Shedding Light On General Anesthesia: Uncovering The Molecular Mechanisms For Propofol And Volatile Anesthetics

General anesthetics have played a pivotal role in the history of medicine. Despite accounts of their use within the earliest of human records, our understanding of anesthetic mechanisms remains unclear. Understanding these molecular mechanisms would be a significant advance toward enhanced drug design and optimal the clinical use of these potentially hazardous agents. Recent advances in chemical and molecular biology, including photoaffinity labeling, have allowed enhanced appreciation of the complex interactions anesthetic’s have with their macromolecular substrates. This work is dedicated to further define the protein interactions of the frequently administered volatile anesthetics sevoflurane and isoflurane, as well as the most commonly used intravenous anesthetic, propofol. A novel photoaffinity ligand for sevoflurane was validated and applied to uncover the unique mechanism of sevoflurane positive modulation of mammalian Shaker Kv1.2 channels. This novel sevoflurane photoaffinity ligand was in addition to a previously developed photoaffinity ligand for isoflurane, further applied to determine the anesthetic binding sites within a vital protein target, synaptic GABAA receptors. The molecular recognition elements for propofol-protein interactions were probed using a novel hydrogen-bond null derivative. It was determined that the propofol 1-hydroxyl is key for molecular interactions that contribute to anesthetic endpoints, such as synaptic GABAA receptor positive modulation, while less significant for other known biological effects like decreasing muscle contractility. The range of propofol-binding proteins within synaptosomes was further defined with the synthesis of a novel photoaffinity tandem click chemistry-active ligand and the development of a quantitative affinity-based protein profiling workflow. Results of the investigation indicated a highly complex pool of propofol-specific proteins including an unbiased, selective binding of specific synaptic GABAA receptor subunits. The likely propofol binding cavities and the underlining molecular recognition features that contribute to the selective GABAA receptor subunit binding were examined using molecular dynamics simulations and photoaffinity protection studies. Together these series of studies suggest that general anesthetics bind a to range of molecular substrates that cumulatively result in general anesthesia phenotypes and that multiple, functionally distinct, binding sites can be present within a single protein target

Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites.

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels. © 2018 Wang et al

Shedding Light on General Anesthesia: Uncovering the Molecular Mechanisms of Propofol and Volatile Anesthestics

General anesthetics have played a pivotal role in the history of medicine. Despite accounts of their use within the earliest of human records, our understanding of anesthetic mechanisms remains unclear. Understanding these molecular mechanisms would be a significant advance toward enhanced drug design and optimal the clinical use of these potentially hazardous agents. Recent advances in chemical and molecular biology, including photoaffinity labeling, have allowed enhanced appreciation of the complex interactions anesthetic’s have with their macromolecular substrates. This work is dedicated to further define the protein interactions of the frequently administered volatile anesthetics sevoflurane and isoflurane, as well as the most commonly used intravenous anesthetic, propofol. A novel photoaffinity ligand for sevoflurane was validated and applied to uncover the unique mechanism of sevoflurane positive modulation of mammalian Shaker Kv1.2 channels. This novel sevoflurane photoaffinity ligand was in addition to a previously developed photoaffinity ligand for isoflurane, further applied to determine the anesthetic binding sites within a vital protein target, synaptic GABAA receptors. The molecular recognition elements for propofol-protein interactions were probed using a novel hydrogen-bond null derivative. It was determined that the propofol 1-hydroxyl is key for molecular interactions that contribute to anesthetic endpoints, such as synaptic GABAA receptor positive modulation, while less significant for other known biological effects like decreasing muscle contractility. The range of propofol-binding proteins within synaptosomes was further defined with the synthesis of a novel photoaffinity tandem click chemistry-active ligand and the development of a quantitative affinity-based protein profiling workflow. Results of the investigation indicated a highly complex pool of propofol-specific proteins including an unbiased, selective binding of specific synaptic GABA A receptor subunits. The likely propofol binding cavities and the underlining molecular recognition features that contribute to the selective GABA A receptor subunit binding were examined using molecular dynamics simulations and photoaffinity protection studies. Together these series of studies suggest that general anesthetics bind a to range of molecular substrates that cumulatively result in general anesthesia phenotypes and that multiple, functionally distinct, binding sites can be present within a single protein target

Shedding Light on General Anesthesia: Uncovering the Molecular Mechanisms of Propofol and Volatile Anesthestics

General anesthetics have played a pivotal role in the history of medicine. Despite accounts of their use within the earliest of human records, our understanding of anesthetic mechanisms remains unclear. Understanding these molecular mechanisms would be a significant advance toward enhanced drug design and optimal the clinical use of these potentially hazardous agents. Recent advances in chemical and molecular biology, including photoaffinity labeling, have allowed enhanced appreciation of the complex interactions anesthetic’s have with their macromolecular substrates. This work is dedicated to further define the protein interactions of the frequently administered volatile anesthetics sevoflurane and isoflurane, as well as the most commonly used intravenous anesthetic, propofol. A novel photoaffinity ligand for sevoflurane was validated and applied to uncover the unique mechanism of sevoflurane positive modulation of mammalian Shaker Kv1.2 channels. This novel sevoflurane photoaffinity ligand was in addition to a previously developed photoaffinity ligand for isoflurane, further applied to determine the anesthetic binding sites within a vital protein target, synaptic GABAA receptors. The molecular recognition elements for propofol-protein interactions were probed using a novel hydrogen-bond null derivative. It was determined that the propofol 1-hydroxyl is key for molecular interactions that contribute to anesthetic endpoints, such as synaptic GABAA receptor positive modulation, while less significant for other known biological effects like decreasing muscle contractility. The range of propofol-binding proteins within synaptosomes was further defined with the synthesis of a novel photoaffinity tandem click chemistry-active ligand and the development of a quantitative affinity-based protein profiling workflow. Results of the investigation indicated a highly complex pool of propofol-specific proteins including an unbiased, selective binding of specific synaptic GABA A receptor subunits. The likely propofol binding cavities and the underlining molecular recognition features that contribute to the selective GABA A receptor subunit binding were examined using molecular dynamics simulations and photoaffinity protection studies. Together these series of studies suggest that general anesthetics bind a to range of molecular substrates that cumulatively result in general anesthesia phenotypes and that multiple, functionally distinct, binding sites can be present within a single protein target

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling

In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.Molecular and Cellular Biolog

Macroscopic and macromolecular specificity of alkylphenol anesthetics for neuronal substrates.

We used a photoactive general anesthetic called meta-azi-propofol (AziPm) to test the selectivity and specificity of alkylphenol anesthetic binding in mammalian brain. Photolabeling of rat brain sections with [(3)H]AziPm revealed widespread but heterogeneous ligand distribution, with [(3)H]AziPm preferentially binding to synapse-dense areas compared to areas composed largely of cell bodies or myelin. With [(3)H]AziPm and propofol, we determined that alkylphenol general anesthetics bind selectively and specifically to multiple synaptic protein targets. In contrast, the alkylphenol anesthetics do not bind to specific sites on abundant phospholipids or cholesterol, although [(3)H]AziPm shows selectivity for photolabeling phosphatidylethanolamines. Together, our experiments suggest that alkylphenol anesthetic substrates are widespread in number and distribution, similar to those of volatile general anesthetics, and that multi-target mechanisms likely underlie their pharmacology

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of gamma-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

International audiencePropofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured approximately 4% of the synaptosomal proteome, including the unbiased capture of five alpha or beta GABAA receptor subunits. Lack of gamma2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for alpha/beta than gamma-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and alpha/beta cavity residues but not gamma cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity

Gel Formulation Containing Mixed Surfactant and Lipids Associating with Carboplatin

The interaction of amphiphilic molecules such as lipids and surfactants with the hydrophilic drug carboplatin was investigated to identify suitable self-assembling components for a potential gel-based delivery formulation. 1H-NMR Studies in sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT, AOT)-based reverse micelles show that carboplatin associates and at least partially penetrates the surfactant interface. Langmuir monolayers formed by dipalmitoyl(phosphatidyl)choline are penetrated by carboplatin. Carboplatin was found to also penetrate the more rigid monolayers containing cholesterol. A combined mixed surfactant gel formulation containing carboplatin and cholesterol for lymphatic tissue targeting was investigated for the intracavitary treatment of cancer. This formulation consists of a blend of the surfactants lecithin and AOT (1?:?3 ratio), an oil phase of isopropyl myristate, and an aqueous component. The phases of the system were defined within a pseudo-ternary phase diagram. At low oil content, this formulation produces a gel-like system over a wide range of H2O content. The carboplatin release from the formulation displays a prolonged discharge with a rate three to five times slower than that of the control. Rheological properties of the formulation exhibit pseudoplastic behavior. Microemulsion and Langmuir monolayer studies support the interactions between carboplatin and amphiphilic components used in this formulation. To target delivery of carboplatin, two formulations containing cholesterol were characterized. These two formulations with cholesterol showed that, although cholesterol does little to alter the phases in the pseudo-ternary system or to increase the initial release of the drug, it contributes significantly to the structure of the formulation under physiological temperature, as well as increases the rate of steady-state discharge of carboplatin