In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol.

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

Fropofol prevents disease progression in mice with hypertrophic cardiomyopathy.

AIMS: Increased myofilament contractility is recognized as a crucial factor in the pathogenesis of hypertrophic cardiomyopathy (HCM). Direct myofilament desensitization might be beneficial in preventing HCM disease progression. Here, we tested whether the small molecule fropofol prevents HCM phenotype expression and disease progression by directly depressing myofilament force development. METHODS AND RESULTS: Force, intracellular Ca2+, and steady-state activation were determined in isolated trabecular muscles from wild-type (WT) and transgenic HCM mice with heterozygous human α-myosin heavy chain R403Q mutation (αMHC 403/+). αMHC 403/+ HCM mice were treated continuously with fropofol by intraperitoneal infusion for 12 weeks. Heart tissue was analysed with histology and real-time PCR of prohypertrophic and profibrotic genes. Fropofol decreased force in a concentration-dependent manner without significantly altering [Ca2+]i in isolated muscles from both WT and αMHC 403/+ HCM mouse hearts. Fropofol also depressed maximal Ca2+-activated force and increased the [Ca2+]i required for 50% activation during steady-state activation. In whole-animal studies, chronic intra-abdominal administration of fropofol prevented hypertrophy development and diastolic dysfunction. Chronic fropofol treatment also led to attenuation of prohypertrophic and profibrotic gene expression, reductions in cell size, and decreases in tissue fibrosis. CONCLUSIONS: Direct inhibition of myofilament contraction by fropofol prevents HCM disease phenotypic expression and progression, suggesting that increased myofilament contractile force is the primary trigger for hypertrophy development and HCM disease progression.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

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.

An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules.

Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule-kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule-kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain-ß-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin-microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.

Identification of General Anesthetic Target Protein-Binding Sites by Photoaffinity Labeling and Mass Spectrometry.

General anesthetics are unique in that they represent a diverse range of chemical structures. Therefore, it is not surprising that the desired and undesired molecular targets, and binding sites therein, are as equally diverse and unique. Photoaffinity labeling has proven to be a valuable strategy for the identification of anesthetic molecular targets, as well as binding sites within those targets. In combination with the advances in mass spectrometry-based proteomics, along with the ability to comprehensively map posttranslational modifications, the method is likely to undergo continued improvement. Here, we provide the fundamentals for the design and development of an anesthetic photolabel. We also outline a protocol for the identification of photolabeled residues by mass spectrometry. The major steps include the photolabeling experiment, sample preparation, high-resolution mass spectrometry, and data analysis. The protocol can be used as a foundation for further optimization for the specific protein of interest and conditions of an experiment. The use of photoaffinity labeling adds an advantageous alternative and/or complementary approach to increase understanding of anesthetic molecular mechanisms.

Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation.

Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type Kv1.2 and the established mutant Kv1.2 G329T. In wild-type Kv1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the Kv1.2 G329T. Mutagenesis of Leu317 in WT Kv1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the Kv1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of Kv channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.

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

Propofol, 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 ∼4% of the synaptosomal proteome, including the unbiased capture of five α or ß GABAA receptor subunits. Lack of γ2 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 α/ß than γ-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 α/ß cavity residues but not γ 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.

Shedding Light on Anesthetic Mechanisms: Application of Photoaffinity Ligands.

Anesthetic photoaffinity ligands have had an increasing presence within anesthesiology research. These ligands mimic parent general anesthetics and allow investigators to study anesthetic interactions with receptors and enzymes; identify novel targets; and determine distribution within biological systems. To date, nearly all general anesthetics used in medicine have a corresponding photoaffinity ligand represented in the literature. In this review, we examine all aspects of the current methodologies, including ligand design, characterization, and deployment. Finally we offer points of consideration and highlight the future outlook as more photoaffinity ligands emerge within the field.

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.

Role for the propofol hydroxyl in anesthetic protein target molecular recognition.

Propofol is a widely used intravenous general anesthetic. We synthesized 2-fluoro-1,3-diisopropylbenzene, a compound that we call "fropofol", to directly assess the significance of the propofol 1-hydroxyl for pharmacologically relevant molecular recognition in vitro and for anesthetic efficacy in vivo. Compared to propofol, fropofol had a similar molecular volume and only a small increase in hydrophobicity. Isothermal titration calorimetry and competition assays revealed that fropofol had higher affinity for a protein site governed largely by van der Waals interactions. Within another protein model containing hydrogen bond interactions, propofol demonstrated higher affinity. In vivo, fropofol demonstrated no anesthetic efficacy, but at high concentrations produced excitatory activity in tadpoles and mice; fropofol also antagonized propofol-induced hypnosis. In a propofol protein target that contributes to hypnosis, α1ß2γ2L GABAA receptors, fropofol demonstrated no significant effect alone or on propofol positive allosteric modulation of the ion channel, suggesting an additional requirement for the 1-hydroxyl within synaptic GABAA receptor site(s). However, fropofol caused similar adverse cardiovascular effects as propofol by a dose-dependent depression of myocardial contractility. Our results directly implicate the propofol 1-hydroxyl as contributing to molecular recognition within protein targets leading to hypnosis, but not necessarily within protein targets leading to side effects of the drug.

Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1ß3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ∼ 50%. When the amino acids photolabeled by [(3)H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the ß3-α1 subunit interface (ß3Met-286 in ß3M3 and α1Met-236 in α1M1), previously photolabeled by [(3)H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of ß3Met-227 in ßM1, the amino acid in the α1-ß3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR ß3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the ß3-ß3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

Mechanisms revealed through general anesthetic photolabeling.

General 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.

Photoaffinity labeling the propofol binding site in GLIC.

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.

Identification of propofol binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [(3)H]AziPm, and labeled amino acids were identified by Edman degradation. [(3)H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18' and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (-agonist) amino acids in the M2 helices (αM2-6', ßM2-6' and -13', and δM2-13') that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10'. Propofol enhanced [(3)H]AziPm photolabeling at αM2-10'. Propofol inhibited [(3)H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 µm) than it inhibited ion channel photolabeling (IC50 = 125 µm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.

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, GABA(A) 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.

Inhalational anesthetic photolabeling.

Photolabeling has allowed considerable progress in the understanding of anesthetic binding to proteins, of target identity, and of site localization. There are, however, few groups doing this work, so this article is an attempt to demystify the method. We will discuss the theory, method, and limitations of this useful experimental approach.

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.