicyclophosphorothionate from the picrotoxin site of GABAA receptors present in rat brain mem-

brane preparations; 2) modulation of GABA currents in cultured rat hippocampal neurons; 3) loss of righting reflex in tadpoles; and 4) loss of righting reflex in mice. The data indicate that 5 -reduced steroids, but not 5 -reduced steroids, show a high degree of enantioselectivity/enantiospecificity in their actions as modulators of GABAA receptors and as anesthetics. For all compounds studied, the effects on GABAA receptor function closely tracked with anesthetic effects. These data show that the anesthetic steroid recognition site is capable of distinguishing enantiomers, suggesting a protein-binding site of specific dimensions and shape. The results are compatible either with a structural model of the binding site that can accommodate 3 5 P, 3 5 P, and ent-3 5 P, but not ent-3 5 P, or with two different binding sites for steroid anesthetics.

The steroids 3 5 P (allopregnan-3 -ol-20-one) and 3 5 P (pregnanolone) are potent anesthetics (Phillipps, 1974). A large amount of data supports the hypothesis that the anesthetic actions of these steroids are correlated with their enhancement of -aminobutyric acid (GABA)A receptor-mediated neuronal inhibition (Lambert et al., 1995). The number of binding sites for these steroids and their location on GABAA receptors are not known. Moreover, whether the steroids share a common site is not known. Molecular-modeling studies suggest that the anesthetic activities of these steroids result from binding at a common binding site (Purdy et al., 1990; Han et al., 1996). However, binding studies for steroid-induced noncompetitive displacement of t-butylbicyclophosphorothionate (TBPS) from the picrotoxin-binding
Received for publication November 17, 1999. 1 This study was supported by U.S. Public Health Service Grant GM47969, Research Scientist Development Award MH00964, and the Bantly Foundation.

site on GABAA receptors indicate that, although these steroids may share a common binding site, multiple binding sites are detectable for some steroids (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998). Because 3 5 P and 3 5 P are molecules that each contain eight chiral centers (C-3, C-5, C-8, C-9, C-10, C-13, C-14, and C-17), their stereoselective modulation of GABAA receptor function can be studied by inverting each, some, or all of the chiral centers in the molecules. Inversion of one center in a molecule containing multiple chiral centers gives compounds called epimers. Epimers are a subset of compounds called diastereomers. Diastereomers are compounds with multiple chiral centers that differ in configuration at one or some, but not all, chiral centers. Studies of the C-3 (inversion of the 3-hydroxyl group) and C-17 (inversion of the acetyl group) epimers of 3 5 P and 3 5 P have been very informative in establishing the structure-activity relationships for anesthetic steroids (Phillipps, 1974). No doubt, studies of addi-

ABBREVIATIONS: 3 5 P, (3 ,5 )-3-hydroxypregnan-20-one; 3 5 P, (3 ,5 )-3-hydroxypregnan-20-one; GABA, -aminobutyric acid; TBPS, t-butylbicyclophosphorothionate; ent-3 5 P, (3 ,5 ,8 ,9 ,10 ,13 ,14 ,17 )-3-hydroxypregnan-20-one; ent-3 5 P, (3 ,5 ,8 ,9 ,10 ,13 , 14 ,17 )-3-hydroxypregnan-20-one; 3 5 ACN, (3 ,5 ,17 )-3-hydroxyandrostane-17-carbonitrile; 3 5 ACN, (3 ,5 ,17 )-3-hydroxyandrostane17-carbonitrile; ent-3 5 ACN, (3 ,5 ,8 ,9 ,10 ,13 ,14 ,17 )-3-hydroxyandrostane-17-carbonitrile; ent-3 5 ACN, (3 ,5 ,8 ,9 , 10 ,13 ,14 ,17 )-3-hydroxyandrostane-17-carbonitrile; DMSO, dimethylsulfoxide; LRR, loss of righting reflex. 1009

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tional diastereomers of 3 5 P and 3 5 P will further define these structure-activity relationships. However, because diastereomers have different physical properties due to the relative positions of some atoms in each diastereomer being different, the membrane-perturbing effects as well as the direct effects of these steroids on GABAA receptor function will be different. Thus, C-3 epimers (3 -OH) of 3 5 P and 3 5 P not only fail to potentiate GABA effects at GABAA receptors but also interact differently with membrane phospholipids (Makriyannis et al., 1991). To avoid the membrane-perturbing effects caused by studying anesthetic steroid diastereomers having different physical properties, we have studied anesthetic steroid enantiomers (Fig. 1). Enantiomers are the stereoisomers of optically active compounds that are mirror images of each other (all chiral centers have the opposite absolute configuration). Enantiomers have identical physical properties because the relative positions of all atoms in each enantiomer are identical. For example, any group having an axial configuration in one enantiomer also has an axial configuration in the other enantiomer. Enantioselectivity of 3 5 P-induced GABAA receptor modulation and anesthesia in Xenopus laevis tadpoles and mice has been reported previously (Wittmer et al., 1996). Herein, we report the corresponding actions of the 3 5 P enantiomers. Additionally, new information for the displacement of TBPS binding by both pairs of enantiomers is reported. Some comparative data for the corresponding enantiomers of the 17-carbonitrile analogs of 3 5 P and 3 5 P also are presented. The new data show that enantioselectivity [comparison of 3 5 P/ent-3 5 P with 3 5 P/ent-3 5 P) and diastereoselectivity (comparison of 3 5 P/3 5 P with ent-3 5 P/ ent-3 5 P) for modulation of GABAA receptor function by these anesthetic steroids are different. The enantioselective actions of 3 5 P are significantly greater than the enantioselective actions of 3 5 P. The diastereoselective actions of the 3 5 P/3 5 P steroid pair are not significant and those of the ent-3 5 P/ent-3 5 P pair are significantly different.

Materials and Methods

Chemicals. The ent-3 5 P, ent-3 5 P, 3 5 ACN [(3 ,5 ,17 )-3hydroxyandrostane-17-carbonitrile], ent-3 5 ACN, and 3 5 ACN [(3 ,5 ,17 )-3-hydroxyandrostane-17-carbonitrile] were prepared and characterized as described previously (Hu et al., 1993, 1997; Han et al., 1996; Nilsson et al., 1998). The ent-3 5 ACN was prepared from ent-(3 ,5 )-3-hydroxyandrostan-17-one with the methods described for the preparation of 3 5 ACN (Han et al., 1996). The infrared, 1H NMR, and 13C NMR spectra of the 3 5 ACN and ent-3 5 ACN were identical. The 3 5 P and 3 5 P were purchased from either Sigma Chemical Co. (St. Louis, MO) or Steraloids (Newport, RI). The [35S]TBPS was purchased from NEN Life Science Products (Boston, MA) and TBPS was purchased from Research Biochemicals International (Natick, MA). [35S]TBPS Binding. Rat brain cortical membranes were prepared with minor modifications of the method previously reported (Hawkinson et al., 1994a). Briefly, frozen rat cerebral cortices (Pelfreez, Rogers, AK) were thawed and homogenized in 10 volumes of ice-cold 0.32 M sucrose with a glass/Teflon pestle. The homogenate was centrifuged at 1500g for 10 min at 4C. The resultant supernatant was centrifuged at 10,000g for 30 min at 4C. The pellet (P2) from this centrifugation was resuspended in 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4, and centrifuged at 10,000g for 20 min at 4C. This washing procedure was done three times, and then pellets were resuspended in buffer ( 4 ml/brain) with a glass/ Teflon pestle. The membrane suspension was aliquoted, frozen in liquid nitrogen, and stored at 80C before use. [35S]TBPS binding assays were done according to the procedure described previously (Hawkinson et al., 1994a) with modifications. Briefly, aliquots of membrane solution (0.5 mg/ml final protein concentration in assay) were incubated with 5 M GABA, 2 nM [35S]TBPS (45120 Ci/ mmol), and 5 l-aliquots of steroid in dimethyl sulfoxide (DMSO) solution (final assay concentrations ranged from 1 nM to 10 M), and brought to a final volume of 1 ml with 200 mM NaCl, 50 mM potassium phosphate buffer, pH 7.4. Control binding was defined as binding observed in the presence of 0.5% DMSO and the absence of steroid. Nonspecific binding was defined as binding observed in the presence of 200 M picrotoxin and ranged from 6.1 to 14.3% of total binding. Assay tubes were incubated for 2 h at room temperature. A Brandel (Gaithersburg, MD) cell harvester was used for filtration of the assay tubes through Whatman/GF/C glass filter paper. Filter paper was rinsed with 4 ml of ice-cold buffer three times. Radioactivity bound to the filters was read by liquid scintillation counter and data were fit with Sigma Plot version 3.0 to the Hill equation

R max/ 1

conc /IC50

Fig. 1. Structures of the four pairs of anesthetic steroid enantiomers studied.

where Rmax is the maximal effect, [conc] is steroid concentration, IC50 is the half-maximal inhibitor concentration, and n is the Hill coefficient. Each data point was determined in triplicate and two or three full concentration-response curves were generated for each steroid. Electrophysiological Recording. Hippocampal neurons were cultured from 1- to 2-day-old Sprague-Dawley albino rats with methods described previously (Thio et al., 1991). After 4 to 7 days in culture, neurons were voltage clamped at 60 mV with whole-cell patch-clamp recording techniques. The extracellular recording solution contained 140 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.3. Recording pipettes were filled with a solution containing 140 mM CsCl, 4 mM NaCl, 0.5 mM CaCl2, 4 mM MgCl2, and 10 mM HEPES, pH 7.3. GABA and steroids were applied for 500 ms with a pressure (20 psi of air) ejection drug delivery system with patch pipettes positioned 5 m from the recorded neuron. This system allowed reliable drug application while minimizing exposure of neurons to steroids. The concentrations reported are those in the pipette and are 2- to 3-fold higher than the concentrations bathing the cell. Steroids were prepared in a DMSO

2000 stock at 10 to 100 mM and diluted so that the final concentration of DMSO was 0.5%. DMSO at this concentration had no effect on GABA responses. Concentration-response data were fit to an equation of the form

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R max

conc n / conc

EC50 n

where Rmax is the maximum effect, [conc] is the steroid concentration, EC50 is the half-maximal effective concentration, and n is the Hill coefficient. Tadpole Loss of Righting Reflex (LRR) Anesthesia Assay. Tadpole LRR was measured as described previously (Wittmer et al., 1996). Briefly, groups of 10 early prelimb-bud stage Xenopus laevis tadpoles (Nasco, Fort Atkinson, WI) were placed in 100 ml of oxygenated Ringers stock solution containing various concentrations of compound. Compounds were added from a 10 mM DMSO stock (final concentration of DMSO in test solutions was 0.1%). After equilibrating at room temperature for 3 h, tadpoles were evaluated with the LRR behavioral endpoint. LRR was defined as failure of the tadpole to right itself within 5 s after being flipped by a smooth glass rod. In all cases, the tadpoles regained their righting reflex when placed in fresh oxygenated Ringers solution. Control beakers containing up to 0.6% DMSO produced no LRR in tadpoles. Concentration-response curves were fit with Sigma Plot version 3.0 to the Hill equation

R max/ 1

conc /EC50

where Rmax is the maximum effect, [conc] is the steroid concentration, EC50 is the half-maximal effective concentration, and n is the Hill coefficient. Mouse Anesthesia Assay. BALB/c mice (Sprague-Dawley; Harlan Breeders, Indianapolis, IN) weighing 20 to 30 g each were placed under a heat lamp for 1 to 2 min. Compounds were injected i.v. through a tail vein with various doses of either 3 5 P or ent-3 5 P in an 8% ethanol, 16% Cremaphor EL (Sigma Chemical Co.) solution at a rate of 50 to 250 l/5 to 10 s. Sleep time was measured from the moment mice displayed LRR until they were able to right themselves. All mice recovered fully without observable neurological deficits. Control solutions without steroids were administered with no observable neurobehavioral effect.

Results
[ S]TBPS Binding. Anesthetic steroids are known to be noncompetitive displacers of [35S]TBPS from the picrotoxinbinding site of GABAA receptors (Majewska et al., 1986). In this study, the enantioselectivity for [35S]TBPS displacement by four pairs of anesthetic steroid enantiomers was examined (Fig. 2). For the two 5 -reduced steroid enantiomer pairs, 3 5 P/ent-3 5 P and 3 5 ACN/ent-3 5 ACN, the natural enantiomers were each 4-fold more potent displacers of [35S]TBPS. For the two 5 -reduced steroid enantiomer pairs, 3 5 P/ent-3 5 P and 3 5 ACN/ent-3 5 ACN, the natural enantiomers were the more potent displacers by factors of 26 and 80, respectively. Thus, enantioselectivity for [35S]TBPS displacement was found for all enantiomer pairs, but the degree of enantioselectivity was much higher for the 5 reduced steroids. Steroid Effects on GABAA Currents. The enantioselectivity found for modulation of GABAA receptor function in cultured rat hippocampal neurons by the enantiomer pairs is shown in Figs. 3 and 4. At a steroid concentration of 10 M, there was no enantioselectivity for potentiation of 2 M GABA-mediated currents by the 3 5 P/ent-3 5 P pair (Fig. 3). In contrast, an 4-fold enantioselectivity for the degree of potentiation under the same experimental conditions was found for the 3 5 P/ent-3 5 P pair (Fig. 3). This last result
35

is in close agreement with results previously reported from a more extensive electrophysiological evaluation of GABAA receptor modulation in these cells by the 3 5 P/ent-3 5 P pair (Wittmer et al., 1996). A concentration-response curve for potentiation of GABAmediated currents by the 3 5 P/ent-3 5 P pair is shown in Fig. 4A. The 3 5 P enantiomer was 3-fold more potent than the ent-3 5 P enantiomer (EC50 1.2 and 3.6 M, respectively). In Fig. 4B, the concentration-response curve for gating of GABAA receptors in the absence of added GABA is shown. The ent-3 5 P gates a current at the same concentration as 3 5 P and does so to a very similar extent. This contrasts with the results previously obtained with ent3 5 P and ent-3 5 ACN, neither of which gates GABAA receptors at a concentration of up to 100 M (Wittmer et al., 1996). Tadpole LRR. The concentration-response relationships for the loss of tadpole LRR were determined for the 3 5 ACN/ent-3 5 ACN and 3 5 P/ent-3 5 P pairs. Little, if any, enantioselectivity was observed for these enantiomer pairs (Fig. 5). The results contrast with the significant degrees of enantioselectivity found previously (Wittmer et al., 1996) for tadpole LRR with the 3 5 ACN/ent-3 5 ACN and 3 5 P/ent-3 5 P pairs (10- and 2.8-fold, respectively). Mouse LRR. When injected into mice, both 3 5 P and ent-3 5 P produced anesthesia (as indicated by LRR) in a dose-dependent manner (Fig. 6). The slopes of the lines from a regression analysis of the dose-response data for this enantiomer pair differ by a factor of 2. The 3 5 ACN/ent3 5 ACN pair was not tested for anesthetic activity in mice because adequate amounts of ent-3 5 ACN were not available. These 3 5 P/ent-3 5 P results for mouse anesthesia are different from those obtained previously for the 3 5 ACN/ent-3 5 ACN pair in this bioassay (Wittmer et al., 1996). In that study, the lowest dose of 3 5 ACN shown to cause LRR was 4 mg/kg; whereas ent-3 5 ACN did not cause LRR at a dose as high as 80 mg/kg.

Discussion
The results from this and our previous enantioselectivity study (Wittmer et al., 1996) provide new information about the enantioselective and diastereoselective interactions of anesthetic steroids with GABAA receptors. Enantioselective interactions can be examined by comparing the results obtained with the 3 5 /ent-3 5 and 3 5 /ent-3 5 steroid pairs. Diastereoselective interactions can be examined by comparing the results obtained for the 3 5 /3 5 and ent3 5 /ent-3 5 steroid pairs because only the chiral center at C-5 is different in each of these steroid pairs. As summarized in Table 1, all pairs of anesthetic steroid enantiomers studied in this and our previous study (Wittmer et al., 1996) exhibit some degree of enantioselectivity in electrophysiology, [35S]TBPS binding, and when performed, the mouse anesthesia tests. Significant enantioselectivity was not found in the tadpole LRR experiments for the 5 -reduced enantiomer pairs. Notably, the degree of enantioselectivity is uniformly greater across the various assays for the steroids in the 5 -reduced series. The consistency of these results is remarkable considering that different species and preparations were used in the bioassays. Of course, differences in species and preparation also limit interpretation of the re-

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Fig. 2. Inhibition of [35S]TBPS binding to rat brain cortical membranes by steroid enantiomers. The enantiomers of 3 5 ACN and 3 5 P show a larger degree of enantioselectivity than do the enantiomers of 3 5 ACN and 3 5 P. The figure shows a representative concentration-response curve for each steroid. The IC50 S.E. values reported are from the fit of data points from two or three experiments. A, the 3 5 ACN (F) and ent-3 5 ACN (E) show an 4-fold difference in IC50 (72 8 versus 292 21 nM) for inhibition of [35S]TBPS binding. B, the 3 5 P (F) and ent-3 5 P (E) enantiomer pair also show an 4-fold difference in IC50 (71 18 versus 276 34 nM) for inhibition of [35S]TBPS binding. C, an 80-fold difference in IC50 (50 5 versus 4020 310 nM) for inhibition of [35S]TBPS binding is demonstrated between 3 5 ACN (F) and ent-3 5 ACN (E). D, an 26-fold difference in IC50 (74 7 versus 1910 270 nM) is seen between 3 5 P (F) and ent-3 5 P (E).

sults. For example, the enantioselectivity for anesthetic steroid effects on TBPS binding may differ for different GABAA receptor subtypes. Additonal studies with different subtypes of GABAA receptors are needed to address this possibility. In the electrophysiological experiments, the enantioselectivity is manifested in different ways for the 5 - and 5 series of compounds. For potentiation of GABA-mediated currents, enantioselectivity is detected as a difference in maximal response for the steroids in the 5 -reduced series, whereas it is detected as a difference in the EC50 values for the 3 5 P/ent-3 5 P enantiomer pair in the 5 -reduced series. Based on prior studies (Wittmer et al., 1996; Zorumski et al., 1998), there are likely to be significant differences in

the potencies of the 5 -reduced enantiomers for potentiating GABA responses, but poor solubility at concentrations 100 M limits the ability to generate accurate concentrationresponse data for the ent-steroids. There is also a marked enantioselectivity difference in the gating of GABAA receptors by the two series of steroids. The ent-steroids in the 5 -reduced series do not gate these ion channels at concentrations up to 100 M, whereas ent-3 5 P was found to gate the ion channels at least as well as 3 5 P over the concentration range of 1 to 100 M. Comparing the IC50 values from the [35S]TBPS binding experiments and the EC50 values from tadpole LRR and electrophysiological experiments shows that the effects of the

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Fig. 3. A, traces show the enhancement of GABA-mediated currents (2 M; EC10) by 10 M 3 5 P and ent-3 5 P in hippocampal neurons recorded at a holding potential of 60 mV. GABA, either alone or in the presence of the steroids, was applied for 500 ms. B, average potentiation of 2 M GABA currents by 10 M enantiomers of 3 5 P (right columns). For comparison, the effects of 10 M 3 5 P enantiomers on currents gated by 2 M GABA are shown (left columns). Results represent the mean S.E. of four to six cells per experimental condition.

compounds occur in the same concentration range throughout these bioassays. However, the enantioselectivity for [35S]TBPS displacement by the 3 5 ACN and 3 5 P enantiomer pairs is much greater than the enantioselectivity found for the anesthetic effects of these compounds in the tadpole LRR test. Additionally, whereas a small degree of enantioselectivity is found for the 5 -reduced steroids in the [35S]TBPS-binding experiments, significant enantioselectivity for these compounds was not found in the tadpole LRR test. The reasons for the failure of the tadpole LRR results to more closely correlate with those of the [35S]TBPS binding experiment are not clear, although the complexity of factors involved in behavioral responses is likely to contribute. Overall the results show that 3 5 -, 3 5 - and ent-3 5 steroids potently modulate GABAA receptors, whereas ent3 5 -steroids do not effectively modulate these receptors. Enantioselectivity for GABAA receptor modulation/anesthesia by anesthetic steroids in the 5 - and 5 -reduced steroid series is different (5 5 ). Because of this enantioselectivity difference, the diastereoselective interactions of the ent3 5 /ent-3 5 and 3 5 /3 5 steroid pairs are also different (ent-pairs natural pairs). These stereoselectivity results raise interesting questions. Do these stereoselectivity differences imply different mechanisms (direct versus indirect) for modulation of GABAA receptor function and the correlated

Fig. 4. Concentration-response curves for the potentiation of currents gated by 1 M ( EC5) GABA (A) and for direct chloride-channel gating in the absence of GABA (B) by 3 5 P (F) and ent-3 5 P (). A, points represent the percentage of increase S.E. in GABA response produced by the steroid for three to five cells per concentration. The solid lines represent the best fit of the logistic concentration-response equation. For 3 5 P, the maximal response, Hill coefficient, and EC50 were 1725 0.4 M, respectively, whereas for ent111%, 1.0 0.3 M, and 1.2 3 5 P these values were 1737 1%, 1.4 0.1 M, and 3.6 0.1 M, respectively. B, responses to the steroids were normalized with respect to the response obtained at 10 M steroid and represent data (points are the mean S.E.) from four or five cells per concentration. The responses to 10 and 100 M 3 5 P were 316 64 pA and 1234 224 pA, respectively (n 4 each). The responses to 10 and 100 M ent-3 5 P were 346 53 pA and 2010 254 pA, respectively, (n 4 each). The solid lines represent best fits with maximal responses of 1429 1 and 1337 1%, Hill coefficients of 0.8 0.01 and 1.0 0.01, and EC50 values of 272 1 and 125 1 M for 3 5 P and ent-3 5 P, respectively. For the best fits, values S.E. given are from the fit of the data points.

anesthetic actions of the two series of anesthetic steroids? Do these stereoselectivity differences suggest different binding sites for the two series of anesthetic steroids on GABAA

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Fig. 5. 3 5 ACN and 3 5 P enantiomers cause tadpole LRR but do not act enantioselectively. Points on the tadpole concentration-response curves represent mean response of 10 to 40 animals, scored quantally. A, concentration-response curves for LRR in tadpoles showing the effects of 3 5 ACN (F) versus ent-3 5 ACN (E). The EC50 values S.E. of the fit data were 80 13 and 115 19 nM, respectively. B, concentration-response curves for LRR in tadpoles showing the effects of 3 5 P (F) versus ent-3 5 P (E). The EC50 values S.E. of the fit data were 61 4 and 58 18 nM, respectively.

Fig. 6. Intravenous injection of 3 5 P (F) or its enantiomer ent-3 5 P (E) each produced dose-dependent LRR (sleep times) in mice. Points S.E. on mouse dose-response curves represent the average sleep time for three or four animals and were fit to a straight line.

receptors? These questions are addressed in the following discussion. Our studies of the enantioselectivity of anesthetic steroid effects on GABAA receptor function were initiated as a way to distinguish between direct (a steroid-binding site on the receptor) and indirect (membrane perturbation) effects of these compounds on receptor function. We define direct effects as those arising from molecular interactions involving the steroid and amino acids of the receptor. We define indirect effects as those arising from molecular interactions involving the steroid and other nonprotein membrane constituents such as cholesterol and phospholipids. The degree to which enantioselectivity can be used to make the distinction between the two types of interactions is dependent on how large a difference is expected for the direct and indirect actions of the steroid on receptor function.

Binding interactions of ligands with receptors are expected to be highly enantioselective because receptor proteins are made of only L-amino acids. For substrates binding to enzymes, where both the initial binding and then a subsequent chemical reaction must occur, the expected result is complete enantioselectivity (i.e., the enzymatic transformation is expected to be enantiospecific). However, there are exceptions to the expected results for these types of interactions. For example, the binding of nornicotine to nicotinic acetylcholine receptors is not enantioselective. Both ( )- and ( )-nornicotine displace ( )-[3H]nicotine with equal potency and efficacy (Zhang and Nordberg, 1993; Badio and Daly, 1994; Abreo et al., 1996). Many examples of esterases that demonstrate varying degrees of enantioselectivity for the hydrolysis of racemic esters are reported in the literature. Indeed, the effective separation of racemic esters by enzymes (one ester enantiomer is preferentially hydrolyzed and readily separated from the unreacted ester enantiomer as the corresponding alcohol enantiomer) as a method for the resolution of the optically active components may require the screening of different esterases to identify the most enantioselective esterase for a particular separation (Chen et al., 1982 and references therein). The implication of these examples for this study is that the varying degrees of enantioselectivity found for the anesthetic steroids are all consistent with a direct steroid-receptor interaction. Indeed, even if enantioselectivity had not been observed in this study, the possibility of a direct steroidreceptor interaction could not be unequivocally ruled out. Based on information currently available, no enantioselectivity would be expected for the indirect modulation of GABAA receptor function by anesthetic steroid enantiomers. This conclusion is based on the following reasoning and supporting evidence. Because enantiomers have identical physical properties, they alter the physical properties of a membrane (e.g., fluidity) in an identical manner unless the

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TABLE 1 Summary of enantioselectivity results

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GABAA Receptor Modulation Enantiomer Pair TBPS IC50 Potentiation 2 M GABA Response Potentiation EC50 M Gating EC50 M Anesthesia Ratio (Mice) Tadpole ED50 nM

nM

3 5 ACN ent-3 5 ACN 3 5 P ent-3 5 P 3 5 ACN ent-3 5 ACN 3 5 P ent-3 5 P

50 4020 (80)d 74 1910 (26) 72 292 (4.1) 71 276 (3.9)

18-folda 2-folda (9) 11-folde 3-folde (3.7) 13-foldf 5-foldf (2.6) 17-foldh 17-foldh (1)

1.4b c N.D. 2.2b c N.D. N.D. N.D. N.D. 1.2h 3.6h (3)

5b 100b ( 20) 23b 100b ( 4.3) g g N.D. i i N.D.

( 20) N.D. N.D. (2.1)j

70b 700b (10) 390b 1100b (2.8) 80 115 (1.4) 61 58 (1.1)

N.D., not determined. a Determined at steroid concentrations of 10 M (data not shown). Values S.E. were 209 9% (n 6; ent-3 5 ACN) and 1829 272% (n 4; 3 5 ACN). Similar results were reported previously (Wittmer et al., 1966). b Based on concentration-response data from the literature (Wittmer et al., 1996). c Value could not be accurately determined because of steroid insolubility. See literature (Wittmer et al., 1996) for details. d Enantioselectivity ratio (higher value/lower value). e Determined at steroid concentrations of 10 M (Fig. 3B). f Determined at steroid concentrations of 10 M (data not shown). Values S.E. were 534 87% (n 5; ent-3 5 ACN) and 1297 251% (n 4; 3 5 ACN). g Both 10 M ent-3 5 ACN and 10 M 3 5 ACN directly gated GABA channels. Values S.E. were 22 8% (n 4; ent-3 5 ACN) and 68 18% (n 4; 3 5 ACN) compared with 2 M GABA-mediated currents. h Based on concentration-response data (Fig. 4A). i The direct gating effects of ent-3 5 P were found to be comparable to those of 3 5 P. See Fig. 4B for details. j Enantioselectivity ratio is based on slopes of graphed dose-response data (Fig. 6).

physical properties of the membrane are already dependent on preexisting enantiospecific interactions between membrane constituents. Most obviously, because the cholesterol and phospholipids found in cell membranes occur in enantiomerically pure form, interactions between these molecules could fulfill the preexisting enantiospecificity requirement. The extent to which the different anesthetic steroid enantiomers altered these preexisting enantiospecific cholesterolphospholipid interactions would then determine the anesthetic steroid enantioselectivity for indirect modulation of channel function. Is there any evidence that enantiospecific cholesterol-phospholipid interactions are determinants of membrane physical properties? Because of the difficulty involved in addressing this question (the unnatural enantiomers of cholesterol and/or a phospholipid are required for the studies), few experiments have been conducted to examine this question. However, the few available experiments (lipid monolayer and NMR studies) provide no evidence that enantiospecific cholesterol-phospholipid interactions have any effect on the physical properties of the membrane (Ghosh et al., 1971; Arnett and Gold, 1982). Thus, we have referred previously to the membrane as a nonchiral environment and we have concluded that no enantioselectivity for anesthetic steroid action is expected if the effects of these compounds are indirectly caused by membrane perturbation (Wittmer et al., 1996). Realizing that this conclusion is based on very limited experimental data, we recently reported a new method for preparing the unnatural enantiomer of cholesterol (Kumar and Covey, 1999) and we plan to address the biological significance of enantiospecific interactions between cholesterol and different classes of phospholipids in future studies. Because, in this study, we find some degree of enantioselectivity for both series of anesthetic steroids, we find no compelling reasons to conclude that either series of anesthetic steroids

modulates GABAA receptor function by an indirect mechanism. Whether the different degrees of enantioselectivity imply different receptor-binding sites for the two series of anesthetic steroids is the final question to address. Although there are data from previous studies suggesting the existence of more than one class of binding sites for some steroids on GABAA receptors (Morrow et al., 1990; Hawkinson et al., 1994a,b, 1998), the enantioselectivity results we have obtained thus far present no new evidence for this phenomenon. Previous data from studies of the diastereoselective modulation of GABAA receptor function by 3 5 P and 3 5 P have been reasonably explained by a common binding-site hypothesis developed from molecular modeling studies (Purdy et al., 1990; Han et al., 1996). The results from this and the previous enantioselectivity study of 5 -reduced anesthetic steroids (Wittmer et al., 1996) establish new criteria that must be satisfied by molecular models of a common binding site for 5 - and 5 -reduced anesthetic steroids. Molecular models of a common binding site for both classes of anesthetic steroids also must be able to explain the greater enantioselectivity of 5 -reduced anesthetic steroids and the greater diastereoselectivity observed for the ent-3 5 P/ent3 5 P steroid pair versus the 3 5 P/3 5 P steroid pair.
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Send reprint requests to: Douglas F. Covey, Ph.D., Department of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: dcovey@molecool.wustl.edu