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.

Etomidate inhibits tumor growth of glioblastoma by regulating M1 macrophage polarization.

Glioblastoma (GBM) is a common primary central nervous system tumor. Although the multimodal integrated treatment for GBM has made great progress in recent years, the overall survival time of GBM is still short. Thus, novel treatments for GBM are worth further investigation and exploration. This study aimed to investigate the effects of etomidate on GBM tumor growth and the underlying mechanism. A xenograft tumor model was established and treated with etomidate to assess tumor growth. Immunohistochemistry (IHC) assay evaluated the positive rate of Ki67 cells in tumor tissues. Cell counting kit (CCK)-8 and EdU assays accessed the cell viability and proliferation. Immunofluorescence (IF) staining detected the distribution of macrophage markers in tumor tissues. The percentages of M1- and M2-like macrophages in tumor-associated macrophages (TAMs) and co-culture system (macrophages and GBM cells) were detected using flow cytometry. Macrophage polarization-related genes were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Etomidate treatment inhibited the tumor growth, and increased the CD86+ cells but decreased the CD206+ cells in TAMs. The gene expression of M1 markers was increased in TAMs of etomidate-treated mice, whereas that of M2 markers was decreased. Moreover, etomidate treatment increased the number of CD86+ M1-like macrophages co-cultured with tumor cells but decreased that of CD206+ M2-like macrophages, with the upregulation of M1 markers and downregulation of M2 markers. Etomidate inhibited GBM tumor growth by promoting M1 macrophage polarization, suggesting a new insight into the clinical treatment of GBM.

Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors.

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1ß2/3γ2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near ßM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1ß3M286Cγ2L receptors to estimate the distance from etomidate to ß3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cß3γ2L receptors; (ii) studies in α1L232Wß3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to ß3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cß3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wß3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1ß3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to ß3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between ß3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane ß+/α- inter-subunit pockets. Comparing results in α1ß3M286Cγ2L and α1L232Wß3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward ß3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.

Influences of Etomidate Combined with Propofol on Cognitive Function, Inflammation and Immunity in Patients Undergoing Gastric Cancer Surgery.

This study examined how etomidate combined with propofol affected cognitive function, inflammation, and immunity in patients undergoing gastric cancer surgery. 182 gastric cancer patients treated in our hospital were enrolled and randomly divided into two groups, namely group A (anesthetized using etomidate) and group B (anesthetized using etomidate combined with propofol). Then the cognitive function, inflammation and immunity indicators were determined in the two groups. Compared with group A, group B exhibited shorter operation duration and hospital stay and smaller bleeding volume (p<0.01). At 3 d after the operation, group B had a higher Ramsay score, but a lower visual analogue scale (VAS) score than group A (p<0.05). Moreover, the mini-mental state examination (MMSE) score was lower in group A than that in group B (p<0.01). At the end of the operation, the heart rate (HR), mean arterial pressure (MAP) and saturation of pulse oxygen (SpO2) were decreased to a great extent in both groups compared with those before anesthesia (p<0.05). Compared with those before anesthesia, the levels of immunoglobulin (Ig)M, IgG and IgA were lower in group A at the end of the operation and 1 and 3 d after the operation (p<0.05), but they were substantially higher in group B than those in group A (p<0.05). At the end of the operation and 1 and 3 d after the operation, the levels of the T-cell subset indicators decreases in group A were greater than those in group B (p<0.05). Etomidate combined with propofol has few influences on the immune and cognitive functions of gastric cancer patients and can effectively lower the expression levels of inflammatory factors in these patients.

Etomidate Depresses Spontaneous Complex Spikes Activity of Cerebellar Purkinje Cells via Cannabinoid 1 Receptor in vivo in Mice.

INTRODUCTION: Complex spikes (CSs) activity of cerebellar Purkinje cells plays critical roles in motor coordination and motor learning by transferring information to cerebellar cortex, which is an accessible and useful model for neurophysiological investigation. Etomidate is an ultrashort-acting nonbarbiturate intravenous anesthetic, which inhibits the spontaneous activity of cerebellar Purkinje cells through activation of GABAA and glycine receptors in vivo in mice. However, the effect of etomidate on the spontaneous CSs activity of cerebellar Purkinje cells in living mouse is not clear. METHODS: We here investigated the effects of etomidate on spontaneous CSs activity of cerebellar Purkinje cell in urethane-anesthetized mice by electrophysiology recording technique and pharmacological methods. RESULTS: Our results showed that cerebellar surface perfusion of etomidate significantly depressed the activity of spontaneous CSs, which exhibited decreases in the number of spikelets and the area under curve (AUC) of the CSs. The etomidate-produced inhibition of CSs activity was persisted in the presence of GABAA and glycine receptors antagonists. However, application of cannabinoid 1 (CB1) receptor antagonist, AM-251, completely blocked the etomidate-induced inhibition of CSs. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease of CSs. Moreover, in the presence of a specific protein kinase A (PKA) inhibitor, KT5720, etomidate failed to produce decreases in the spikelets number and the AUC of the spontaneous CSs. CONCLUSION: These results indicate that cerebellar surface application of etomidate facilitates CB1 receptor activity resulting in a depression of spontaneous CSs activity of Purkinje cells via PKA signaling pathway in mouse cerebellar cortex. Our present results suggest that the etomidate administration may impair the function of cerebellar cortical neuronal circuitry by inhibition of the climbing fiber - Purkinje cells synaptic transmission through activation of CB1 receptors in vivo in mice.

The effect-site concentration of remifentanil blunting endotracheal intubation responses during anesthesia induction with etomidate: a dose-finding study.

BACKGROUND: Remifentanil can inhibit the hemodynamic responses caused by endotracheal intubation, but the effect-site concentration of it required to control intubation responses when combined with etomidate has not been demonstrated. The purpose of this study was to determine the effect-site concentration of remifentanil blunting tracheal intubation responses in 50% and 95% of patients (EC50 and EC95) during etomidate anesthesia. METHODS: American Society of Anesthesiologists physical status (ASA) I-II elective surgical patients receiving target-controlled infusion (TCI) of remifentanil, followed by etomidate and rocuronium for anesthesia were enrolled. The Belive Drive A2 monitor was used to calculate the MGRSSI (Maygreen Sedative state index) of hypnotic effect and the MGRNOX (Maygreen Nociception index) of nociception. The MGRSSI and the MGRNOX value were generated every 1 s. Mean arterial pressure (MAP) and heart rate (HR) were measured every minute, noninvasively. Using the modified Dixon's up-and-down method, the concentration of remifentanil was determined based on the intubation response of the previous patient. The cardiovascular response during endotracheal intubation was defined as positive when MAP or HR is 20% higher than the pre-intubation value. A probit analysis was used for calculating EC50, EC95 and 95% confidence interval (CI). RESULTS: The EC50 and EC95 of remifentanil blunting tracheal intubation responses were found to be 7.731 ng/ml (95%CI: 7.212-8.278 ng/ml) and 8.701 ng/ml (95%CI: 8.199-11.834 ng/ml). There were statistically significant increases in HR, MGRSSI and MGRNOX value to tracheal intubation in the positive responses group compared to the negative group. The most common adverse event was postoperative nausea and vomiting, which occurred in 3 patients. CONCLUSION: Remifentanil effect-site concentration of 7.731 ng/ml is effective in blunting sympathetic responses to tracheal intubation in 50% of patients when combined with etomidate anesthesia. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trials Registry ( www.chictr.org.cn , registration number: ChiCTR2100054565, date of registration: 20/12/2021).

Effect of vitamin C on adrenal suppression following etomidate for rapid sequence induction in trauma patients: a randomized clinical trial.

BACKGROUND: Etomidate is an imidazole derivative that is widely used in the emergency department for Rapid Sequence Intubation (RSI). Although it has a safe hemodynamic profile, there are some concerns about its suppressant effects on the adreno-cortical axis. Vitamin C, as an antioxidant, can play a protective role in this issue. METHOD: In a controlled clinical trial, we studied adult traumatic patients who needed RSI with etomidate. In one group underwent RSI with etomidate and cortisol levels were measured three hours later. In the other group, we administered one gram of vitamin C before etomidate administration, and the cortisol level was measured three hours later. RESULTS: Fifty-one patients have been studied. The serum cortisol level was significantly lower after RSI with etomidate in both groups. In the Vitamin C group, there was a significantly higher cortisol level after RSI in comparison to the control group. CONCLUSION: Etomidate can suppress the cortisol level in trauma patients who undergo RSI. Vitamin C can reduce this suppressant effect of etomidate. TRIAL REGISTRATION: IRCT registration number: IRCT20090923002496N11, URL of trial registry record: https://en.irct.ir/trial/34586 , Date of trial registration: 19/04/2019. Full date of the first registration: 30/05/2019.

Requirement for Discharge in the Care of a Responsible Adult in Procedural Sedation in the Emergency Department: Necessity or Potential Barrier to Health Equity?

BACKGROUND: Procedural sedation is commonly practiced by emergency physicians to facilitate patient care in the emergency department (ED). Although various guidelines have modernized our approach to procedural sedation, many procedural sedation guidelines and practices still often require that patients be discharged into the care of a responsible adult. DISCUSSION: Such requirement for discharge often cannot be met by underserved and undomiciled patients. Benzodiazepines, opioids, propofol, ketamine, "ketofol," etomidate, and methohexital have all been utilized for procedural sedation in the ED. For patients who may require discharge without the presence of an accompanying responsible adult, ketamine, propofol, methohexital, "ketofol," and etomidate are ideal agents for procedural sedation given rapid onsets, short durations of action, and rapid recovery times in patients without renal or hepatic impairment. Proper pre- and postprocedure protocols should be utilized when performing procedural sedation to ensure patient safety. Through the use of appropriate medications and observation protocols, patients can safely be discharged 2 to 4 h postprocedure. CONCLUSION: There is no pharmacodynamic or pharmacokinetic basis to require discharge in the care of a responsible adult after procedural sedation. Thoughtful medication selection and the use of evidence-based pre- and postprocedure protocols in the ED can help circumvent this requirement, which likely disproportionally impacts patients who are of low socioeconomic status or undomiciled.

Changes in Memory, Sedation, and Receptor Kinetics Imparted by the ß2-N265M and ß3-N265M GABAA Receptor Point Mutations.

Point mutations in the ß2 (N265S) and ß3 (N265M) subunits of γ-amino butyric acid type A receptors (GABAARs) that render them insensitive to the general anesthetics etomidate and propofol have been used to link modulation of ß2-GABAARs to sedation and ß3-GABAARs to surgical immobility. These mutations also alter GABA sensitivity, and mice carrying the ß3-N265M mutation have been reported to have impaired baseline memory. Here, we tested the effects of the ß2-N265M and ß3-N265M mutations on memory, movement, hotplate sensitivity, anxiety, etomidate-induced sedation, and intrinsic kinetics. We found that both ß2-N265M and ß3-N265M mice exhibited baseline deficits in the Context Preexposure Facilitation Effect learning paradigm. Exploratory activity was slightly greater in ß2-N265M mice, but there were no changes in either genotype in anxiety or hotplate sensitivity. ß2-N265M mice were highly resistant to etomidate-induced sedation, and heterozygous mice were partially resistant. In rapid solution exchange experiments, both mutations accelerated deactivation two- to three-fold compared to wild type receptors and prevented modulation by etomidate. This degree of change in the receptor deactivation rate is comparable to that produced by an amnestic dose of etomidate but in the opposite direction, indicating that intrinsic characteristics of GABAARs are optimally tuned under baseline conditions to support mnemonic function.

General intravenous anesthetics - pharmacodynamics, pharmacokinetics and chiral properties.

In continuation of our published review on general inhalational anesthetics, the current article presents a survey of intravenous agents for general anaesthesia. From chemical point of view these compounds belong to structurally diverse categories, such as barbiturates - thiopental (Sodium pentothal®, Trapanal®, Pentothal®), methohexital (Brevital®), and hexobarbital (Evipan®, Hexenal®, Citopan®, Tobinal®); non-barbiturate derivatives - ketamine (Ketalar® Ketaset®), esketamine (Ketanest®), and etomidate (Amidate®, Hypnomidate®), phenolic derivatives - propofol (Diprivan®); steroid derivatives - mixture of alfadolone and alfaxalone (Althesin® in human and Saffan® in veterinary anesthesia); and derivatives of phenylacetic acid - propanidid (Epontol®, Sombrevin®). Most of these compounds are chiral, with the exception of propofol and propanidid. Apart from etomidate and esketamine, they are used in the form of their racemates. Besides their characteristics and mechanism of action, attention is centred also on their chiral properties.

Competitive Interactions Between Propofol and Diazepam: Studies in GABAA Receptors and Zebrafish.

Although propofol is among the most commonly administered general anesthetics, its mechanism of action is not fully understood. It has been hypothesized that propofol acts via a similar mechanism as (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate) by binding within the GABAA receptor transmembrane receptor domain at the two ß +/α - subunit interfaces with resultant positive allosteric modulation. To test this hypothesis, we leveraged the ability of diazepam to bind to those sites and act as a competitive antagonist. We used oocyte-expressed α 1 ß 3 γ 2L GABAA receptors to define the actions of diazepam (± flumazenil) on currents activated or potentiated by propofol and a zebrafish activity assay to define the impact of diazepam and flumazenil on propofol-induced anesthesia. We found that diazepam increased the amplitudes of GABAA receptor-mediated currents at nanomolar concentrations but reduced them at micromolar concentrations. The current amplitude changes produced by nanomolar diazepam concentrations were inhibited by flumazenil whereas those produced by micromolar diazepam concentrations were not. Studies of agonist potentiation showed that the micromolar inhibitory action of diazepam was surmountable by high concentrations of propofol and produced a rightward shift in the propofol concentration-response curve characterized by a Schild slope not statistically significantly different from 1, consistent with competition between diazepam and propofol. Although micromolar concentrations of diazepam (plus flumazenil) similarly reduced GABAA receptor currents modulated by propofol and etomidate, it only reduced the anesthetic actions of etomidate. We conclude that while both propofol and etomidate can modulate GABAA receptors by binding to the ß +/α - subunit interfacial sites, propofol-induced anesthesia likely involves additional target sites. SIGNIFICANCE STATEMENT: Although the drug combination of diazepam and flumazenil reverses the GABAA receptor positive modulatory actions of both propofol and (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate), it only reverses the in vivo anesthetic actions of etomidate. These results strongly suggest that distinct mechanisms of action account for the anesthetic actions of these two commonly administered anesthetic agents.

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.

Effects of adding low-dose ketamine to etomidate on serum cortisol levels in critically ill cardiac patients: a randomized clinical trial.

BACKGROUND: Etomidate was associated with an inhibition of adrenal steroid synthesis. This study aimed to evaluate the effects of adding low-dose ketamine to etomidate to minimize the decrease in serum cortisol level in critically ill cardiac patients. METHODS: Sixty adult cardiac patients, ≥ 18 years, who underwent upper endoscopy and Colonoscopy to manage acute anemia in the cardiac intensive care units were enrolled. Patients were randomly divided into two groups: (group (E): n = 30) received etomidate 0.2 mg/kg IV followed by etomidate 0.05 mg/kg IV, and (group (KE): n = 30) received ketamine 0.5 mg/kg IV, then etomidate 0.1 mg/kg IV, followed by etomidate 0.05 mg/kg IV. The primary outcome was Serum cortisol level at 6 h after the procedure. RESULTS: The mean postoperative cortisol level was significantly lower in group E (295.60 ± 49.218 nmol/L) versus group KE (461.00 ± 67.946 nmol/L), with 95% CI = 351.94 to 404.66; p = 0.000. In addition, the estimated serum cortisol reduction level was also significant between groups; In group E, the estimated cortisol level decreased nearly 53% from 632.40 ± 35.066 nmol/L to 295.60 ± 49.218 nmol/L 6 hours postoperative. While in group KE, the estimated cortisol level decreased only 27% from 639.13 ± 43.035 nmol/L to 461.00 ± 67.946 nmol/L. CONCLUSIONS: Single-dose ketamine (0.5 mg/kg) was helpful to decrease the total dose of etomidate and hence decreased the percentage of serum cortisol level in such critically ill patients with preservation of patient satisfaction. TRIAL REGISTRATION: This study is registered on ClinicalTrials.gov ( NCT04857450 ; principal investigator: Mostafa Mohammed Elsaid Elhamamsy; registration date: 23/04/ 2021).

The influence of anaesthetic choice on seizure duration of electroconvulsive therapy; etomidate versus methohexital.

BACKGROUND: Many of the anaesthetic drugs used for electroconvulsive therapy have anticonvulsant properties and may influence efficacy of electroconvulsive therapy. With this study we aim to provide more information on the effect of etomidate and methohexital on seizure duration. We explore the relationship between induction drug, motor and electroencephalography seizure duration. Moreover, we study the relationship of seizure duration and number of therapies. METHODS: In this retrospective study we collected data from patient records from 2005 until 2016. Inclusion criteria were the use of etomidate and/or methohexital and documentation of dosage, electroconvulsive therapy dosage and seizure duration. Exclusion criteria were missing data on either induction drug, dosage or seizure duration. RESULTS: Thirty seven patients were analysed. The mean age was 52 years and seventy six percent were female. Most patients were suffering from affective disorders (81%). Motor and electroencephalography seizure duration were analysed in 679 and 551 electroconvulsive therapies, respectively. Compared to methohexital, motor and electroencephalography seizures under etomidate were 7 and 13 s longer, respectively. Furthermore, there was a negative association between seizure duration and number of treatment and a negative association between seizure duration and electroconvulsive therapy dosage. CONCLUSIONS: This study demonstrates significant longer motor and electroencephalography seizure duration using etomidate compared to methohexital. Etomidate might therefore increase the effectiveness of electroconvulsive therapy. Moreover, we observed a negative association between seizure duration, number of treatment and electroconvulsive therapy dosage. With this study we contribute to the available literature comparing methohexital and etomidate as induction agents for electroconvulsive therapy.

A newly developed anesthetic based on a unique chemical core.

Intravenous anesthetic agents are associated with cardiovascular instability and poorly tolerated in patients with cardiovascular disease, trauma, or acute systemic illness. We hypothesized that a new class of intravenous (IV) anesthetic molecules that is highly selective for the slow type of γ-aminobutyric acid type A receptor (GABAAR) could have potent anesthetic efficacy with limited cardiovascular effects. Through in silico screening using our GABAAR model, we identified a class of lead compounds that are N-arylpyrrole derivatives. Electrophysiological analyses using both an in vitro expression system and intact rodent hippocampal brain slice recordings demonstrate a GABAAR-mediated mechanism. In vivo experiments also demonstrate overt anesthetic activity in both tadpoles and rats with a potency slightly greater than that of propofol. Unlike the clinically approved GABAergic anesthetic etomidate, the chemical structure of our N-arylpyrrole derivative is devoid of the chemical moieties producing adrenal suppression. Our class of compounds also shows minimal to no suppression of blood pressure, in marked contrast to the hemodynamic effects of propofol. These compounds are derived from chemical structures not previously associated with anesthesia and demonstrate that selective targeting of GABAAR-slow subtypes may eliminate the hemodynamic side effects associated with conventional IV anesthetics.

Substituted Cysteine Modification and Protection with n-Alkyl- Methanethiosulfonate Reagents Yields a Precise Estimate of the Distance between Etomidate and a Residue in Activated GABA Type A Receptors.

The anesthetic etomidate modulates synaptic α1ß2/3γ2 GABAA receptors via binding sites located in transmembrane ß+/α- interfaces. Various approaches indicate that etomidate binds near ß2/3M286 side chains, including recent cryogenic electron microscopy images in α1ß2γ2L receptors under nonphysiologic conditions with ∼3.5-Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized n-alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and ß3M286 side chains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1ß3M286Cγ2L GABAA receptors, we measured functional changes after exposing GABA-activated receptors to n-alkyl-MTS reagents, from methyl-MTS to n-decyl-MTS. Based on previous studies using a large sulfhydryl reagent, we anticipated that cysteine modifications large enough to overlap etomidate sites would cause persistently increased GABA sensitivity and decreased etomidate modulation and that etomidate would hinder these modifications, reducing effects. Based on altered GABA or etomidate sensitivity, ethyl-MTS and larger n-alkyl-MTS reagents modified GABA-activated α1ß3M286Cγ2L GABAA receptors. Receptor modification by n-propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked ß3M286C modification by n-propyl-MTS, n-butyl-MTS, and n-hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, and ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and n-propyl-MTS, from which we infer that -S-(n-propyl) is the smallest ß3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and -S-ethyl and -S-(n-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the ß3M286 side chain. SIGNIFICANCE STATEMENT: Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. This study combined electrophysiology, a cysteine substitution, and n-alkyl-methanethiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1ß3γ2L GABA type A receptor residue and etomidate bound in the transmembrane ß+/α- interface.

Molecular Determinants Underlying Delta Selective Compound 2 Activity at δ-Containing GABAA Receptors.

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 ß 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αßγ 2 receptors, and two sites in the transmembrane domain (TMD) - one in the α 4 (+) ß 1 (-) and one in the α 4 (-) ß 1 (+) interface, with the α 4 (-) ß 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) ß 1 (-) interface, either α 4(S303L) of the α 4 (+) side or ß 1(I289Q) of the ß 1 (-) side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD. SIGNIFICANCE STATEMENT: δ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.

Hippocampal ß2-GABAA receptors mediate LTP suppression by etomidate and contribute to long-lasting feedback but not feedforward inhibition of pyramidal neurons.

The general anesthetic etomidate, which acts through γ-aminobutyric acid type A (GABAA) receptors, impairs the formation of new memories under anesthesia. This study addresses the molecular and cellular mechanisms by which this occurs. Here, using a new line of genetically engineered mice carrying the GABAA receptor (GABAAR) ß2-N265M mutation, we tested the roles of receptors that incorporate GABAA receptor ß2 versus ß3 subunits to suppression of long-term potentiation (LTP), a cellular model of learning and memory. We found that brain slices from ß2-N265M mice resisted etomidate suppression of LTP, indicating that the ß2-GABAARs are an essential target in this model. As these receptors are most heavily expressed by interneurons in the hippocampus, this finding supports a role for interneuron modulation in etomidate control of synaptic plasticity. Nevertheless, ß2 subunits are also expressed by pyramidal neurons, so they might also contribute. Therefore, using a previously established line of ß3-N265M mice, we also examined the contributions of ß2- versus ß3-GABAARs to GABAA,slow dendritic inhibition, because dendritic inhibition is particularly well suited to controlling synaptic plasticity. We also examined their roles in long-lasting suppression of population activity through feedforward and feedback inhibition. We found that both ß2- and ß3-GABAARs contribute to GABAA,slow inhibition and that both ß2- and ß3-GABAARs contribute to feedback inhibition, whereas only ß3-GABAARs contribute to feedforward inhibition. We conclude that modulation of ß2-GABAARs is essential to etomidate suppression of LTP. Furthermore, to the extent that this occurs through GABAARs on pyramidal neurons, it is through modulation of feedback inhibition.NEW & NOTEWORTHY Etomidate exerts its anesthetic actions through GABAA receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that ß2-GABAARs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.

A Novel Etomidate Analog EL-0052 Retains Potent Hypnotic Effect and Stable Hemodynamics without Suppressing Adrenocortical Function.

Etomidate is a potent and rapidly acting anesthetic with high therapeutic index (TI) and superior hemodynamic stability. However, side effect of suppressing adrenocortical function limits its clinical use. To overcome this side effect, we designed a novel etomidate analog, EL-0052, aiming to retain beneficial properties of etomidate and avoid its disadvantage of suppressing adrenocortical steroid synthesis. Results exhibited that EL-0052 enhanced GABAA receptors currents with a concentration for EC50 of 0.98 ± 0.02 µM, which was about three times more potent than etomidate (3.07 ± 1.67 µM). Similar to hypnotic potency of etomidate, EL-0052 exhibited loss of righting reflex with ED50s of 1.02 (0.93-1.20) mg/kg in rats and 0.5 (0.45-0.56) mg/kg in dogs. The TI of EL-0052 in rats was 28, which was higher than 22 of etomidate. There was no significant difference in hypnotic onset time, recovery time, and walking time between EL-0052 and etomidate in rats. Both of them had minor effects on mean arterial pressure in dogs. EL-0052 had no significant effect on adrenocortical function in dogs even at a high dose (4.3 × ED50), whereas etomidate significantly inhibited corticosteroid secretion. The inhibition of cortisol synthesis assay showed that EL-0052 had a weak inhibition on cortisol biosynthesis in human H259 cells with an IC50 of 1050 ± 100 nM, which was 2.09 ± 0.27 nM for etomidate. EL-0052 retains the favorable properties of etomidate, including potent hypnotic effect, rapid onset and recovery, stable hemodynamics, and high therapeutic index without suppression of adrenocortical function. SIGNIFICANCE STATEMENT: The novel etomidate analog EL-0052 retains the favorable properties of etomidate without suppressing adrenocortical function and provides a new strategy to optimize the structure of etomidate.

Electroencephalographic dynamics of etomidate-induced loss of consciousness.

BACKGROUND: Highly structured electroencephalography (EEG) oscillations can occur in adults during etomidate-induced general anesthesia, but the link between these two phenomena is poorly understood. Therefore, in the present study, we investigated the electroencephalogram dynamics of etomidate-induced loss of consciousness (LOC) in order to understand the neurological mechanism of etomidate-induced LOC. METHODS: This study is a prospective observational study. Etomidate-induced anesthesia was performed on eligible patients undergoing elective surgery. We analyzed EEG data from 20 patients who received etomidate for the induction of general anesthesia. We used power spectra and coherence methods to process and analyze the EEG data. Our study was based on 4-channel EEG recordings. RESULTS: Compared with the baseline (awake period), etomidate induced an increase in power in delta, theta, alpha and beta waves during LOC. Compared with the awake period, the delta-wave (1-4 Hz), alpha-wave(8-13 Hz), and theta-wave(4-8 Hz) coherence increased significantly during LOC, while the slow-wave (< 1 Hz) coherence decreased. However, the delta wave (1.0-4.0 Hz) during etomidate-induced LOC was more coherent than during the awake period (1.86-3.17 Hz, two-group test for coherence, p < 0.001). CONCLUSIONS: The neural circuit mechanism of etomidate-induced LOC is closely related to the induction of oscillation in delta, theta, alpha and beta waves and the enhancement of delta-wave coherence. TRIAL REGISTRATION: ChiCTR1800017110.