Identification of Anesthetic Binding Sites on Human Serum Albumin Using a Novel Etomidate Photolabel
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m700479200
ISSN1083-351X
AutoresDamian P. Bright, Sara D. Adham, Lucienne C.J.M. Lemaire, Rodrigo Benavides, Marco Gruß, Graham W. Taylor, Edward H. Smith, Nicholas P. Franks,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoWe have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on α1β2γ2s GABAA receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (β2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds ∼75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABAA receptor or other putative targets. We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on α1β2γ2s GABAA receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (β2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds ∼75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABAA receptor or other putative targets. Although it is now widely accepted that general anesthetics exert their effects by binding directly to their protein targets (1Campagna J.A. Miller K.W. Forman S.A. N. Engl. J. Med. 2003; 348: 2110-2124Crossref PubMed Scopus (637) Google Scholar, 2Franks N.P. Br. J. Pharmacol. 2006; 147: S72-S81Crossref PubMed Scopus (298) Google Scholar, 3Rudolph U. Antkowiak B. Nat. Rev. Neurosci. 2004; 5: 709-720Crossref PubMed Scopus (622) Google Scholar), information on the precise molecular locations of these binding sites has been slow in coming. Most information has been derived from in vitro electrophysiological experiments in which putative anesthetic targets, ion channels, or receptors, are genetically modified. What this approach provides is information on the molecular determinants of anesthetic sensitivity, but it is usually impossible to tell whether these determinants represent portions of anesthetic binding sites or regions of the channel or receptor that are responsible for transducing anesthetic binding into changes in channel gating. In principle, the most direct approach would be to determine a high resolution crystal structure of the ion channel or receptor in question in the presence and absence of anesthetics. Unfortunately, because of the difficulties in crystallizing membrane proteins, such experiments are still some way off. An alternative strategy that has recently had some success is to use anesthetics that have been modified so that they contain photoactivatable groups. When these anesthetic analogs are illuminated with a bright light, they are converted into highly reactive intermediates, which then, hopefully, bind irreversibly to their targets. The idea of using photoaffinity labeling to identify anesthetic binding sites was first implemented using the volatile anesthetic halothane (4Eckenhoff R.G. J. Biol. Chem. 1996; 271: 15521-15526Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Eckenhoff R.G. Shuman H. Anesthesiology. 1993; 79: 96-106Crossref PubMed Scopus (76) Google Scholar). Halothane breaks down to form highly reactive ethane radicals when illuminated with ultraviolet light at wavelengths around 250 nm, but at these wavelengths there is likely to be some damage to the protein targets themselves. Subsequent work has attempted to improve on these pioneering studies by synthesizing anesthetic analogs with incorporated diazirine groups (6Eckenhoff R.G. Knoll F.J. Greenblatt E.P. Dailey W.P. J. Med. Chem. 2002; 45: 1879-1886Crossref PubMed Scopus (17) Google Scholar, 7Husain S.S. Forman S.A. Kloczewiak M.A. Addona G.H. Olsen R.W. Pratt M.B. Cohen J.B. Miller K.W. J. Med. Chem. 1999; 42: 3300-3307Crossref PubMed Scopus (43) Google Scholar). The diazirine moiety absorbs light at longer wavelengths and has been widely used in photolabeling studies (8Blencowe A. Hayes W. Soft Matter. 2005; 1: 178-205Crossref PubMed Scopus (124) Google Scholar) and a number of different photoactivatable anesthetics have been synthesized. A diazirine alcohol derivative, 3-(2-hydroxylethyl)-3-n-pentyldiazirin (azioctanol) has been synthesized and characterized (7Husain S.S. Forman S.A. Kloczewiak M.A. Addona G.H. Olsen R.W. Pratt M.B. Cohen J.B. Miller K.W. J. Med. Chem. 1999; 42: 3300-3307Crossref PubMed Scopus (43) Google Scholar) and shown to label the nicotinic acetylcholine receptor from Torpedo (9Pratt M.B. Husain S.S. Miller K.W. Cohen J.B. J. Biol. Chem. 2000; 275: 29441-29451Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Two regio-isomers of octanol bearing a diazirine group on the third and seventh carbon (3- and 7-azioctanol, respectively) have been used to locate and delineate an anesthetic binding site on adenylate kinase (10Addona G.H. Husain S.S. Stehle T. Miller K.W. J. Biol. Chem. 2002; 277: 25685-25691Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and protein kinase Cδ (11Das J. Addona G.H. Sandberg W.S. Husain S.S. Stehle T. Miller K.W. J. Biol. Chem. 2004; 279: 37964-37972Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). A diazirine derivative of the intravenous anesthetic etomidate has also been synthesized (12Husain S.S. Ziebell M.R. Ruesch D. Hong F. Arevalo E. Kosterlitz J.A. Olsen R.W. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2003; 46: 1257-1265Crossref PubMed Scopus (81) Google Scholar). This photolabel has been shown to bind to the nicotinic acetylcholine receptor from Torpedo (12Husain S.S. Ziebell M.R. Ruesch D. Hong F. Arevalo E. Kosterlitz J.A. Olsen R.W. Forman S.A. Cohen J.B. Miller K.W. J. Med. Chem. 2003; 46: 1257-1265Crossref PubMed Scopus (81) Google Scholar, 13Ziebell M.R. Nirthanan S. Husain S.S. Miller K.W. Cohen J.B. J. Biol. Chem. 2004; 279: 17640-17649Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) as well as to the GABAA 7The abbreviations used are: GABAA, γ-aminobutyric acid, type A; HSA, human serum albumin; HPLC, high performance liquid chromatography; MS, mass spectrometry; SIM, selected ion mode. receptor (14Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (253) Google Scholar). In the latter case, two particular methionine residues were shown to be labeled. Other workers have synthesized a diazirine derivative of a neuroactive steroid 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), which was shown to label the mitochondrial voltage-dependent anion channel VDAC in rat brain membranes (15Darbandi-Tonkabon R. Hastings W.R. Zeng C.M. Akk G. Manion B.D. Bracamontes J.R. Steinbach J.H. Mennerick S.J. Covey D.F. Evers A.S. J. Biol. Chem. 2003; 278: 13196-13206Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Despite the widespread use of diazirines, other photolabeling approaches are possible and in this article we describe the use of a novel etomidate analog, which incorporates an acyl azide moiety. The azide group produces a highly reactive nitrene on illumination (as opposed to a carbene as is the case for diazirines) and has been used as the photoactivatable group in a number of studies (16Fleming S.A. Tetrahedron. 1995; 51: 12479-12520Crossref Scopus (290) Google Scholar). We show that the etomidate analog behaves essentially identically to the parent compound etomidate in terms of its interactions with the GABAA receptor and its behavior as a general anesthetic in animals. We test the ability of this novel analog to label proteins using human serum albumin (HSA) as a target protein. Albumin is the most abundant protein in human plasma (600 μm) and is known to bind a very large variety of endogenous and exogenous ligands (17Curry S. Brick P. Franks N.P. Biochim Biophys Acta. 1999; 1441: 131-140Crossref PubMed Scopus (462) Google Scholar, 18Ghuman J. Zunszain P.A. Petitpas I. Bhattacharya A.A. Otagiri M. Curry S. J. Mol. Biol. 2005; 353: 38-52Crossref PubMed Scopus (1488) Google Scholar). In the case of etomidate, it is estimated that over 75% of the drug is bound to plasma proteins following an intravenous injection (19Meuldermans W.E. Heykants J.J. Arch. Int. Pharmacodyn Ther. 1976; 221: 150-162PubMed Google Scholar); the locations of the binding sites on HSA, however, are unknown. Using our novel etomidate analog we identify two specific binding sites on human serum albumin. Synthesis of Acyl Azide Etomidate—Fig. 1 shows the molecular structure of the acyl azide analogs of etomidate and the parent compound etomidate, together with the reaction scheme for their synthesis. Both isomers (R(+) and S(-)) of this compound were produced using the same synthetic route (see Fig. 1B) but with starting material (methyl benzylamine) of the appropriate chirality in each case. Methyl benzylamine (I) was treated with triethyl amine and ethyl chloroacetate in dimethylformamide. The resulting methylbenzyl glycine ethyl ester was reacted with formic acid in xylene. The crude methylbenzyl-N-formylglycine ethyl ester (II) was distilled to furnish pure product as a yellow oil (53% yield, b.p. 154–160 °C/0.63 mm Hg). This was then treated with freshly prepared sodium methoxide and methyl formate in tetrahydrofuran at 10 °C, followed by reaction of the resulting c-formyl derivative with hydrochloric acid-potassium thiocyanate resulting in the formation of the thiol (III). Oxidative desulfurization of the thiol (III) in the presence of nitric acid-sodium nitrite afforded the desired methyl ester (IV) in 74% yield as a viscous orange oil, which was used without further purification. This was refluxed for 24 h with four equivalents of hydrazine hydrate in ethanol to yield the hydrazide (V) as a pale yellow oil (83% yield). Subsequent reaction of this hydrazide with nitrous acid resulted in the desired product acyl azide (VI) in moderate yield (40%) as an off-white solid (m.p. 58 °C). If too much hydrochloric acid was used in this last step the hydrochloride salt of the acyl azide, m.p. 108–110°, was isolated. The free acyl azide could be obtained from this by treatment with di-isopropylamine. Ultraviolet absorption spectra of compounds were obtained using a Beckman DU650 spectrophotometer. Octanol-water partition coefficients were measured by weighing compounds directly into disposable UV-transparent cuvettes (Brand, Wertheim, Germany) before addition of 2 ml water and 0.5 ml octanol. The cuvette was then inverted repeatedly over 2–3 min, before separation of the two phases by centrifugation. Concentrations in the two phases were then determined spectrophotometrically. Solutions—R(+)-etomidate was obtained as Hypnomidate (2 mg ml-1 in 35% propylene glycol; Janssen-Cilag Ltd, High Wycombe, UK). S(-)-etomidate was synthesized according to previously described procedures (20Godefroi E.F. Janssen P.A. Vandereycken C.A. Vanheertum A.H. Niemegeers C.J. J. Med. Chem. 1965; 8: 220-223Crossref PubMed Scopus (70) Google Scholar) with minor modifications. For all experiments, S(-)-etomidate solutions were made up using a stock solution of 2 mg ml-1 in 35% propylene glycol. For the electrophysiological and photolabeling experiments, acyl azide solutions were made up using concentrated stocks in ethanol. The highest concentration of ethanol used (34 mm) was found to have no effect on GABA-activated currents. In the photolabeling experiments, an equivalent amount of ethanol was added to the control solutions. For the in vivo potency measurements, solutions containing the acyl azide were made up from 2 mg ml-1 stock solutions in 35% propylene glycol (tadpole experiments) or from stocks in 100% propylene glycol (upto 6 mg ml-1; rat experiments). In both sets of in vivo experiments, the carrier was found to have no effect by itself. Solutions containing GABA were prepared using concentrated stocks made up in saline on the day of the experiment. All chemicals were obtained from Sigma unless otherwise stated. Cell Culture—Modified HEK-293 cells (tsA 201) were maintained in 5% CO2, 95% air in a humidified incubator at 37 °C in growth media (89% Dulbecco's modified Eagle's medium; 10% heat-inactivated fetal bovine serum; 1% penicillin (10,000 units ml-1) and streptomycin (10 mg ml-1)). When the tsA cells were 80% confluent, they were split and plated for transfection onto glass coverslips coated with poly-d-lysine to ensure good cell adhesion. The HEK-293 cells were transiently transfected with human cDNAs coding for α1, β2, and γ2s GABAA subunits using the calcium phosphate method. The coding sequences for these three subunits were subcloned into pcDNA3.1 (Invitrogen, Paisley, UK), a vector designed for high expression in mammalian cells. 1 μg of cDNA encoding each subunit was added to each 35-mm diameter well, and 1 μg of a plasmid encoding the cDNA of green fluorescent protein was included to identify cells expressing GABAA receptor cDNAs. Following a 24-h incubation period at 3% CO2 the cells were rinsed with saline and fresh growth medium was added to the wells. The cells were incubated at 37 °C with 5% CO2/95% air for 12–72 h before electrophysiological measurements were made. Electrophysiology—Ionic currents evoked by the application of GABA were recorded using the whole cell patch-clamp technique with an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Recording pipettes were pulled from thin-walled borosilicate glass capillaries (Harvard Apparatus, Edenbridge, Kent, UK) using a two-stage vertical puller (Narishige, Tokyo, Japan). After brief fire-polishing, pipettes were back-filled with 0.2 μm-filtered intracellular solution (140 mm CsCl, 1 mm MgCl2, 11 mm EGTA, 10 mm HEPES, titrated to pH 7.2 with CsOH). These pipettes, with typical resistances of 2.5–4 MΩ, readily formed “giga-ohm” seals with the cells, which upon establishing the whole cell configuration were voltage-clamped at -60 mV. Series resistance was compensated by 80–90%. During recording, cells were bathed in an extracellular solution containing 145 mm NaCl, 3 mm KCl, 1 mm MgCl2, 1.5 mm CaCl2, 5.5 mm d-glucose, 10 mm HEPES, (titrated to pH 7.4 with NaOH). All drugs were applied locally to the cell via a double-barreled glass capillary tube as described previously (21Downie D.L. Hall A.C. Lieb W.R. Franks N.P. Br. J. Pharmacol. 1996; 118: 493-502Crossref PubMed Scopus (131) Google Scholar). Recordings were filtered at 50 Hz (-3 dB) using an 8-pole Bessel filter (Frequency Devices, Lyons Instruments, Hertfordshire, UK) and digitized at 200 Hz before being stored on a computer hard disk. Data were subsequently analyzed offline using Clampfit software (Axon Instruments, Union City, CA). All electrophysiological measurements were carried out at room temperature (22 ± 1 °C). Agonist-containing test solutions were typically applied to the cell for 2–5 s. GABA concentration-response data were then fitted using a least squares method to the Hill equation shown in Equation 1.I=Imax[GABA]nH[GABA]nH+EC50nH(Eq.1) For experiments involving anesthetics, test solutions contained the appropriate concentrations of GABA and the anesthetic. High concentrations of anesthetic were found to directly activate receptors in the absence of GABA and therefore to separate this GABA-mimetic effect from the GABA-modulatory effect, the anesthetic was applied before, as well as during, the application of GABA. Anesthetics were typically pre-applied for at least 30 s to ensure that a stable baseline current had been obtained, before application of the GABA-containing test solution. At low concentrations (3 μm or less for R(+)-etomidate), there was no GABA-mimetic effect and pre-application of anesthetics was found to be unnecessary. GABA concentration-response curves in the presence of anesthetic were constructed that were fitted with a Hill equation as before. Anesthetic potentiation (P) was defined by Equation 2 where I0 is the peak of the control GABA-induced current, and I is the peak of the GABA-induced current in the presence of anesthetic.p(%)=100×(I-I0)I0(Eq.2) Values throughout this article are given as means ± S.E. In Vivo Anesthetic Potency Measurements—All experiments were performed in compliance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Potency Measurements in Tadpoles—General anesthetic potencies of compounds were determined for 4–6-week-old Rana temporaria tadpoles (Blades Biological, Cowden, Kent, UK) in the pre-limb-bud stage of development. Tadpoles were maintained in an aerated aquarium at 20–22 °C. Groups of 8–12 tadpoles were placed in 400-ml glass beakers containing 300 ml of the anesthetic solution. Stock solutions of acyl azide etomidate were usually made up in the same carrier as used clinically i.e. 2 mg ml-1 in 35% propylene glycol. However, for the highest concentrations of some compounds, a more concentrated stock, 3 mg ml-1, was utilized. The highest concentration of propylene glycol used was 71 mm, which was found to have no effect by itself. The anesthetic end point was defined as the lack of a purposeful and sustained swimming response after a gentle inversion with a smooth glass rod. This was assessed at 10-min intervals over a period of 60 min by which time equilibrium had been reached. After the experiment, tadpoles were returned to fresh tap water where their recovery was monitored. In most cases, normal swimming activity was restored within 1 h. Tadpoles that did not recover were excluded from the analysis. Tadpole concentration-response data were fitted according to the method of Waud (22Waud D.R. J. Pharmacol. Exp. Ther. 1972; 183: 577-607PubMed Google Scholar) with a logistic equation of the form in Equation 3,p=100CnCn+(EC50)n(Eq.3) where p is the percentage anesthetized, C is the anesthetic concentration, n is the slope parameter, and EC50 is the concentration required for a half-maximal effect. Potency Measurements in Rats—Experiments were performed on male Sprague-Dawley rats (B & K Universal, London, UK) weighing ∼300 g. Rats were given free access to food and water and were maintained on a 12 h light-dark cycle. Anesthetic solutions were prepared in the same carrier as used clinically, i.e. 35% propylene glycol in water. Anesthetics were administered intravenously through one of the two lateral tail veins using a 24-gauge, 19-mm cannula (Becton Dickinson, Oxford, UK). The cannula was connected to a short length of PTFE tubing using a specially designed stainless steel fitting to ensure a minimal “dead volume” ( 320 nm band-pass filter (Lot-Oriel). Exposures were controlled using a manual shutter. Samples were continuously mixed during exposure using a small magnetic stirrer. Trypsin Digestion—Following UV exposure, 0.5 ml of the sample was dried down to a volume of about 50 μl using a rotary evaporator (DNA Plus; Jouan Nordic, Denmark), before addition of 0.5 ml of denaturing buffer (6 m guanidine HCl, 0.3 m Tris-HCl, 2 mm EDTA, pH 8.0) and incubation for 2 h at 37 °C. The protein was then reduced by addition of 10 μl dithiothreitol (50 mg/ml in denaturing buffer) and incubation at 37 °C for 1 h. Finally, the protein was alkylated to prevent the reformation of disulfide bonds by addition of 80 μl of iodoacetamide (250 mg/ml in denaturing buffer) and incubation at room temperature for a further 30 min. The reduced and alkylated protein was subsequently dialyzed against 0.1 m ammonium bicarbonate (pH 7.35) at room temperature for ∼24 h. Protein samples were then digested by addition of 100 μg of “sequencing grade” trypsin and incubation at 37 °C for ∼24 h. Samples were then taken to dryness in a rotary evaporator to yield a white powdery pellet. The ammonium bicarbonate was removed by addition of 5% acetic acid (0.5 ml), after which the sample was again dried down to yield a pellet. This pellet was then resuspended in 5% acetic acid for loading onto HPLC. Analysis by High Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS)—Analysis of tryptic peptides of photolabeled HSA was performed using an Agilent 1100 Series HPLC/MS. Generally, a sample volume of 15 μl was used. Peptides were separated on a Vydac C18 column (2.1 mm inner diameter, 250 mm length, Grace Vydac, Strathaven, Lanarkshire, UK) at a flow rate of 0.2 ml/min at 25 °C. A gradient elution was employed using two solvents, solvent A (0.06% trifluoroacetic acid in water) and solvent B (0.056% trifluoroacetic acid in 80% acetonitrile). The elution profile used was 100% solvent A/0% solvent B to 63% solvent A/37% solvent B over 63 min, then to 25% A/75% B over the next 32 min and then to 2% A/98% B over the next 10 min. The eluent was monitored using a photodiode detector array at wavelengths of 214 nm, 245 nm, and 280 nm. In addition, the output from the HPLC was coupled to an Agilent VL series mass spectrometer, configured to operate in electrospray ionization (ESI) mode. This allowed the detection of positively charged species with mass to charge (m/z) ratios in the range 150–1500. The mass spectrometer was usually configured to run in “scan” mode, whereby the entire m/z range was scanned. However, a “selected ion mode” (SIM) was also used, whereby a defined set of m/z could be searched for selectively, leading to an enhanced signal to noise ratio. HSA Binding Measurements—The extent of binding of etomidate and acyl azide etomidate to HSA was determined by measuring the free aqueous concentration AF of the ligand as a function of total protein concentration PT. Mixtures of HSA and ligand were pre-equilibrated and spun through centrifugal filters with a 10,000 molecular weight cut-off (Centricon YM-10, Millipore, Bedford, MA). The concentration of ligand in the initial spin-though was taken as an estimate of the free aqueous concentration AF. It is easy to show Equation 4,AF=-(PT-AT+K)+(PT-AT+K)2+4KAT2(Eq.4) where K is the ligand dissociation constant and AT is the total ligand concentration. A least-squares fit of the observed data to this equation gave an estimate of the dissociation constant K. Both enantiomers of acyl azide etomidate were synthesized to a high degree of purity (>99%) and the ultraviolet spectra showed a strong absorption band centered at 270 nm (Fig. 1C). (This is in contrast to etomidate which shows a strong absorption band at 240 nm.) The extinction coefficient at the maximum absorption was ϵ270 = 15,500 m-1 cm-1. The absorption at longer wavelengths was still substantial (e.g. ϵ320 = 120 m-1 cm-1) and illumination using a band-pass filter (λ > 320 nm) caused rapid photolysis (Fig. 1C). The time course of photoconversion was roughly exponential with a half-time τ ∼ 8 s. Acyl azide etomidate was stable in aqueous solution, with less than 10% hydrolysis after 4 h. The replacement of the ethyl ether on etomidate for the acyl azide group had very little effect on the overall polarity of the molecule, and the octanol/water partition coefficients were comparable. Table 1 gives the octanol/water partition coefficients for etomidate, acyl azide etomidate, and some related analogs for comparison.TABLE 1Octanol-water partition coefficients and melting points for etomidate and related analogs Open table in a new tab Acyl azide etomidate also displayed a similar in vivo pharmacology to that of the parent compound. This is illustrated by the data in Fig. 2A, which show that the EC50 concentration for loss of righting reflex in tadpoles is 11 ± 2 μm for the R(+) enantiomer and significantly greater for the S(-) enantiomer (36 ± 7 μm), as is the case for etomidate (23Tomlin S.L. Jenkins A. Lieb W.R. Franks N.P. Anesthesiology. 1998; 88: 708-717Crossref PubMed Scopus (138) Google Scholar). The R(+) acyl azide etomidate also caused rapid and reversible loss of righting reflex in rats (Fig. 2B) with an ED50 dose of 1.7 ± 0.2 mg kg-1. The in vitro pharmacology of acyl azide etomidate was virtually indistinguishable from that of etomidate. This is illustrated by the data in Fig. 3 for α1β2γ2s GABAA receptors expressed in HEK-293 cells. Both R(+) etomidate and R(+) acyl azide etomidate caused a marked leftwards shift in GABA concentration-response curves. A Hill equation fitted to the control data gave an EC50 of 56 ± 3 μm and a Hill coefficient of nH = 1.3 ± 0.1. These parameters were 11 ± 4 μm and 0.6 ± 0.1 in the presence of 3 μm R(+) etomidate and 11 ± 5 μm and 0.6 ± 0.2 in the presence of 3 μm R(+) acyl azide etomidate. The S(-) enantiomers had no significant effect on the GABA concentration-response curve (Fig. 3, A and B). Both R(+) etomidate and R(+) acyl azide etomidate caused a concentration-dependent and saturable potentiation of GABA-evoked currents (Fig. 3, C and D), which was abolished in receptors which carried the N265M mutation in the β2 subunit (insets to Fig. 3, C and D). This mutation has been shown to block the potentiating action of etomidate (24McGurk K.A. Pistis M. Belelli D. Hope A.G. Lambert J.J. Br. J. Pharmacol. 1998; 124: 13-20Crossref PubMed Scopus (34) Google Scholar). Finally, both R(+) etomidate and R(+) acyl azide etomidate caused a concentration-dependent activation of the α1β2γ2s GABAA receptor in the absence of GABA. This GABA-mimetic effect showed no signs of saturating at high concentrations (Fig. 3, E and F). Having established that R(+) etomidate and R(+) acyl azide etomidate shared very similar in vivo and in vitro pharmacological profiles, we went on to test the ability of R(+) acyl azide etomidate to photolabel a protein target, human serum albumin. This plasma protein is known to play an important role in the pharmacokinetics of etomidate, and ∼75% of etomidate binds to plasma proteins following intravenous injection (19Meuldermans W.E. Heykants J.J. Arch. Int. Pharmacodyn Ther. 1976; 221: 150-162PubMed Google Scholar). We first determined the extent of binding of R(+) etomidate and R(+) acyl azide etomidate to HSA and measured dissociation constants of 176 ± 6 μm and 121 ± 5 μm, respectively. We next developed a protocol using HPLC/MS that would allow the separation a
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