Etomidate Improves the Antidepressant Effect of Electroconvulsive Therapy by Suppressing Hippocampal Neuronal Ferroptosis via Upregulating BDNF/Nrf2.

Electroconvulsive therapy (ECT) performed under general anesthesia is an effective treatment for severe depression. Etomidate is an intravenous anesthetic that shows beneficial effects on ECT. However, the potential mechanisms have rarely been reported. In this study, male rats were exposed to chronic unpredictable mild stress for 4 weeks, followed by ECT for 10 days, with or without intervention with ferrostatin-1 (2 mg/kg) or all-trans retinoic acid (ATRA, 5 mg/kg). Rats subjected to etomidate (20 mg/kg) or propofol (120 mg/kg) treatment were administered with designated anesthetic before ECT. Compared to depressive rats without ECT, those who received ECT showed increased numbers of hippocampal neurons, increased expression of negative regulators of ferroptosis including glutathione peroxidase 4, ferritin heavy chain 1, and ferroptosis suppressor protein 1, upregulation of brain-derived neurotrophic factor and nuclear factor erythroid 2-related factor, and downregulation of acyl-CoA synthetase long-chain family member 4, a positive regulator of ferroptosis in the hippocampus. Additionally, compared with propofol, etomidate used in ECT resulted in higher upregulation of BDNF/Nrf2 and inhibited neuronal ferroptosis in hippocampus. These results showed etomidate may enhance the antidepressant effect of ECT by protecting hippocampal neurons against ferroptosis.

Propofol attenuates kinesin-mediated axonal vesicle transport and fusion.

Propofol is a widely used general anesthetic, yet the understanding of its cellular effects is fragmentary. General anesthetics are not as innocuous as once believed and have a wide range of molecular targets that include kinesin motors. Propofol, ketamine, and etomidate reduce the distances that Kinesin-1 KIF5 and Kinesin-2 KIF3 travel along microtubules in vitro. These transport kinesins are highly expressed in the CNS, and their dysfunction leads to a range of human pathologies including neurodevelopmental and neurodegenerative diseases. While in vitro data suggest that general anesthetics may disrupt kinesin transport in neurons, this hypothesis remains untested. Here we find that propofol treatment of hippocampal neurons decreased vesicle transport mediated by Kinesin-1 KIF5 and Kinesin-3 KIF1A ∼25-60%. Propofol treatment delayed delivery of the KIF5 cargo NgCAM to the distal axon. Because KIF1A participates in axonal transport of presynaptic vesicles, we tested whether prolonged propofol treatment affects synaptic vesicle fusion mediated by VAMP2. The data show that propofol-induced transport delay causes a significant decrease in vesicle fusion in distal axons. These results are the first to link a propofol-induced delay in neuronal trafficking to a decrease in axonal vesicle fusion, which may alter physiological function during and after anesthesia.

Photomotor Responses in Zebrafish and Electrophysiology Reveal Varying Interactions of Anesthetics Targeting Distinct Sites on γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Etomidate, barbiturates, alfaxalone, and propofol are anesthetics that allosterically modulate γ-aminobutyric acid type A (GABAA) receptors via distinct sets of molecular binding sites. Two-state concerted coagonist models account for anesthetic effects and predict supra-additive interactions between drug pairs acting at distinct sites. Some behavioral and molecular studies support these predictions, while other findings suggest potentially complex anesthetic interactions. We therefore evaluated interactions among four anesthetics in both animals and GABAA receptors. METHODS: The authors used video assessment of photomotor responses in zebrafish larvae and isobolography to evaluate hypnotic drug pair interactions. Voltage clamp electrophysiology and allosteric shift analysis evaluated coagonist interactions in α1ß3γ2L receptors activated by γ-aminobutyric acid (GABA) versus anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA. RESULTS: In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-5-allyl-1-methyl m-trifluoromethyl mephobarbital (R-mTFD-MPAB; mean ± SD α = 1.0 ± 0.07 and 0.96 ± 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean α range 0.76 to 0.89). Electrophysiologic allosteric shifts revealed that both propofol and R-mTFD-MPAB modulated etomidate-activated receptors much less than GABA-activated receptors [log(d, AN):log(d, GABA) ratios = 0.09 ± 0.021 and 0.38 ± 0.024, respectively], while alfaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alfaxalone was supra-additive (n = 6; P = 0.023 by paired t test), but etomidate plus R-mTFD-MPAB or propofol was not. CONCLUSIONS: In both zebrafish and GABAA receptors, anesthetic drug pairs interacted variably, ranging from additivity to synergy. Pairs including etomidate displayed corresponding interactions in animals and receptors. Some of these results challenge simple two-state coagonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.

Antifungal activity of etomidate against growing biofilms of fluconazole-resistant Candida spp. strains, binding to mannoproteins and molecular docking with the ALS3 protein.

This study evaluated the effect of etomidate against biofilms of Candida spp. and analysed through molecular docking the interaction of this drug with ALS3, an important protein for fungal adhesion. Three fluconazole-resistant fungi were used: Candida albicans, Candida parapsilosis and Candida tropicalis. Growing biofilms were exposed to etomidate at 31.25-500 µg ml-1. Then, an ALS3 adhesive protein from C. albicans was analysed through a molecular mapping technique, composed of a sequence of algorithms to perform molecular mapping simulation based on classic force field theory. Etomidate showed antifungal activity against growing biofilms of resistant C. albicans, C. parapsilosis and C. tropicalis at all concentrations used in the study. The etomidate coupling analysis revealed three interactions with the residues of interest compared to hepta-threonine, which remained at the ALS3 site. In addition, etomidate decreased the expression of mannoproteins on the surface of C. albicans. These results revealed that etomidate inhibited the growth of biofilms.

Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Binding site location on GABAA receptors determines whether mixtures of intravenous general anaesthetics interact synergistically or additively in vivo.

BACKGROUND AND PURPOSE: General anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R-mTFD-MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site-selective agents binding to the same or to different sites would combine additively or synergistically respectively. EXPERIMENTAL APPROACH: The potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods. KEY RESULTS: The potencies of combinations of two or three site-selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site-selective anaesthetics that all bound selectively to different sites always interacted synergistically. The strength of the synergy increased with the number of separate sites involved such that the percentage of each agent's EC50 required to cause anaesthesia was just 35% and 14% for two or three sites respectively. Propofol, which binds non-selectively to the etomidate and R-mTFD-MPAB sites, interacted synergistically with each of these agents. CONCLUSIONS AND IMPLICATIONS: The established pharmacology of the three anaesthetic binding sites on synaptic GABAA receptors was sufficient to predict whether a mixture of anaesthetics interacted additively or synergistically to cause loss of righting reflexes in vivo. The principles established here have implications for clinical practice.

Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands.

INTRODUCTION: Primary aldosteronism accounts for 6-15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. METHODS: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites. RESULTS: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. CONCLUSIONS: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

Pretreatment with Oxycodone Simultaneously Reduces Etomidate-Induced Myoclonus and Rocuronium-Induced Withdrawal Movements During Rapid-Sequence Induction.

BACKGROUND Etomidate and rocuronium are often paired in rapid-sequence anesthesia induction. However, the effect of pretreatment with oxycodone on myoclonic and withdrawal movements has not been previously investigated. The aim of this study was to evaluate the effects of oxycodone on the incidence and severity of etomidate-induced myoclonus and rocuronium-induced nociceptive withdrawal movements during rapid-sequence anesthesia induction. MATERIAL AND METHODS We randomly divided 120 patients into the saline group (group S) and the oxycodone group (group O) (n=60 in each group). Patients received 0.05 mg/kg oxycodone or saline intravenously 2 min before administration of 0.3 mg/kg etomidate. The occurrence and severity of myoclonus were assessed after administration of etomidate, then rocuronium was injected, followed by evaluation of withdrawal movements. RESULTS The total frequency of involuntary movements following sequential administration of etomidate and rocuronium was significantly lower in Group O than in Group S (28.3% vs. 90%, p<0.001). The total frequency and grade 3 severity of myoclonus following etomidate injection in Group O was significantly lower than in Group S (25.0% vs. 63.3% for total frequency; 0 vs. 10 for grade 3 severity, P<0.001). The total frequency and grade 3 intensity of withdrawal movements were significantly less in Group O than in Group S (6.7% vs. 73.3% for total frequency; 0 vs. 11 for grade 3 intensity, P<0.001). CONCLUSIONS Oxycodone is effective for simultaneously preventing etomidate-induced myoclonus and rocuronium-induced withdrawal movements during general anesthesia induction.

Competitive Antagonism of Anesthetic Action at the γ-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy.

BACKGROUND: The authors characterized the γ-aminobutyric acid type A receptor pharmacology of the novel etomidate analog naphthalene-etomidate, a potential lead compound for the development of anesthetic-selective competitive antagonists. METHODS: The positive modulatory potencies and efficacies of etomidate and naphthalene-etomidate were defined in oocyte-expressed α1ß3γ2L γ-aminobutyric acid type A receptors using voltage clamp electrophysiology. Using the same technique, the ability of naphthalene-etomidate to reduce currents evoked by γ-aminobutyric acid alone or γ-aminobutyric acid potentiated by etomidate, propofol, pentobarbital, and diazepam was quantified. The binding affinity of naphthalene-etomidate to the transmembrane anesthetic binding sites of the γ-aminobutyric acid type A receptor was determined from its ability to inhibit receptor photoaffinity labeling by the site-selective photolabels [H]azi-etomidate and R-[H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS: In contrast to etomidate, naphthalene-etomidate only weakly potentiated γ-aminobutyric acid-evoked currents and induced little direct activation even at a near-saturating aqueous concentration. It inhibited labeling of γ-aminobutyric acid type A receptors by [H]azi-etomidate and R-[H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid with similar half-maximal inhibitory concentrations of 48 µM (95% CI, 28 to 81 µM) and 33 µM (95% CI, 20 to 54 µM). It also reduced the positive modulatory actions of anesthetics (propofol > etomidate ~ pentobarbital) but not those of γ-aminobutyric acid or diazepam. At 300 µM, naphthalene-etomidate increased the half-maximal potentiating propofol concentration from 6.0 µM (95% CI, 4.4 to 8.0 µM) to 36 µM (95% CI, 17 to 78 µM) without affecting the maximal response obtained at high propofol concentrations. CONCLUSIONS: Naphthalene-etomidate is a very low-efficacy etomidate analog that exhibits the pharmacology of an anesthetic competitive antagonist at the γ-aminobutyric acid type A receptor.

Contrasting Effects of the γ-Aminobutyric Acid Type A Receptor ß3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB.

BACKGROUND: Previous studies have shown that etomidate modulates γ-aminobutyric acid type A receptors by binding at the ß-α subunit interface within the transmembrane domain of receptors that incorporate ß2 or ß3 subunits. Introducing an asparagine-to-methionine (N265M) mutation at position 265 of the ß3 subunit, which sits within the etomidate-binding site, attenuates the hypnotic effect of etomidate in vivo. It was reported recently that the photoactivatable barbiturate R-mTFD-MPAB also acts on γ-aminobutyric acid type A receptors primarily by binding to a homologous site at the γ-ß interface. Given this difference in drug-binding sites established by the in vitro experiments, we hypothesized that the ß3-N265M-mutant mice would not be resistant to the anesthetic effects of R-mTFD-MPAB in vivo, whereas the same mutant mice would be resistant to the anesthetic effects of R-etomidate. METHODS: We measured the effects of IV injection of etomidate and R-mTFD-MPAB on loss and recovery of righting reflex in wild-type mice and in mice carrying the ß3-N265M mutation. RESULTS: Etomidate-induced hypnosis, as measured by the duration of loss of righting reflex, was attenuated in the N265M knock-in mice, confirming prior results. By contrast, recovery of balance and coordinated movement, as measured by the ability to maintain all 4 paws on the ground, was unaffected by the mutation. Neither hypnosis nor impairment of coordinated movement produced by the barbiturate R-mTFD-MPAB was affected by the mutation. CONCLUSIONS: The findings confirmed our hypothesis that mutating the etomidate-binding site would not alter the response to the barbiturate R-mTFD-MPAB. Furthermore, we confirmed previous studies indicating that etomidate-induced hypnosis is mediated in part by ß3-containing receptors. We also extended previous findings by showing that etomidate-impaired balance and coordinated movement are not mediated by ß3-containing receptors, thus implicating ß2-containing receptors in this end point.

Distinct Hypnotic Recoveries After Infusions of Methoxycarbonyl Etomidate and Cyclopropyl Methoxycarbonyl Metomidate: The Role of the Metabolite.

BACKGROUND: Methoxycarbonyl etomidate (MOC-etomidate) and cyclopropyl methoxycarbonyl metomidate (CPMM) are rapidly metabolized "soft" etomidate analogs. CPMM's duration of hypnotic effect is context insensitive, whereas MOC-etomidate's is not. In this study, we tested the hypothesis that CPMM's effect is context insensitive because, unlike MOC-etomidate, its metabolite fails to reach physiologically important concentrations in vivo even with prolonged continuous infusion. METHODS: We compared the potencies with which MOC-etomidate and CPMM activate α1(L264T)ß3γ2 γ-aminobutyric acid type A receptors and induce loss-of-righting reflexes (i.e., produce hypnosis) in tadpoles with those of their metabolites (MOC-etomidate's carboxylic acid metabolite [MOC-ECA] and CPMM's carboxylic acid metabolite [CPMM-CA], respectively). We measured metabolite concentrations in the blood and cerebrospinal fluid of Sprague-Dawley rats on CPMM infusion and compared them with those achieved with MOC-etomidate infusion. We measured the rates with which brain tissue from Sprague-Dawley rats metabolize MOC-etomidate and CPMM. RESULTS: Both analogs and their metabolites enhanced γ-aminobutyric acid type A receptor function and induced loss-of-righting reflexes in a concentration-dependent manner. However, in these 2 assays, CPMM-CA's potency relative to its parent hypnotic was approximately 1:4900 and 1:1900, respectively, whereas MOC-ECA's was only approximately 1:415 and 1:390, respectively. With 2-hour CPMM infusions, CPMM-CA reached respective concentrations in the blood and cerebrospinal fluid that were 2 and >3 orders of magnitude lower than that which produced hypnosis. CPMM was metabolized by the brain tissue at a rate that is approximately 1/15th that of MOC-etomidate. CONCLUSIONS: Hypnotic recovery after CPMM administration is context insensitive because its metabolite does not accumulate to hypnotic levels in the central nervous system. This reflects the very large potency ratio between CPMM and CPMM-CA and the resistance of CPMM to metabolism by esterases present in the brain.

Biophysical study on the interaction of etomidate and the carrier protein in vitro.

Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K = 3.55 × 10(5 )M(-1), 295 K), the number of binding sites (n = 1.16), thermodynamic parameters (ΔG = 3.13 × 10(4 )J·mol(-1), ΔS = 364 J·mol(-1)·K(-1) and ΔH = -6.85 × 10(5 )J·mol(-1)) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate-HSA interactions which illustrates the complex nature of this subject.

Etomidate produces similar allosteric modulation in α1ß3δ and α1ß3γ2L GABA(A) receptors.

BACKGROUND AND PURPOSE: Neuronal GABA(A) receptors are pentameric chloride ion channels, which include synaptic αßγ and extrasynaptic αßδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αßγ receptors is established as ß-α-γ-ß-α, that of αßδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, ß3 and δ or γ2L subunits and concatenated ß3-α1-δ and ß3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between ß3 and α1. EXPERIMENTAL APPROACH: GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model. KEY RESULTS: ETO (3.2 µM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1ß3(1:1), α1ß3δ(1:1:1) and α1ß3δ(1:1:3), but this potentiation by ETO was significantly greater for ß3-α1-δ/ß3-α1(1:1) receptors. Reducing the amount of α1ß3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with ß3-α1-δ/ß3-α1(1:1, 1 ng). ETO similarly reduced EC50s and enhanced maxima of GABA concentration-response curves for both α1ß3δ and ß3-α1-δ/ß3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1ß3γ2L receptors. CONCLUSION AND IMPLICATIONS: Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1ß3γ2/δ GABA(A) receptors.

Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1ß3γ2 γ-aminobutyric acid type A (GABAA) receptor.

GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the ß(+)-α(-) subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1ß3γ2 GABAARs. Protein microsequencing revealed that R-[(3)H]mTFD-MPAB did not photolabel the etomidate sites at the ß(+)-α(-) subunit interfaces. Instead, it photolabeled sites at the α(+)-ß(-) and γ(+)-ß(-) subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (-)-side, ß3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the ß(+)-α(-) interface relative to the α(+)-ß(-)/γ(+)-ß(-) interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.

Carboetomidate: an analog of etomidate that interacts weakly with 11ß-hydroxylase.

BACKGROUND: Carboetomidate is a pyrrole etomidate analog that is 3 orders of magnitude less potent an inhibitor of in vitro cortisol synthesis than etomidate (an imidazole) and does not inhibit in vivo steroid production. Although carboetomidate's reduced functional effect on steroid synthesis is thought to reflect lower binding affinity to 11ß-hydroxylase, differential binding to this enzyme has never been experimentally demonstrated. In the current study, we tested the hypothesis that carboetomidate and etomidate bind with differential affinity to 11ß-hydroxylase by comparing their abilities to inhibit photoaffinity labeling of purified enzyme by a photoactivatable etomidate analog and to modify the enzyme's absorption spectrum in a way that is indicative of ligand binding. In addition, we made a preliminary exploration of the manner in which etomidate and carboetomidate might differentially interact with this site using spectroscopic methods as well as molecular modeling techniques to better understand the structural basis for their selectivity. METHODS: The ability of azi-etomidate to inhibit cortisol synthesis was tested by assessing its ability to inhibit cortisol synthesis by H295R cells. The binding affinities of etomidate and carboetomidate to 11ß-hydroxylase were compared by assessing their abilities to (1) inhibit photoincorporation of the photolabile etomidate analog [(3)H]azi-etomidate into the enzyme and (2) modify the absorption spectrum of the enzyme's heme group. In silico docking studies of etomidate, carboetomidate, and azi-etomidate binding to 11ß-hydroxylase were performed using the computer software GOLD. RESULTS: Similar to etomidate, azi-etomidate potently inhibits in vitro cortisol synthesis. Etomidate inhibited [(3)H]azi-etomidate photolabeling of 11ß-hydroxylase in a concentration-dependent manner. At a concentration of 40 µM, etomidate reduced photoincorporation of [(3)H]azi-etomidate by 96% ± 1% whereas carboetomidate had no experimentally detectable effect. On addition of etomidate to 11ß-hydroxylase, a type 2 difference spectrum was produced indicative of etomidate complexation with the enzyme's heme iron; carboetomidate had no effect whereas azi-etomidate produced a reverse type 1 spectrum. Computer modeling studies predicted that etomidate, carboetomidate, and azi-etomidate can fit into the heme-containing pocket that forms 11ß-hydroxylase's active site and pose with their carbonyl oxygens interacting with the heme iron and their phenyl rings stacking with phenylalanine-80. However, additional unique poses were identified for etomidate and azi-etomidate that likely account for their higher affinities. CONCLUSIONS: Carboetomidate's reduced ability to suppress in vitro and in vivo steroid synthesis as compared with etomidate reflects its lower binding affinity to 11ß-hydroxylase and may be attributed to carboetomidate's inability to form a coordination bond with the heme iron located at the enzyme's active site.

[(3)H]metyrapol and 4-[(131)i]iodometomidate label overlapping, but not identical, binding sites on rat adrenal membranes.

Metyrapone, metyrapol, and etomidate are competitive inhibitors of 11-deoxycorticosterone hydroxylation by 11ß-hydroxylase. [(3)H]Metyrapol and 4-[(131)I]iodometomidate bind with high affinity to membranes prepared from bovine and rat adrenals. Here we report inhibitory potencies of several compounds structurally related to one or both of these adrenostatic drugs, against the binding of both radioligands to rat adrenal membranes. While derivatives of etomidate inhibited the binding of both radioligands with similar potencies, derivatives of metyrapone inhibited the binding of 4-[(131)I]iodometomidate about 10 times weaker than the binding of [(3)H]metyrapol. By X-ray structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; Ki = 6 nM), 2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as new high affinity ligands for the metyrapol binding site on 11ß-hydroxylase and discuss our results in relation to a proposed active site model of 11ß-hydroxylase.

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.

Mapping general anesthetic binding site(s) in human α1ß3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue.

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and ßMet-286 in the αM1 and ßM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the ß and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1ß3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner ß3Val-290, in the ß3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and ßM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of ß3Met-227 in ßM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the ß3-ß3 subunit interface in the α1ß3 GABA(A)R.

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.