Jin Xi

m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol.

Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABAA receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABAA receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABAA receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

Scalable Production of Highly Sensitive Nanosensors Based on Graphene Functionalized with a Designed G Protein-Coupled Receptor

We have developed a novel, all-electronic biosensor for opioids that consists of an engineered μ-opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ∼1500 cm2 V–1 s–1 and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

Identification of a fluorescent general anesthetic, 1-aminoanthracene

We identified a fluorophore, 1-aminoanthracene (1-AMA), that is anesthetic, potentiates GABAergic transmission, and gives an appropriate dissociation constant, Kd ≈ 0.1 mM, for binding to the general anesthetic site in horse spleen apoferritin (HSAF). 1-AMA fluorescence is enhanced when bound to HSAF. Thus, displacement of 1-AMA from HSAF by other anesthetics attenuates the fluorescence signal and allows determination of Kd, as validated by isothermal titration calorimetry. This provides a unique fluorescence assay for compound screening and anesthetic discovery. Additional electrophysiology experiments in isolated cells indicate that 1-AMA potentiates chloride currents elicited by GABA, similar to many general anesthetics. Furthermore, 1-AMA reversibly immobilizes stage 45–50 Xenopus laevis tadpoles (EC50 = 16 μM) and fluorescence micrographs show 1-AMA localized to brain and olfactory regions. Thus, 1-AMA provides an unprecedented opportunity for studying general anesthetic distribution in vivo at the cellular and subcellular levels.

Azi-isoflurane, a Photolabel Analog of the Commonly Used Inhaled General Anesthetic Isoflurane.

Volatility and low-affinity hamper an ability to define molecular targets of the inhaled anesthetics. Photolabels have proven to be a useful approach in this regard, although none have closely mimicked contemporary drugs. We report here the synthesis and validation of azi-isoflurane, a compound constructed by adding a diazirinyl moiety to the methyl carbon of the commonly used general anesthetic isoflurane. Azi-isoflurane is slightly more hydrophobic than isoflurane, and more potent in tadpoles. This novel compound inhibits Shaw2 K+ channel currents similarly to isoflurane and binds to apoferritin with enhanced affinity. Finally, when irradiated at 300 nm, azi-isoflurane adducts to residues known to line isoflurane-binding sites in apoferritin and integrin LFA-1, the only proteins with isoflurane binding sites defined by crystallography. This reagent should allow rapid discovery of isoflurane molecular targets and binding sites within those targets.

A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor

G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d  = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.

Inhaled anesthetics elicit region-specific changes in protein expression in mammalian brain

Inhaled anesthetics bind specifically to many proteins in the mammalian brain. Within the subgroup of proteins whose activity is substantially modulated by anesthetic binding, it is reasonable to expect anesthetic‐induced alterations in host expression level. Thus, in an attempt to define the group of functional targets for these commonly used drugs, we examined changes in protein expression after anesthetic exposure in both intact rodent brains and in neuronal cell culture. Differential in‐gel electrophoresis was used to minimize variance, in order to detect small changes. Quantitative analysis shows that 5 h exposures to 1 minimum alveolar concentration (1 MAC) halothane caused changes in the expression of ∼2% of detectable proteins, but only at 2–24 h after awakening, and only in the cortex. An equipotent concentration of isoflurane altered the expression of only ∼1% of detectable proteins, and only in the hippocampus. Primary cortical neurons were exposed to three‐fold higher concentrations of anesthetics with no evidence of cytotoxicity. Small changes in protein expression were elicited by both drugs. Despite the fact that anesthetics produce profound changes in neurobiology and behavior, we found only minor changes in brain protein expression. A pronounced degree of regional selectivity was noted, indicating an under appreciated degree of specificity for these promiscuous drugs.

Scalable Production of Molybdenum Disulfide Based Biosensors

We demonstrate arrays of opioid biosensors based on chemical vapor deposition grown molybdenum disulfide (MoS2) field effect transistors (FETs) coupled to a computationally redesigned, water-soluble variant of the μ-opioid receptor (MOR). By transferring dense films of monolayer MoS2 crystals onto prefabricated electrode arrays, we obtain high-quality FETs with clean surfaces that allow for reproducible protein attachment. The fabrication yield of MoS2 FETs and biosensors exceeds 95%, with an average mobility of 2.0 cm(2) V(-1) s(-1) (36 cm(2) V(-1) s(-1)) at room temperature under ambient (in vacuo). An atomic length nickel-mediated linker chemistry enables target binding events that occur very close to the MoS2 surface to maximize sensitivity. The biosensor response calibration curve for a synthetic opioid peptide known to bind to the wild-type MOR indicates binding affinity that matches values determined using traditional techniques and a limit of detection ∼3 nM (1.5 ng/mL). The combination of scalable array fabrication and rapid, precise binding readout enabled by the MoS2 transistor offers the prospect of a solid-state drug testing platform for rapid readout of the interactions between novel drugs and their intended protein targets.

Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1

We previously demonstrated that isoflurane targets lymphocyte function‐associated antigen‐1 (LFA‐1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA‐1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA‐1 were probed by photolabeling using photoactivatable isoflurane (azi‐isoflurane). The adducted residues were determined by liquid chromatography/mass spectrometry analysis. Separately, docking simulations were performed to predict binding sites. Point mutations were introduced around isoflurane binding sites. The significance of isofluranes effect was assessed in both intracellular adhesion molecule‐1 (ICAM‐1) binding assays and epitope mapping of activation‐sensitive antibodies using flow cytometry. Two isoflurane binding sites were identified using photolabeling and were further validated by the docking simulation: one at the hydrophobic pocket in the ICAM‐1 binding domain (the αI domain); the other at the βI domain. Mutagenesis of the α′1 helix showed that isoflurane binding sites at the βI domain were significantly important in modulating LFA‐1 function and conformation. Epitope mapping using activation‐sensitive antibodies suggested that isoflurane stabilized LFA‐1 in the closed conformation. This study suggested that isoflurane binds to both the αI and βI domains allosteric to the ICAM‐1 binding site, and that isoflurane binding stabilizes LFA‐1 in the closed conformation.—Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., Eckenhof, R.G. Isoflurane binds and stabilizes a closed conformation of the leukocyte function‐associated antigen‐1. FASEB J. 26, 4408–4417 (2012). www.fasebj.org

A High-Throughput Approach for Identification of Novel General Anesthetics

Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC1280 library of drug-like compounds. A raw hit rate of ∼15% was reduced through the use of computational filters to yield an overall hit rate of ∼1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.