S. Shaukat Husain

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 (GABAARs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([3H]azietomidate), which retains anesthetic potency in vivo and enhances GABAAR function in vitro, to identify directly, for the first time, amino acids that contribute to a GABAAR anesthetic binding site. For GABAARs purified by affinity chromatography from detergent extracts of bovine cortex, [3H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC50 = 30 μm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the αM1 transmembrane helix at α1Met-236 (and/or the homologous methionines in α2,3,5), not previously implicated in etomidate function, and one within the βM3 transmembrane helix at β3Met-286 (and/or the homologous methionines in β1,2), 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 β and α subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ∼50 Å 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.

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 &ggr;-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human &agr;1&bgr;2&ggr;2l 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 &ggr;-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.

Identification of Sites of Incorporation in the Nicotinic Acetylcholine Receptor of a Photoactivatible General Anesthetic

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[3H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 μm, 3-[3H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the α-subunit in the desensitized state. The incorporation into the α-subunit was mapped to two large proteolytic fragments. One fragment of ∼20 kDa (αV8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of ∼10 kDa (αV8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within αV8-20, the primary site of incorporation was αGlu-262 at the C-terminal end of αM2, labeled preferentially in the desensitized state. The incorporation at αGlu-262 approached saturation between 1 μm, with ∼6% labeled, and 275 μm, with ∼30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at ∼1% of the levels in αGlu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.

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 11&bgr;-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 11&bgr;-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 &agr;1&bgr;2&ggr;2l and etomidate-insensitive mutant &agr;1&bgr;2M286W&ggr;2l human &ggr;-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 &ggr;-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.

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 γ-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 μm. 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 μm) increased the submaximal response to acetylcholine (10 μm) with a 2.5-fold increase at 60 μm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [3H]tetracaine and [3H]phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0. 8 mm). nAChR-rich membranes were photolabeled with [3H]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 (δLeu-265) and M2-13 (αVal-255 and δVal-269), whereas labeling of αM2-10 (αSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in γM3 (γMet-299), a residue that contributes to the same pocket in the nAChR structure as αM2-10. 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 α and γ subunits (labeling of αM2-10 and γMet-299) likely to be a site for positive allosteric modulation.

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, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]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 IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]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, [3H]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).

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 β3 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 &bgr;3 subunit point mutation in GABAA 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 &bgr;3 subunit of the GABAA 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.

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 latters 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 &ggr;-aminobutyric acid type A (GABAA) receptor modulatory potencies of MOC-ECA and MOC-etomidate were compared by defining the concentrations of each required to directly activate &agr;1(L264T)&bgr;2&ggr;2L GABAA 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-ECAs 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 &mgr;M for MOC-etomidate. The 50% effective concentrations for direct activation of GABAA receptors were 3.5 ± 0.63 mM for MOC-ECA versus 10 ± 2.5 &mgr;M for MOC-etomidate. The half-maximal inhibitory concentration for inhibiting in vitro cortisol production by adrenocortical cells was 30 ± 7 &mgr;M for MOC-ECA versus 0.10 ± 0.02 &mgr;M for MOC-etomidate. CONCLUSIONS:In all 3 biological assays, MOC-ECAs potency was approximately 300-fold lower than that of MOC-etomidate.

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.