Anesthetic binding in a pentameric ligand-gated ion channel: GLIC.

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K(D)) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.

Hydrogenative desulphurization of thienopyrrolizinones: an easy and selective access to (Z)-phenethylidenepyrrolizinones with in vitro cytotoxic activity.

Attempts in view to dearomatize some previously reported tripentones with potent antineoplastic activities led in thiophene series to an unexpected hydrogenative desulphurization reaction. The resulting (Z)-phenethylidenepyrrolizinones were tested in vitro over human epidermoid carcinoma KB cell line. The results of this biological evaluation indicated that the tricyclic core of our model can be cleaved with a partial respect of the activity.

Total synthesis of brevenal.

A total synthesis of brevenal is described. The pentacyclic ether core was constructed by the intramolecular allylation of alpha-acetoxy ether and subsequent ring-closing metathesis. Both of the diene side chains were introduced by Wittig olefination and a Horner-Wadsworth-Emmons reaction, respectively, in a highly stereoselective manner.

Brevisamide: an unprecedented monocyclic ether alkaloid from the dinoflagellate Karenia brevis that provides a potential model for ladder-frame initiation.

The dinoflagellate Karenia brevis is known for the production of brevetoxins, a family of polycyclic ether toxins, as well as their antagonist brevenal. Further examination of organic extracts of K. brevis has uncovered yet another unprecedented cyclic ether alkaloid named brevisamide. This report describes the structure elucidation of brevisamide based on detailed MS and NMR spectral analysis, and the importance of this new compound in shedding light on the biogenesis of fused polyethers is discussed.

Abeta peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: a NMR study.

Abeta peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Abeta peptide and induce Abeta oligomerization. The molecular mechanism of Abeta peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly (15)N labeled Abeta40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Abeta peptide located in the hinge region leading to Abeta oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Abeta oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Abeta oligomerization was found. Perturbation of these specific Abeta residues (G29, A30 and I31) by different anesthetics could play an important role to induce Abeta oligomerization.

Total synthesis of (-)-brevenal: a concise synthetic entry to the pentacyclic polyether core.

Total synthesis of (-)-brevenal, a novel marine polycyclic ether natural product, is described. Highly efficient and scalable entries to the AB-ring exo-olefin and the DE-ring enol phosphate and a rapid construction of the C-ring by means of our Suzuki-Miyaura coupling-based strategy realized a concise synthesis of the pentacyclic skeleton of (-)-brevenal. The present synthesis is considerably more efficient than our previous synthesis (longest linear sequence: 50 steps from 2-deoxy-d-ribose).

Convergent strategies for the total synthesis of polycyclic ether marine metabolites.

Marine polycyclic ether natural products continue to fascinate chemists and biologists due to their exceptionally large and complex molecular architectures and potent and diverse biological activities. Tremendous progress has been made over the past decade toward the total synthesis of marine polycyclic ether natural products. In this area, a convergent strategy for assembling small fragments into an entire molecule always plays a key role in successful total synthesis. This review describes our efforts to develop convergent strategies for the synthesis of polycyclic ethers and their application to the total synthesis of gambierol, gymnocin-A, and brevenal, and to the partial synthesis of the central part of ciguatoxins and the nonacyclic polyether skeleton of gambieric acids.

A minimal physiological model of thiopental distribution kinetics based on a multiple indicator approach.

Currently available models of thiopental disposition kinetics using only plasma concentration-time data neglect the influence of intratissue diffusion and provide no direct information on tissue partitioning in individual subjects. Our approach was based on a lumped-organ recirculatory model that has recently been applied to unbound compounds. The goal was to find the simplest model that accounts for the heterogeneity in tissue partition coefficients and accurately describes initial distribution kinetics of thiopental in dogs. To ensure identifiability of the underlying axially distributed capillary-tissue exchange model, simultaneously measured disposition data of the vascular indicator, indocyanine green, and the marker of whole body water, antipyrine, were analyzed together with those of thiopental. A model obtained by grouping the systemic organs in two subsystems containing fat and nonfat tissues, successfully described all data and allowed an accurate estimation of model parameters. The estimated tissue partition coefficients were in accordance with those measured in rats. Because of the effect of tissue binding, the diffusional equilibration time characterizing intratissue distribution of thiopental is longer than that of antipyrine. The approach could potentially be used in clinical pharmacokinetics and could increase our understanding of the effect of obesity on the disposition kinetics of lipid-soluble drugs.

Molecular understanding of Abeta peptide interaction with isoflurane, propofol, and thiopental: NMR spectroscopic study.

Abeta peptide is the major component of senile plaques (SP), which accumulate in the brain of a patient with Alzheimer's disease (AD). A recent report indicated that isoflurane enhanced Abeta oligomerization (micro-aggregation) and subsequent cytotoxicity of the Abeta peptide. A separate study showed that a clinically relevant concentration of isoflurane induces apoptosis and increases Abeta production in a human neuroglioma cell line. In vitro studies have indicated that halothane interacts specifically with Abeta peptide to induce oligomerization and that Abeta42 oligomerizes faster than Abeta40. The specific interactions of isoflurane, propofol, and thiopental with uniformly 15N labeled Abeta40 and Abeta42 peptide were investigated using multidimensional nuclear magnetic resonance (NMR) experiments. We found that isoflurane and propofol (at higher concentration) interact with Abeta40 peptides and induce Abeta oligomerization. Thiopental does not interact with specific residues (G29, A30, and I31) of Abeta40; hence, the peptide remains in the monomeric form. On the basis of our NMR study, thiopental does not oligomerize Abeta40 even at higher concentrations.

Total synthesis, structure revision, and absolute configuration of (-)-brevenal.

Total synthesis of structure 1 originally proposed for brevenal, a nontoxic polycyclic ether natural product isolated from the Florida red tide dinoflagellate, Karenia brevis, was accomplished. The key features of the synthesis involved (i) convergent assembly of the pentacyclic polyether skeleton based on our developed Suzuki-Miyaura coupling chemistry and (ii) stereoselective construction of the multi-substituted (E,E)-dienal side chain by using copper(I) thiophen-2-carboxylate (CuTC)-promoted modified Stille coupling. The disparity of NMR spectra between the synthetic material and the natural product required a revision of the proposed structure. Detailed spectroscopic comparison of synthetic 1 with natural brevenal, coupled with the postulated biosynthetic pathway for marine polyether natural products, suggested that the natural product was most likely represented by 2, the C26 epimer of the proposed structure 1. The revised structure was finally validated by completing the first total synthesis of (-)-2, which also unambiguously established the absolute configuration of the natural product.

Comparison of intracarotid anesthetics for EEG silence.

The goal of this study was to compare systemic and cerebrovascular effects of three anesthetic drugs (etomidate, thiopental, and propofol) when delivered by intracarotid and intravenous routes in doses that produce electrocerebral silence (electroencephalography [EEG]). EEG activity, mean arterial pressure (MAP), and laser Doppler flow as a proxy of cerebral blood flow (CBF) of 24 anesthetized New Zealand white rabbits were continuously recorded. Data were compared at three timepoints: baseline, during EEG silence, and after recovery of EEG activity. Drugs were randomly injected via the carotid artery to produce 10 minutes of EEG silence. After 30 minutes of rest, intravenous boluses of the same drug were injected to achieve 10 minutes of EEG silence. During EEG silence, transient hypotension was seen with intracarotid propofol, but there was no decrease in CBF. MAP and CBF did not decrease with either intracarotid etomidate or thiopental during EEG silence. Intracarotid/intravenous dose ratio of propofol (26%+/-22%; n=8, P<0.02) was much higher than that of etomidate and thiopental (14%+/-2% and 19%+/-11%, respectively; NS). Collectively, these results suggest intracarotid etomidate and thiopental are more useful than propofol in producing EEG silence because they offer better dose advantage and are less likely to impair cerebral or systemic hemodynamics.

Inhibition of bacterial growth by different mixtures of propofol and thiopentone.

Propofol is, as a result of its formulation, an ideal bacterial and yeast culture medium. An outbreak of sepsis in humans and an increase in wound infections in dogs has been ascribed to the use of propofol. It has been previously reported that a 1:1 mixture of propofol and thiopentone has bactericidal properties. This study was undertaken to determine if further serial mixtures of propofol and thiopentone maintained the bactericidal properties. Mixtures of 1:1 (solution A), 5:1 (solution B), 10:1 (solution C), 50:1 (solution D) and 100:1 (solution E) of 1% propofol to 2.5 % thiopentone, 2.5% thiopentone (solution T), 1% propofol (solution P) and saline (solution S) were prepared and inoculated with between 10(5) and 10(6) colony-forming units of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. A sample was withdrawn from each solution at 0, 1, 6, 12, 48 and 120 hours after inoculation and a bacterial count was performed. This study showed that thiopentone and solution A behaved in similar fashion by inhibiting bacterial growth and was bactericidal after 48 hours. Solution B was not bactericidal against S. aureus and C. albicans. Propofol and solutions D and E all supported growth of all the organisms tested. These data indicate that mixtures of propofol and thiopentone at a ratio less than 1:1 do not maintain the bactericidal properties.

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system.

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.

Membrane sensors for the selective determination of thiopental.

Novel miniaturized polyurethane (PU) membrane sensors in an all-solid state graphite support were developed, electrochemically evaluated and used for the assay of thiopental drug. The thiopental (T) sensors are based on the formation of ion-association complexes of thiopental with copper(II) and cobalt(II)-bathophenanthroline (bphen) counter anions as electroactive materials dispersed in a polyurethane matrix. The sensors show a linear response for thiopental over the range of 1 x 10(-1) - 5 x 10(-5) M thiopental at 25 degrees C over the pH range 6 - 11 with anionic slopes of -28.7 and -28.3 mV decade(-1) with Cu- and Co-bphen thiopental membrane sensors, respectively. These sensors exhibit a fast response time (25 - 45 s), a low detection limit (5 x 10(-6) M), a long lifetime (7 weeks) and good stability. The selectivity coefficients for thiopental sensors relative to the number of interfering anions, were investigated. These sensors were used for the direct potentiometry of thiopental in a pharmaceutical formulation and human serum. Results with mean accuracy of 99.8 +/- 0.5% of nominal were obtained, which compare well with data obtained using spectrophotometric (UV-Vis) and British Pharmacopoeia (BP) methods.

Brevenal is a natural inhibitor of brevetoxin action in sodium channel receptor binding assays.

1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.

Electroencephalographic effects of thiopentone and its enantiomers in the rat.

Electrophysiological studies with some chiral barbiturates have shown that one enantiomer can be excitant while the other is depressant. Thiopentone, a chiral barbiturate, has both differences in potency between enantiomers and biphasic effects on the electroencephalogram (EEG). This study investigated whether a differential EEG activity between the enantiomers of thiopentone could account for the biphasic effects. Rats were administered rac-, R- or S-thiopentone to determine the nature and time course of quantitative EEG effects. Two studies using computer-controlled i.v. infusions of the three drugs were performed in groups of animals previously prepared with EEG electrodes and/or arterial blood sampling cannulae. Study 1 used several stepwise increments in plasma drug concentration over 35 min, followed by washout. Study 2 used a 4 min period of constant plasma drug concentration, followed by washout. In both studies, both enantiomers and racemate caused an initial EEG activation followed by deactivation. Quantitative enantioselectivity was found for depression. The extent of depression was significantly less for R-thiopentone (P=0.008) and racthiopentone (P=0.038) than for S-thiopentone; recovery from depression appeared to be faster for R-thiopentone than either rac- or S-thiopentone. Fatality was only found with S-thiopentone (3/7 animals in Study 2). R-thiopentone plasma concentrations were approximately 8% less than those of S-thiopentone in rats treated with racthiopentone. Although small differences in clearance between enantiomers were found that may influence recovery, they were not large enough to account for the reported differences in potency between the two enantiomers.