Sedative-hypnotic Binding to 11ß-hydroxylase.

BACKGROUND: Etomidate potently suppresses adrenocortical steroid synthesis with potentially deleterious consequences by binding to 11ß-hydroxylase and inhibiting its function. The authors hypothesized that other sedative-hypnotics currently in clinical use or under development (or their metabolites) might bind to the same site at clinically relevant concentrations. The authors tested this hypothesis by defining etomidate's affinity for this site and the potencies with which other sedative-hypnotics (and their metabolites) inhibit etomidate binding. METHODS: H-etomidate's binding to adrenal membranes from Sprague-Dawley rats was characterized with a filtration assay, and its dissociation constant was defined using saturation and homologous ligand competition approaches. Half-inhibitory concentrations of sedative-hypnotics and metabolites were determined from the reduction in specific H-etomidate binding measured in the presence of ranging sedative-hypnotic and metabolite concentrations. RESULTS: Saturation and homologous competition studies yielded H-etomidate dissociation constants of 40 and 21 nM, respectively. Half-inhibitory concentrations of etomidate and cyclopropyl methoxycarbonyl metomidate (CPMM) differed significantly (26 vs. 143 nM, respectively; P < 0.001), and those of the carboxylic acid (CA) metabolites etomidate-CA and CPMM-CA were greater than or equal to 1,000× higher than their respective parent hypnotics. The half-inhibitory concentration of dexmedetomidine was 2.2 µM, whereas those of carboetomidate, ketamine, and propofol were greater than or equal to 50 µM. CONCLUSION: Etomidate's in vitro dissociation constant for 11ß-hydroxylase closely approximates its in vivo adrenocortical half-inhibitory concentration. CPMM produces less adrenocortical suppression than etomidate not only because it is metabolized faster but also because it binds to 11ß-hydroxylase with lower affinity. Other sedative-hypnotics and metabolites bind to 11ß-hydroxylase and inhibit etomidate binding only at suprahypnotic concentrations.

γ-Aminobutyric Acid Type A Receptor Modulation by Etomidate Analogs.

BACKGROUND: Etomidate is a highly potent anesthetic agent that is believed to produce hypnosis by enhancing γ-aminobutyric acid type A (GABAA) receptor function. The authors characterized the GABAA receptor and hypnotic potencies of etomidate analogs. The authors then used computational techniques to build statistical and graphical models that relate the potencies of these etomidate analogs to their structures to identify the specific molecular determinants of potency. METHODS: GABAA receptor potencies were defined with voltage clamp electrophysiology using α1ß3γ2 receptors harboring a channel mutation (α1[L264T]) that enhances anesthetic sensitivity (n = 36 to 60 measurements per concentration-response curve). The hypnotic potencies of etomidate analogs were defined using a loss of righting reflexes assay in Sprague Dawley rats (n = 9 to 21 measurements per dose-response curve). Three-dimensional quantitative structure-activity relationships were determined in silico using comparative molecular field analysis. RESULTS: The GABAA receptor and hypnotic potencies of etomidate and the etomidate analogs ranged by 91- and 53-fold, respectively. These potency measurements were significantly correlated (r = 0.72), but neither measurement correlated with drug hydrophobicity (r = 0.019 and 0.005, respectively). Statistically significant and predictive comparative molecular field analysis models were generated, and a pharmacophore model was built that revealed both the structural elements in etomidate analogs associated with high potency and the interactions that these elements make with the etomidate-binding site. CONCLUSIONS: There are multiple specific structural elements in etomidate and etomidate analogs that mediate GABAA receptor modulation. Modifying any one element can alter receptor potency by an order of magnitude or more.

Analogues of etomidate: modifications around etomidate's chiral carbon and the impact on in vitro and in vivo pharmacology.

BACKGROUND: R-etomidate possesses unique desirable properties but potently suppresses adrenocortical function. Consequently, efforts are being made to define structure-activity relationships with the goal of designing analogues with reduced adrenocortical toxicity. The authors explored the pharmacological impact of modifying etomidate's chiral center using R-etomidate, S-etomidate, and two achiral etomidate analogues (cyclopropyl etomidate and dihydrogen etomidate). METHODS: The γ-aminobutyric acid type A receptor modulatory potencies of drugs were assessed in oocyte-expressed α1(L264T)ß3γ2L and α1(L264T)ß1γ2L γ-aminobutyric acid type A receptors (for each drug, n = 6 oocytes per subtype). In rats, hypnotic potencies and durations of action were measured using a righting reflex assay (n = 26 to 30 doses per drug), and adrenocortical potencies were quantified by using an adrenocorticotropic hormone stimulation test (n = 20 experiments per drug). RESULTS: All four drugs activated both γ-aminobutyric acid type A receptor subtypes in vitro and produced hypnosis and suppressed adrenocortical function in rats. However, drug potencies in each model ranged by 1 to 2 orders of magnitude. R-etomidate had the highest γ-aminobutyric acid type A receptor modulatory, hypnotic, and adrenocortical inhibitory potencies. Respectively, R-etomidate, S-etomidate, and cyclopropyl etomidate were 27.4-, 18.9-, and 23.5-fold more potent activators of receptors containing ß3 subunits than ß1 subunits; however, dihydrogen etomidate's subunit selectivity was only 2.48-fold and similar to that of propofol (2.08-fold). S-etomidate was 1/23rd as potent an adrenocortical inhibitor as R-etomidate. CONCLUSION: The linkage between the structure of etomidate's chiral center and its pharmacology suggests that altering etomidate's chiral center may be used as part of a strategy to design analogues with more desirable adrenocortical activities and/or subunit selectivities.

Electroencephalographic and hypnotic recoveries after brief and prolonged infusions of etomidate and optimized soft etomidate analogs.

BACKGROUND: Methoxycarbonyl etomidate is the prototypical soft etomidate analog. Because it has relatively low potency and is extremely rapidly metabolized, large quantities must be infused to maintain hypnosis. Consequently with prolonged infusion, metabolite reaches sufficient concentrations to delay recovery. Dimethyl-methoxycarbonyl metomidate (DMMM) and cyclopropyl-methoxycarbonyl metomidate (CPMM) are methoxycarbonyl etomidate analogs with higher potencies and slower clearance. Because of these properties, we hypothesized that dosing would be lower and electroencephalographic and hypnotic recoveries would be faster - and less context-sensitive - with DMMM or CPMM versus methoxycarbonyl etomidate or etomidate. METHODS: Etomidate, DMMM, and CPMM where infused into rats (n = 6 per group) for either 5 min or 120 min. After infusion termination, electroencephalographic and hypnotic recovery times were measured. The immobilizing ED50 infusion rates were determined using a tail clamp assay. RESULTS: Upon terminating 5-min infusions, electroencephalographic and hypnotic recovery times were not different among hypnotics. However, upon terminating 120-min infusions, recovery times varied significantly with respective values (mean ± SD) 48 ± 13 min and 31 ± 6.5 min (etomidate), 17 ± 7.0 min and 14 ± 3.4 min (DMMM), and 4.5 ± 1.1 min and 4.2 ± 1.6 min (CPMM). The immobilizing ED50 infusion rates were (mean ± SD) 0.19 ± 0.03 mg · kg · min (etomidate), 0.60 ± 0.12 mg · kg · min (DMMM), and 0.89 ± 0.18 mg · kg · min (CPMM). CONCLUSIONS: Electroencephalographic and hypnotic recoveries following prolonged infusions of DMMM and CPMM are faster than those following methoxycarbonyl etomidate or etomidate. In the case of CPMM infusion, recovery times are 4 min and context-insensitive.

Modifying methoxycarbonyl etomidate inter-ester spacer optimizes in vitro metabolic stability and in vivo hypnotic potency and duration of action.

BACKGROUND: Methoxycarbonyl etomidate is the prototypical very rapidly metabolized etomidate analog. Initial studies suggest that it may be too short acting for many clinical uses. We hypothesized that its duration of action could be lengthened and clinical utility broadened by incorporating specific aliphatic groups into the molecule to sterically protect its ester moiety from esterase-catalyzed hydrolysis. To test this hypothesis, we developed a series of methoxycarbonyl etomidate analogs (spacer-linked etomidate esters) containing various aliphatic-protecting groups and spacer lengths. METHODS: Spacer-linked etomidate esters were synthesized and their hypnotic potencies and durations of action following bolus administration were measured in rats using a loss-of-righting reflexes assay. Octanol:water partition coefficients and metabolic half-lives in pooled rat blood were determined chromatographically. RESULTS: All spacer-linked etomidate esters produced hypnosis rapidly and in a dose-dependent manner. ED50s for loss of righting reflexes ranged from 0.69 ± 0.04 mg/kg for cyclopropyl-methoxycarbonyl metomidate to 11.1 ± 0.8 mg/kg for methoxycarbonyl metomidate. The slope of a plot of the duration of loss of righting reflexes versus the logarithm of the dose ranged 12-fold among spacer-linked etomidate esters, implying widely varying brain clearance rates. The in vitro metabolic half-lives of these compounds in rat blood varied by more than two orders of magnitude and were diastereometrically selective. CONCLUSIONS: We created 13 new analogs of methoxycarbonyl etomidate and identified two that have significantly higher potency and potentially address the too-brief duration of action for methoxycarbonyl etomidate. This work may provide a blueprint for optimizing the pharmacological properties of other soft drugs.

Electroencephalographic recovery, hypnotic emergence, and the effects of metabolite after continuous infusions of a rapidly metabolized etomidate analog in rats.

BACKGROUND: Methoxycarbonyl etomidate is an ultrarapidly metabolized etomidate analog. It is metabolized to methoxycarbonyl etomidate carboxylic acid (MOC-ECA), which has a hypnotic potency that is 350-fold less than that of methoxycarbonyl etomidate. The authors explored the relationships between methoxycarbonyl etomidate infusion duration, recovery time, metabolite concentrations in blood and cerebrospinal fluid (CSF), and methoxycarbonyl etomidate metabolism in brain tissue and CSF to test the hypothesis that rapid metabolism of methoxycarbonyl etomidate may lead to sufficient accumulation of MOC-ECA in the brain to produce a pharmacologic effect. METHODS: A closed-loop system with burst suppression ratio feedback was used to administer methoxycarbonyl etomidate infusions of varying durations to rats. After infusion, recovery of the electroencephalogram and righting reflexes were assessed. MOC-ECA concentrations were measured in blood and CSF during and after methoxycarbonyl etomidate infusion, and the in vitro half-life of methoxycarbonyl etomidate was determined in rat brain tissue and CSF. RESULTS: Upon termination of continuous methoxycarbonyl etomidate infusions, the burst suppression ratio recovered in a biexponential manner with fast and slow components having time constants that differed by more than 100-fold and amplitudes that varied inversely with infusion duration. MOC-ECA concentrations reached hypnotic concentrations in the CSF with prolonged methoxycarbonyl etomidate infusion and then decreased during a period of several hours after infusion termination. The metabolic half-life of methoxycarbonyl etomidate in brain tissue and CSF was 11 and 20 min, respectively. CONCLUSION: In rats, methoxycarbonyl etomidate metabolism is sufficiently fast to produce pharmacologically active MOC-ECA concentrations in the brain with prolonged methoxycarbonyl etomidate infusion.

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3)H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

Pharmacological studies of methoxycarbonyl etomidate's carboxylic acid metabolite.

BACKGROUND: Methoxycarbonyl etomidate (MOC-etomidate) is a rapidly metabolized and ultrashort-acting etomidate analog that does not produce prolonged adrenocortical suppression after bolus administration. Its metabolite (MOC-ECA) is a carboxylic acid whose pharmacology is undefined. We hypothesized that MOC-ECA possesses significantly lower pharmacological activity than MOC-etomidate, accounting for the latter's very brief duration of hypnotic action and inability to produce prolonged adrenocortical suppression after bolus administration. To test this hypothesis, we compared the potencies of MOC-ECA and MOC-etomidate in 3 biological assays. METHODS: The hypnotic potency of MOC-ECA was assessed in tadpoles using a loss-of-righting reflexes assay. The γ-aminobutyric acid type A (GABA(A)) receptor modulatory potencies of MOC-ECA and MOC-etomidate were compared by defining the concentrations of each required to directly activate α(1)(L264T)ß(2)γ(2L) GABA(A) receptors. The adrenocortical inhibitory potencies of MOC-ECA and MOC-etomidate were compared by defining the concentrations of each required to inhibit in vitro cortisol production by adrenocortical cells. RESULTS: MOC-ECA's 50% effective concentration for loss-of-righting reflexes in tadpoles was 2.8 ± 0.64 mM as compared with a previously reported value of 8 ± 2 µM for MOC-etomidate. The 50% effective concentrations for direct activation of GABA(A) receptors were 3.5 ± 0.63 mM for MOC-ECA versus 10 ± 2.5 µM for MOC-etomidate. The half-maximal inhibitory concentration for inhibiting in vitro cortisol production by adrenocortical cells was 30 ± 7 µM for MOC-ECA versus 0.10 ± 0.02 µM for MOC-etomidate. CONCLUSIONS: In all 3 biological assays, MOC-ECA's potency was approximately 300-fold lower than that of MOC-etomidate.

Closed-loop continuous infusions of etomidate and etomidate analogs in rats: a comparative study of dosing and the impact on adrenocortical function.

BACKGROUND: Etomidate is a sedative-hypnotic that is often given as a single intravenous bolus but rarely as an infusion because it suppresses adrenocortical function. Methoxycarbonyl etomidate and (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate) are etomidate analogs that do not produce significant adrenocortical suppression when given as a single bolus. However, the effects of continuous infusions on adrenocortical function are unknown. In this study, we compared the effects of continuous infusions of etomidate, methoxycarbonyl etomidate, and carboetomidate on adrenocortical function in a rat model. METHODS: A closed-loop system using the electroencephalographic burst suppression ratio as the feedback was used to administer continuous infusions of etomidate, methoxycarbonyl etomidate, or carboetomidate to Sprague-Dawley rats. Adrenocortical function was assessed during and after infusion by repetitively administering adrenocorticotropic hormone 1-24 and measuring serum corticosterone concentrations every 30 min. RESULTS: The sedative-hypnotic doses required to maintain a 40% burst suppression ratio in the presence of isoflurane, 1%, and the rate of burst suppression ratio recovery on infusion termination varied (methoxycarbonyl etomidate > carboetomidate > etomidate). Serum corticosterone concentrations were reduced by 85% and 56% during 30-min infusions of etomidate and methoxycarbonyl etomidate, respectively. On infusion termination, serum corticosterone concentrations recovered within 30 min with methoxycarbonyl etomidate but persisted beyond an hour with etomidate. Carboetomidate had no effect on serum corticosterone concentrations during or after continuous infusion. CONCLUSIONS: Our results suggest that methoxycarbonyl etomidate and carboetomidate may have clinical utility as sedative-hypnotic maintenance agents when hemodynamic stability is desirable.

Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 µM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).

p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels.

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidate's, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidate's novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

Carboetomidate: a pyrrole analog of etomidate designed not to suppress adrenocortical function.

BACKGROUND: Etomidate is a sedative hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However, it also binds with high affinity to 11beta-hydroxylase, potently suppressing the synthesis of steroids by the adrenal gland that are necessary for survival. The authors report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analog of etomidate specifically designed not to bind with high affinity to 11beta-hydroxylase. METHODS: The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type alpha1beta2gamma2l and etomidate-insensitive mutant alpha1beta2M286Wgamma2l human gamma-aminobutyric acid type A receptor activities was assessed using electrophysiologic techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. RESULTS: Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type but not etomidate-insensitive mutant gamma-aminobutyric acid type A receptors. Carboetomidate was a three orders of magnitude less-potent inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. CONCLUSIONS: Carboetomidate is an etomidate analog that retains many beneficial properties of etomidate, but it is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.

Methoxycarbonyl-etomidate: a novel rapidly metabolized and ultra-short-acting etomidate analogue that does not produce prolonged adrenocortical suppression.

BACKGROUND: Etomidate is a rapidly acting sedative-hypnotic that provides hemodynamic stability. It causes prolonged suppression of adrenocortical steroid synthesis; therefore, its clinical utility and safety are limited. The authors describe the results of studies to define the pharmacology of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to be susceptible to ultra-rapid metabolism. METHODS: The gamma-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human alpha1beta2gamma2l receptors. MOC-etomidate's hypnotic concentration was determined in tadpoles by using a loss of righting reflex assay. Its in vitro metabolic half-life was measured in human liver S9 fraction, and the resulting metabolite was provisionally identified by using high-performance liquid chromatography/mass spectrometry techniques. The hypnotic and hemodynamic actions of MOC-etomidate, etomidate, and propofol were defined in rats. The abilities of MOC-etomidate and etomidate to inhibit corticosterone production were assessed in rats. RESULTS: MOC-etomidate potently enhanced gamma-aminobutyric acid type A receptor function and produced loss of righting reflex in tadpoles. Metabolism in human liver S9 fraction was first-order, with an in vitro half-life of 4.4 min versus more than 40 min for etomidate. MOC-etomidate's only detectable metabolite was a carboxylic acid. In rats, MOC-etomidate produced rapid loss of righting reflex that was extremely brief and caused minimal hemodynamic changes. Unlike etomidate, MOC-etomidate produced no adrenocortical suppression 30 min after administration. CONCLUSIONS: MOC-etomidate is an etomidate analogue that retains etomidate's important favorable pharmacological properties. However, it is rapidly metabolized, ultra-short-acting, and does not produce prolonged adrenocortical suppression after bolus administration.

Time-resolved photolabeling of the nicotinic acetylcholine receptor by [3H]azietomidate, an open-state inhibitor.

Azietomidate is a photoreactive analog of the general anesthetic etomidate that acts as a nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist. We used rapid perfusion electrophysiological techniques to characterize the state dependence and kinetics of azietomidate inhibition of Torpedo californica nAChRs and time-resolved photolabeling to identify the nAChR binding sites occupied after exposure to [(3)H]azietomidate and agonist for 50 ms (open state) or at equilibrium (desensitized state). Azietomidate acted primarily as an open channel inhibitor characterized by a bimolecular association rate constant of k(+) = 5 x 10(5) M(-1) s(-1) and a dissociation rate constant of <3s(-1). Azietomidate at 10 microM, when perfused with acetylcholine (ACh), inhibited the ACh response by approximately 50% after 50 ms; when preincubated for 10 s, it decreased the peak initial response by approximately 15%. Comparison of the kinetics of recovery of ACh responses after exposure to ACh and azietomidate or to ACh alone indicated that at subsecond times, azietomidate inhibited nAChRs without enhancing the kinetics of agonist-induced desensitization. In nAChRs frozen after 50-ms exposure to agonist and [(3)H]azietomidate, amino acids were photolabeled in the ion channel [position M2-20 (alphaGlu-262, betaAsp-268, deltaGln-276)], in deltaM1 (deltaCys-236), and in alphaMA/alphaM4 (alphaGlu-390, alphaCys-412) that were also photolabeled in nAChRs in the equilibrium desensitized state at approximately half the efficiency. These results identify azietomidate binding sites at the extracellular end of the ion channel, in the delta subunit helix bundle, and in the nAChR cytoplasmic domain that seem similar in structure and accessibility in the open and desensitized states of the nAChR.

Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog.

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([(3)H]azietomidate), which retains anesthetic potency in vivo and enhances GABA(A)R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA(A)R anesthetic binding site. For GABA(A)Rs purified by affinity chromatography from detergent extracts of bovine cortex, [(3)H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC(50) = 30 microm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the alphaM1 transmembrane helix at alpha1Met-236 (and/or the homologous methionines in alpha2,3,5), not previously implicated in etomidate function, and one within the betaM3 transmembrane helix at beta3Met-286 (and/or the homologous methionines in beta1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between beta and alpha subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket approximately 50 A below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels.

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

R (+) etomidate and the photoactivable R (+) azietomidate have comparable anesthetic activity in wild-type mice and comparably decreased activity in mice with a N265M point mutation in the gamma-aminobutyric acid receptor beta3 subunit.

A photoactivable diazirine derivative of etomidate, azietomidate, shares many actions of etomidate, including a capacity to abolish the righting reflexes in tadpoles and enhance gamma-aminobutyric acid (GABA)-induced currents. Azietomidate's usefulness in studies of mechanisms of anesthesia depends on the assumption that it shares a site of action with etomidate. Mice bearing an N265M beta3 subunit point mutation in GABA(A) receptors have a markedly decreased sensitivity to loss of righting reflexes induced by etomidate over a range of doses. Accordingly, in the present study we measured the time to recovery of righting reflexes of wild type and mutant mice as a function of dose given as an IV bolus. Analysis of the data for azietomidate yielded mean times to recovery of righting reflexes at a dose of 7.5 mg/kg of 10.0 +/- 0.9 min and 3.0 +/- 1.6 min for wild type and mutant mice, respectively (mean +/- sd). A similar analysis for etomidate yielded mean times to recovery of righting reflexes at a dose of 7.5 mg/kg of 12.0 +/- 1.3 min and 4.0 +/- 0.7 min for wild type and mutant mice respectively. Thus, at this dose a single mutation, N265M on the beta3 subunit of the GABA(A) receptor, approximately halved the time to recovery of righting reflexes for both etomidate and azietomidate (by 7.6 +/- 1.5 min and 7.2 +/- 1.8 min, respectively), emphasizing the contribution of this residue as a determinant of a behavioral response of azietomidate in mice.

Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta.

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic.

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.