Brian P. Weiser

Mechanisms Revealed Through General Anesthetic Photolabeling

AbstractGeneral anesthetic photolabels are used to reveal molecular targets and molecular binding sites of anesthetic ligands. After identification, the relevance of anesthetic substrates or binding sites can be tested in biological systems. Halothane and photoactive analogs of isoflurane, propofol, etomidate, neurosteroids, anthracene, and long chain alcohols have been used in anesthetic photolabeling experiments. Interrogated protein targets include the nicotinic acetylcholine receptor, GABAA receptor, tubulin, leukocyte function-associated antigen-1, and protein kinase C. In this review, we summarize insights revealed by photolabeling these targets, as well as general features of anesthetics, such as their propensity to partition to mitochondria and bind voltage-dependent anion channels. The theory of anesthetic photolabel design and the experimental application of photoactive ligands are also discussed.

In vivo activation of azipropofol prolongs anesthesia and reveals synaptic targets.

Background: Azipropofol is a photoactive analog of the general anesthetic propofol. Results: In vivo photolabeling of tadpoles results in covalent ligand binding to neuronal proteins and prolongation of anesthesia. Conclusion: Reconciling time-resolved gel proteomics with behavioral state allows identification of potential anesthetic targets. Significance: In vivo activation of efficacious photolabels provides a novel approach to investigate mechanisms of general anesthesia. General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed “optoanesthesia.” Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics.

Computational investigation of cholesterol binding sites on mitochondrial VDAC.

The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs. We docked cholesterol to specific sites on VDAC that were previously identified with NMR, and we tested the reliability of multiple docking results in each site with simulations. The most favorable binding modes were used to build a VDAC model with cholesterol occupying five unique sites, and during multiple 100 ns simulations, cholesterol stably and reproducibly remained bound to the protein. For comparison, VDAC was simulated in systems with identical components but with cholesterol initially unbound. The dynamics of loops that connect adjacent β-strands were most affected by bound cholesterol, with the averaged root-mean-square fluctuation (RMSF) of multiple residues altered by 20–30%. Cholesterol binding also stabilized charged residues inside the channel and localized the surrounding electrostatic potentials. Despite this, ion diffusion through the channel was not significantly affected by bound cholesterol, as evidenced by multi-ion potential of mean force measurements. Although we observed modest effects of cholesterol on the open channel, our model will be particularly useful in experiments that investigate how cholesterol affects VDAC function under applied electrochemical forces and also how other ligands and proteins interact with the channel.

Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

Background:The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods:Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene–apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for &ggr;-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results:From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusion:The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Sites and functional consequence of VDAC–alkylphenol anesthetic interactions

General anesthetics have previously been shown to bind mitochondrial VDAC. Here, using a photoactive analog of the anesthetic propofol, we determined that alkylphenol anesthetics bind to Gly56 and Val184 on rat VDAC1. By reconstituting rat VDAC into planar bilayers, we determined that propofol potentiates VDAC gating with asymmetry at the voltage polarities; in contrast, propofol does not affect the conductance of open VDAC. Additional experiments showed that propofol also does not affect gramicidin A properties that are sensitive to lipid bilayer mechanics. Together, this suggests propofol affects VDAC function through direct protein binding, likely at the lipid‐exposed channel surface, and that gating can be modulated by ligand binding to the distal ends of VDAC β‐strands where Gly56 and Val184 are located.

Propofol inhibits SIRT2 deacetylase through a conformation-specific, allosteric site.

Background: The general anesthetic propofol binds many on-pathway and off-pathway proteins. Results: The deacetylase SIRT2 is a target of propofol, and propofol can inhibit the enzymatic function of the protein. Conclusion: SIRT2 is a novel, presumably off-pathway protein target of the general anesthetic propofol. Significance: Propofol might affect cellular events that are regulated by the acetylation state of proteins. meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [3H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr139, Phe190, and Met206) that are located in a single allosteric protein site, and propofol inhibited [3H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [3H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [3H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD+ during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state.

Disinhibition of Histaminergic Neurons: Lack of Effect on Arousal Switch Following Propofol Hypnosis

Transitions between wake and sleep are a routine and essential part of our lives. The bistable “switch” model of consciousness considers these two mutually exclusive states to be governed by interconnected subcortical nuclei ([Saper et al., 2010][1]). Wake- or sleep-promoting nuclei enforce

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 [3H]AziPm revealed widespread but heterogeneous ligand distribution, with [3H]AziPm preferentially binding to synapse-dense areas compared to areas composed largely of cell bodies or myelin. With [3H]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 [3H]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.