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Stuart A. Forman - One of the best experts on this subject based on the ideXlab platform.
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substituted cysteine modification and protection with n alkyl mts reagents yields a precise estimate of the distance between Etomidate and a residue in activated gaba type a receptors
Molecular Pharmacology, 2021Co-Authors: Ryan J Fantasia, Anahita Nourmahnad, Elizabeth Halpin, Stuart A. FormanAbstract:The anesthetic Etomidate modulates synaptic α1β2/3γ2 GABAA receptors via binding sites located in transmembrane β+/α- interfaces. Various approaches indicate that Etomidate binds near β2/3M286 sidechains, including recent cryogenic electron microscopy images in α1β2γ2L receptors under non-physiologic conditions with ~3.5 A 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 β3M286 sidechains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1β3M286Cγ2L 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 α1β3M286Cγ2L GABAA receptors. Receptor modification by n-propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased Etomidate modulation. Receptor-bound Etomidate blocked β3M286C 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, while 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 β3M286C 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 A from the β3M286 sidechain. Significance Statement Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. We combined electrophysiology, a cysteine substitution, and n-alkyl-methanthiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1β3γ2L GABA type A receptor residue and Etomidate bound in the transmembrane β+/α- interface.
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cysteine substitutions define Etomidate binding and gating linkages in the α m1 domain of γ aminobutyric acid type a gabaa receptors
Journal of Biological Chemistry, 2013Co-Authors: Deirdre S. Stewart, Rooma Desai, Mayo Hotta, David C Chiara, Richard W Olsen, Stuart A. FormanAbstract:Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive Etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to Etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and Etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if Etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected Etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to Etomidate binding. Thus, Etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of Etomidate approximately orthogonal to the transmembrane axis.
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state dependent Etomidate binding in gabaa receptors probed with cysteine substitution and protection from modification
Biophysical Journal, 2013Co-Authors: Deirdre S. Stewart, Mayo Hotta, Stuart A. FormanAbstract:Background: A central tenet of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. This is a difficult concept to test experimentally, because in most agonist-receptor systems, the act of assessing agonist binding affinity results in receptor activation. In α1β2γ2L GABAA receptors, the anesthetic Etomidate is an allosteric agonist at high concentrations, and potentiates GABA activation at low concentrations. Sites mediating both actions were photolabeled with azi-Etomidate, identifying the β2M286 sidechain as a contact point. We used a cysteine substitution and sulfhydryl modification to study Etomidate interactions with this sidechain. Methods: using two-microelectrode voltage clamp electrophysiology in Xenopus oocytes, we characterized Etomidate agonism and potentiation of GABA activation in α1β2M286Cγ2L GABAA receptors. We studied covalent modification of the β2M286C sidechain by a sulfhydryl-selective reagent, para-chloromercuribenzenesulfonate (pCMBS) with and without GABA. Etomidate-dependent protection of the sulfhydryl was also assessed. Results: Oocyte-expressed α1β2M286Cγ2L receptors displayed reduced sensitivity to GABA, and no agonism by Etomidate. However, Etomidate still enhanced GABA-activated currents from α1β2M286Cγ2L receptors. Exposure of α1β2M286Cγ2L receptors to pCMBS irreversibly increased activation by low (EC10) GABA. The apparent rate of pCMBS modification increased with addition of GABA. Etomidate in a concentration-dependent manner reduced the rate of β2M286C modification by pCMBS. Etomidate protection at β2M286C was enhanced in the presence of GABA. Conclusions: Etomidate, like azi-Etomidate, binds next to the β2M286 residue. Based on its apparent protectant potency, the affinity of Etomidate for GABA-bound receptors is greater than that for resting receptors, as expected. Our results have important implications for both designing and interpreting experiments that use mutations to map allosteric modulator/agonist sites.Support: NIH (R01GM89745 and P01GM58448)
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pharmacological studies of methoxycarbonyl Etomidate s carboxylic acid metabolite
Anesthesia & Analgesia, 2012Co-Authors: Ervin Pejo, Shaukat S Husain, Stuart A. Forman, Marian Haburcak, Douglas E RainesAbstract: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)β(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.
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Mutations in the GABAA receptor that mimic the allosteric ligand Etomidate.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Stuart A. Forman, Deirdre S. StewartAbstract:Etomidate is a hydrophobic molecule, a potent general anesthetic, and the best understood drug in this group. Etomidate's target molecules are GABA(A) receptors, its site of action has been identified with photolabeling, and a quantitative allosteric coagonist model has emerged for Etomidate effects on GABA(A) receptors. We have shown that when methionine residues that are thought to be adjacent to the Etomidate site are mutated to tryptophan, that the bulky hydrophobic side-chains alter mutant GABA(A) receptor function in ways that mimic the effects of Etomidate binding to wild-type receptors. Furthermore, these mutations reduce receptor modulation by Etomidate. Both of these observations support the hypothesis that these methionine residues form part of the Etomidate binding pocket.
Douglas E Raines - One of the best experts on this subject based on the ideXlab platform.
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Behavioral and steroidogenic pharmacology of phenyl ring substituted Etomidate analogs in rats
BMC pharmacology & toxicology, 2019Co-Authors: Megan Mcgrath, Alissa Hofmann, Douglas E RainesAbstract:Cushing’s syndrome is an endocrine disorder characterized by the overproduction of adrenocortical steroids. Steroidogenesis enzyme inhibitors are the mainstays of pharmacological treatment. Unfortunately, they produce significant side effects. Among the most potent inhibitors is the general anesthetic Etomidate whose GABAA receptor-mediated sedative-hypnotic actions restrict use. In this study, we defined the sedative-hypnotic and steroidogenesis inhibiting actions of Etomidate and four phenyl-ring substituted Etomidate analogs (dimethoxy-Etomidate, isopropoxy-Etomidate, naphthalene-Etomidate, and naphthalene (2)-Etomidate) that possess negligible GABAA receptor modulatory activities. In the first set of experiments, male Sprague-Dawley rats were assessed for loss of righting reflexes (LoRR) after receiving intravenous boluses of either Etomidate (1 mg/kg) or an Etomidate analog (40 mg/kg). In the second set of experiments, rats were assessed for LoRR and their abilities to produce adrenocortical and androgenic steroids after receiving 2-h infusions (0.5 mg kg− 1 min− 1) of either Etomidate or an Etomidate analog. All rats that received Etomidate boluses or infusions had LoRR that persisted for minutes or hours, respectively. In contrast, no rat that received an Etomidate analog had LoRR. Compared to rats in the vehicle control group, rats that received Etomidate analog infusions had plasma corticosterone and aldosterone concentrations that were reduced by 80–84% and 68–94%, respectively. Rats that received Etomidate infusions had plasma corticosterone and aldosterone concentrations that were also significantly reduced (by 92 and 96%, respectively). Rats that received Etomidate or isopropoxy-Etomidate had significant reductions (90 and 57%, respectively) in plasma testosterone concentrations whereas those that received naphthalene-Etomidate had significant increases (1400%) in plasma dehydroepiandrosterone concentrations. Neither Etomidate nor any Etomidate analog significantly affected plasma androstenedione and dihydrotestosterone concentrations. Our studies demonstrate that the four phenyl-ring substituted Etomidate analogs form a novel class of compounds that are devoid of sedative-hypnotic activities and suppress plasma concentrations of adrenocortical steroids but vary in their effects on plasma concentrations of androgenic steroids.
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Etomidate and Etomidate Analog Binding and Positive Modulation of γ-Aminobutyric Acid Type A Receptors: Evidence for a State-dependent Cutoff Effect.
Anesthesiology, 2018Co-Authors: Megan Mcgrath, Keith W. Miller, Xiaojuan Zhou, Selwyn S. Jayakar, Mansi Tolia, Jonathan B. Cohen, Douglas E RainesAbstract:WHAT WE ALREADY KNOW ABOUT THIS TOPIC WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Naphthalene-Etomidate, an Etomidate analog containing a bulky phenyl ring substituent group, possesses very low γ-aminobutyric acid type A (GABAA) receptor efficacy and acts as an anesthetic-selective competitive antagonist. Using Etomidate analogs containing phenyl ring substituents groups that range in volume, we tested the hypothesis that this unusual pharmacology is caused by steric hindrance that reduces binding to the receptor's open state. METHODS The positive modulatory potencies and efficacies of Etomidate and phenyl ring-substituted Etomidate analogs were electrophysiology defined in oocyte-expressed α1β3γ2L GABAA receptors. Their binding affinities to the GABAA receptor's two classes of transmembrane anesthetic binding sites were assessed from their abilities to inhibit receptor labeling by the site-selective photolabels [H]azi-Etomidate and tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS The positive modulatory activities of Etomidate and phenyl ring-substituted Etomidate analogs progressively decreased with substituent group volume, reflecting significant decreases in both potency (P = 0.005) and efficacy (P < 0.0001). Affinity for the GABAA receptor's two β - α anesthetic binding sites similarly decreased with substituent group volume (P = 0.003), whereas affinity for the receptor's α - β/γ - β sites did not (P = 0.804). Introduction of the N265M mutation, which is located at the β - α binding sites and renders GABAA receptors Etomidate-insensitive, completely abolished positive modulation by naphthalene-Etomidate. CONCLUSIONS Steric hindrance selectively reduces phenyl ring-substituted Etomidate analog binding affinity to the two β - α anesthetic binding sites on the GABAA receptor's open state, suggesting that the binding pocket where Etomidate's phenyl ring lies becomes smaller as the receptor isomerizes from closed to open.
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dimethoxy Etomidate a nonhypnotic Etomidate analog that potently inhibits steroidogenesis
Journal of Pharmacology and Experimental Therapeutics, 2018Co-Authors: Megan Mcgrath, Douglas E RainesAbstract:Cushing’s syndrome is characterized by the overproduction of adrenocortical steroids. Steroidogenesis inhibitors are mainstays of medical therapy for Cushing’s syndrome; unfortunately, adverse side effects and treatment failures are common with currently available drugs. The general anesthetic induction agent Etomidate is among the most potent inhibitors of adrenocortical steroidogenesis. However, its use as a treatment for Cushing’s syndrome is complicated by its sedative-hypnotic activity and ability to produce myoclonus, central nervous system actions thought to be mediated by the GABAA receptor. Here, we describe the pharmacology of the novel Etomidate analog (R)-ethyl 1-(1-(3,5-dimethoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (dimethyoxy-Etomidate). In contrast to Etomidate, dimethoxy-Etomidate minimally enhanced GABA-evoked GABAA receptor-mediated currents even at a near-saturating aqueous concentration. In Sprague-Dawley rats, dimethoxy-Etomidate’s potency for producing loss of righting reflexes – an animal model of sedation/hypnosis – was two orders of magnitude lower than that of Etomidate, and it did not produce myoclonus. However similar to Etomidate, dimethoxy-Etomidate potently suppressed adrenocortical steroid synthesis primarily by inhibiting 11β-hydroxylase. [3H]-Etomidate binding to rat adrenocortical membranes was inhibited by dimethoxy-Etomidate in a biphasic manner with IC50’s of 8.2 nM and 3970 nM whereas that by Etomidate was monophasic with an IC50 of 22 nM. Our results demonstrate that similar to Etomidate, dimethoxy-Etomidate potently and dose-dependently suppresses adrenocortical steroid synthesis by inhibiting 11β-hydroxylase. However, it is essentially devoid of Etomidate’s GABAA receptor positive modulatory and sedative-hypnotic activities and produces no myoclonus, providing proof-of-concept for the design of Etomidate analogs without important central nervous system actions for the pharmacologic treatment of Cushing’s syndrome.
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Dimethoxy-Etomidate: A Nonhypnotic Etomidate Analog that Potently Inhibits Steroidogenesis.
The Journal of pharmacology and experimental therapeutics, 2017Co-Authors: Megan Mcgrath, Douglas E RainesAbstract:Cushing’s syndrome is characterized by the overproduction of adrenocortical steroids. Steroidogenesis inhibitors are mainstays of medical therapy for Cushing’s syndrome; unfortunately, adverse side effects and treatment failures are common with currently available drugs. The general anesthetic induction agent Etomidate is among the most potent inhibitors of adrenocortical steroidogenesis. However, its use as a treatment of Cushing’s syndrome is complicated by its sedative-hypnotic activity and ability to produce myoclonus, central nervous system actions thought to be mediated by the GABAA receptor. Here, we describe the pharmacology of the novel Etomidate analog (R)-ethyl 1-(1-(3,5-dimethoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (dimethoxy-Etomidate). In contrast to Etomidate, dimethoxy-Etomidate minimally enhanced GABA-evoked GABAA receptor–mediated currents even at a near-saturating aqueous concentration. In Sprague-Dawley rats, dimethoxy-Etomidate’s potency for producing loss of righting reflexes—an animal model of sedation/hypnosis—was 2 orders of magnitude lower than that of Etomidate, and it did not produce myoclonus. However, similar to Etomidate, dimethoxy-Etomidate potently suppressed adrenocortical steroid synthesis primarily by inhibiting 11β-hydroxylase. [3H]Etomidate binding to rat adrenocortical membranes was inhibited by dimethoxy-Etomidate in a biphasic manner with IC50 values of 8.2 and 3970 nM, whereas that by Etomidate was monophasic with an IC50 of 22 nM. Our results demonstrate that, similar to Etomidate, dimethoxy-Etomidate potently and dose-dependently suppresses adrenocortical steroid synthesis by inhibiting 11β-hydroxylase. However, it is essentially devoid of Etomidate’s GABAA receptor positive modulatory and sedative-hypnotic activities and produces no myoclonus, providing proof of concept for the design of Etomidate analogs without important central nervous system actions for the pharmacologic treatment of Cushing’s syndrome.
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distinct hypnotic recoveries after infusions of methoxycarbonyl Etomidate and cyclopropyl methoxycarbonyl mEtomidate the role of the metabolite
Anesthesia & Analgesia, 2016Co-Authors: Ervin Pejo, Jifeng Liu, Xiangjie Lin, Douglas E RainesAbstract: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 effe
Qingquan Lian - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells
2016Co-Authors: Hua-cheng Liu, Hongguo Guan, Zhichuan Chen, Danyan Zhu, Han Lin, Qingquan LianAbstract:Background Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomi-date is the reduced plasma concentration of corticosteroids, leading to the abnormal reac-tion of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of eto-midate on Leydig cell steroidogenesis during the cell maturation process is not well established. Methodology Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM Etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progester-one, androstenedione, testosterone and dihydrotestosterone, respectively. The concentra-tions of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidat
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Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells
PLOS ONE, 2015Co-Authors: Chan Wang, Hongguo Guan, Zhichuan Chen, Yuanyuan Hu, Senlin Li, Cong Hu, Qingquan LianAbstract:Background Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of Etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of Etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Methodology Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM Etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of Etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of Etomidate on CYP11A1 and HSD3B1 activity. Results and Conclusions In intact Leydig cells, 30 μM Etomidate significantly inhibited androgen synthesis. Further studies showed that Etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, Etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, Etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, Etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, Etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.
Solimanian Seyed Sedigheh - One of the best experts on this subject based on the ideXlab platform.
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Sedation with Etomidate-fentanyl versus propofol-fentanyl in colonoscopies: A prospective randomized study
caspian journal of internal medicine, 2015Co-Authors: Banihashem Nadia, Basirat Majid, Shokri Shirvany Javad, Kashifard Mehrdad, Taheri Hasan, Savadkohi Shahriyar, Hosseini Vahid, Alijanpour Ebrahim, Solimanian Seyed SedighehAbstract:BACKGROUND: The combination of propofol-fentanyl for sedation during colonoscopy is characterized by high prevalence of side effects. Etomidate-fentanyl provides fewer hemodynamic and respiratory complications. The aim of our study was to compare the safety and efficacy of propofol-fentanyl and Etomidate-fentanyl for conscious sedation in elective colonoscopy. METHODS: This double-blind clinical trial was conducted on 90 patients aged between 18- 55 years old who were candidates for elective colonoscopy. Patients were randomized to receive sedation with fentanyl plus propofol or Etomidate. Two minutes after injecting 1 micro/kg of fentanyl, the patients received 0.5mg/kg propofol by infusion (25 µ/kg/min) or 0.1 mg/kg etmoidate (15 µ/kg/min). Pulse rate, mean arterial blood pressure, respiratory rate, and saturation of peripheral oxygen (SPO2) were monitored. In addition, the patient and colonoscopist satisfaction, the recovery time, sedation and pain score in both groups were assessed. RESULTS: Sedation score in propofol group was higher. Pain score as well as the physician and patient satisfaction showed no significant difference between the two study groups. Hemodynamic changes and arterial saturation were the same in both groups. The duration of recovery was 1.27±0.82 minutes in the Etomidate group; versus 2.57±2.46 minutes in the propofol group (P=0.001). Recovery time in the etmoid group was 2.68±3.14 minutes and in the propofol group was 5.53±4.67 minutes (p=0.001). CONCLUSION: The combination of fentanyl and Etomidate provides an acceptable alternative to sedation with fentanyl and propofol with the advantage of significantly faster recovery time, in the outpatient setting.
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Sedation with Etomidate-fentanyl versus propofol-fentanyl in colonoscopies: A prospective randomized study
caspian journal of internal medicine, 2015Co-Authors: Banihashem Nadia, Basirat Majid, Shokri Shirvany Javad, Kashifard Mehrdad, Taheri Hasan, Savadkohi Shahriyar, Hosseini Vahid, Alijanpour Ebrahim, Solimanian Seyed SedighehAbstract:BACKGROUND: The combination of propofol-fentanyl for sedation during colonoscopy is characterized by high prevalence of side effects. Etomidate-fentanyl provides fewer hemodynamic and respiratory complications. The aim of our study was to compare the safety and efficacy of propofol-fentanyl and Etomidate-fentanyl for conscious sedation in elective colonoscopy. METHODS: This double-blind clinical trial was conducted on 90 patients aged between 18- 55 years old who were candidates for elective colonoscopy. Patients were randomized to receive sedation with fentanyl plus propofol or Etomidate. Two minutes after injecting 1 micro/kg of fentanyl, the patients received 0.5mg/kg propofol by infusion (25 µ/kg/min) or 0.1 mg/kg etmoidate (15 µ/kg/min). Pulse rate, mean arterial blood pressure, respiratory rate, and saturation of peripheral oxygen (SPO2) were monitored. In addition, the patient and colonoscopist satisfaction, the recovery time, sedation and pain score in both groups were assessed. RESULTS: Sedation score in propofol group was higher. Pain score as well as the physician and patient satisfaction showed no significant difference between the two study groups. Hemodynamic changes and arterial saturation were the same in both groups. The duration of recovery was 1.27±0.82 minutes in the Etomidate group; versus 2.57±2.46 minutes in the propofol group (P=0.001). Recovery time in the etmoid group was 2.68±3.14 minutes and in the propofol group was 5.53±4.67 minutes (p=0.001). CONCLUSION: The combination of fentanyl and Etomidate provides an acceptable alternative to sedation with fentanyl and propofol with the advantage of significantly faster recovery time, in the outpatient setting.
Deirdre S. Stewart - One of the best experts on this subject based on the ideXlab platform.
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cysteine substitutions define Etomidate binding and gating linkages in the α m1 domain of γ aminobutyric acid type a gabaa receptors
Journal of Biological Chemistry, 2013Co-Authors: Deirdre S. Stewart, Rooma Desai, Mayo Hotta, David C Chiara, Richard W Olsen, Stuart A. FormanAbstract:Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive Etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to Etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and Etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if Etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected Etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to Etomidate binding. Thus, Etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of Etomidate approximately orthogonal to the transmembrane axis.
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state dependent Etomidate binding in gabaa receptors probed with cysteine substitution and protection from modification
Biophysical Journal, 2013Co-Authors: Deirdre S. Stewart, Mayo Hotta, Stuart A. FormanAbstract:Background: A central tenet of ligand-receptor theory is that agonists bind more tightly to active than to inactive receptors. This is a difficult concept to test experimentally, because in most agonist-receptor systems, the act of assessing agonist binding affinity results in receptor activation. In α1β2γ2L GABAA receptors, the anesthetic Etomidate is an allosteric agonist at high concentrations, and potentiates GABA activation at low concentrations. Sites mediating both actions were photolabeled with azi-Etomidate, identifying the β2M286 sidechain as a contact point. We used a cysteine substitution and sulfhydryl modification to study Etomidate interactions with this sidechain. Methods: using two-microelectrode voltage clamp electrophysiology in Xenopus oocytes, we characterized Etomidate agonism and potentiation of GABA activation in α1β2M286Cγ2L GABAA receptors. We studied covalent modification of the β2M286C sidechain by a sulfhydryl-selective reagent, para-chloromercuribenzenesulfonate (pCMBS) with and without GABA. Etomidate-dependent protection of the sulfhydryl was also assessed. Results: Oocyte-expressed α1β2M286Cγ2L receptors displayed reduced sensitivity to GABA, and no agonism by Etomidate. However, Etomidate still enhanced GABA-activated currents from α1β2M286Cγ2L receptors. Exposure of α1β2M286Cγ2L receptors to pCMBS irreversibly increased activation by low (EC10) GABA. The apparent rate of pCMBS modification increased with addition of GABA. Etomidate in a concentration-dependent manner reduced the rate of β2M286C modification by pCMBS. Etomidate protection at β2M286C was enhanced in the presence of GABA. Conclusions: Etomidate, like azi-Etomidate, binds next to the β2M286 residue. Based on its apparent protectant potency, the affinity of Etomidate for GABA-bound receptors is greater than that for resting receptors, as expected. Our results have important implications for both designing and interpreting experiments that use mutations to map allosteric modulator/agonist sites.Support: NIH (R01GM89745 and P01GM58448)
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Mutations in the GABAA receptor that mimic the allosteric ligand Etomidate.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Stuart A. Forman, Deirdre S. StewartAbstract:Etomidate is a hydrophobic molecule, a potent general anesthetic, and the best understood drug in this group. Etomidate's target molecules are GABA(A) receptors, its site of action has been identified with photolabeling, and a quantitative allosteric coagonist model has emerged for Etomidate effects on GABA(A) receptors. We have shown that when methionine residues that are thought to be adjacent to the Etomidate site are mutated to tryptophan, that the bulky hydrophobic side-chains alter mutant GABA(A) receptor function in ways that mimic the effects of Etomidate binding to wild-type receptors. Furthermore, these mutations reduce receptor modulation by Etomidate. Both of these observations support the hypothesis that these methionine residues form part of the Etomidate binding pocket.
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p-Trifluoromethyldiazirinyl-Etomidate: a potent photoreactive general anesthetic derivative of Etomidate that is selective for ligand-gated cationic ion channels
Journal of medicinal chemistry, 2010Co-Authors: S. Shaukat Husain, Joseph F Cotten, Deirdre S. Stewart, Rooma Desai, Elizabeth Kelly, Ayman K. Hamouda, Zuzana Dostalova, Xiaojuan Zhou, Douglas A. RainesAbstract:We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-Etomidate), or TFD-Etomidate, a novel photoactivable derivative of the stereoselective general anesthetic Etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to Etomidate’s, but stereoselectivity was reversed and attenuated. Relative to Etomidate, TFD-Etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((α1)2β1δ1γ1) and 5-HT3AR (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-Etomidate enhanced currents elicited from inhibitory α1β2γ2L GABAARs by low concentrations of GABA, but with a lower efficacy than R-Etomidate, and site-directed mutagenesis suggests they act at different sites. [3H]TFD-Etomidate photolabeled the α-subunit of the nAChR in a manner allosterically regulated by agoni...
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MTS-Etomidate Selectively Reacts Within the GABAA Receptor Etomidate Binding Site
Biophysical Journal, 2009Co-Authors: Deirdre S. Stewart, S. Shaukat Husain, Keith W. Miller, Stuart A. FormanAbstract:The general anesthetic Etomidate exerts its major clinical actions through potentiation of GABAA receptor activation. GABAA receptors are pentameric, usually consisting of combinations of alpha, beta, and gamma subunits. A photoreactive structural analog of etomdiate ([3H]aziEtomidate) labels amino acids on transmembrane domains in both alpha (M236) and beta (M286) subunits. This suggests the presence of two interfacial anesthetic binding sites per GABAA receptor, consistent with receptor structural homology models based on Torpedo nicotinic acetylcholine receptor. To further characterize the Etomidate binding site, we have created a number of Cys substitutions within the α/β intersubunit region and have synthesized a novel Etomidate derivative, 2-(methylsulfonyl) thio-Etomidate (MTS-Etomidate), designed to covalently modify cysteines. Human GABAA receptors (α1, β2, γ2L) were expressed in Xenopus oocytes and current responses were measured using two-electrode voltage clamp. Using pCMBS and MTSEA, we found that cysteine substitutions at both α1M236 and β2M286, were accessible to modification. Cysteine modification was also evident with pCMBS at α1L232C, one helical turn above α1M236 in TM1. In contrast, modification by MTS-Etomidate was evident only at α1M236C, but not at β2M286C or, α1L232C. These results suggest that MTS-Etomidate orients itself in a precise conformation within its binding pocket and acts as a highly selective structural probe, yielding information not only about the residues with which it interacts but also about the orientation of Etomidate within the biding pocket consistent with its mechanism of action.