Weiming Bu

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

Significance How general anesthetics modulate the function of voltage-gated sodium (NaV) channels remains a mystery. Here, strategic placements of 19F probes, guided by molecular dynamics simulations, allowed for high-resolution NMR quantitation of the volatile anesthetic isoflurane binding to the bacterial Nav channel NaChBac. The data provided experimental evidence showing that channel blockade at the base of the ion selectivity filter and the restricted pivot motion at the S4–S5 linker and the P2–S6 helix hinge underlie the action of isoflurane on NaChBac. The results contribute to a better understanding of the molecular mechanisms of general anesthesia. Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4–S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4–S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

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

Ferritin couples iron and fatty acid metabolism

A physiological relationship between iron, oxidative injury, and fatty acid metabolism exists, but transduction mechanisms are unclear. We propose that the iron storage protein ferritin contains fatty acid binding sites whose occupancy modulates iron uptake and release. Using isothermal microcalorimetry, we found that arachidonic acid binds ferritin specifically and with 60 μM affinity. Arachidonate binding by ferritin enhanced iron mineralization, decreased iron release, and protected the fatty acid from oxidation. Cocrystals of arachidonic acid and horse spleen apoferritin diffracted to 2.18 Å and revealed specific binding to the 2‐fold intersubunit pocket. This pocket shields most of the fatty acid and its double bonds from solvent but allows the arachidonate tail to project well into the ferrihydrite mineralization site on the ferritin L‐subunit, a structural feature that we implicate in the effects on mineralization by demonstrating that the much shorter saturated fatty acid, caprylate, has no significant effects on mineralization. These combined effects of arachidonate binding by ferritin are expected to lower both intracellular free iron and free arachidonate, thereby providing a previously unrecognized mechanism for limiting lipid peroxidation, free radical damage, and proinflammatory cascades during times of cellular stress.—Bu, W., Liu, R., Cheung‐Lau, J. C., Dmochowski, I. J., Loll, P. J., Eckenhoff, R. G. Ferritin couples iron and fatty acid metabolism. FASEB J. 26, 2394‐2400 (2012). www.fasebj.org

Volatile anesthetics, not intravenous anesthetic propofol bind to and attenuate the activation of platelet receptor integrin αIIbβ3.

Background In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3. Methods The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis. Results Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation. Conclusions Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.

Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

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 Shares the Binding Site with Isoflurane and Sevoflurane on Leukocyte Function-Associated Antigen-1

BACKGROUND:We previously demonstrated that propofol interacted with the leukocyte adhesion molecule leukocyte function–associated antigen-1 (LFA-1) and inhibited the production of interleukin-2 via LFA-1 in a dependent manner. However, the binding site(s) of propofol on LFA-1 remains unknown. METHODS:First, the inhibition of LFA-1’s ligand binding by propofol was confirmed in an enzyme-linked immunosorbent assay (ELISA) ELISA-type assay. The binding site of propofol on LFA-1 was probed with a photolabeling experiment using a photoactivatable propofol analog called azi-propofol-m. The adducted residues of LFA-1 by this compound were determined using liquid chromatography–mass spectrometry. In addition, the binding of propofol to the ligand-binding domain of LFA-1 was examined using 1-aminoanthracene (1-AMA) displacement assay. Furthermore, the binding site(s) of 1-AMA and propofol on LFA-1 was studied using the docking program GLIDE. RESULTS:We demonstrated that propofol impaired the binding of LFA-1 to its ligand intercellular adhesion molecule-1. The photolabeling experiment demonstrated that the adducted residues were localized in the allosteric cavity of the ligand-binding domain of LFA-1 called “lovastatin site.” The shift of fluorescence spectra was observed when 1-AMA was coincubated with the low-affinity conformer of LFA-1 ligand-binding domain (wild-type [WT] &agr;L I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT &agr;L I domain. In the 1-AMA displacement assay, propofol decreased 1-AMA fluorescence signal (at 520 nm), suggesting that propofol competed with 1-AMA and bound to the WT &agr;L I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment. CONCLUSIONS:We demonstrated that propofol bound to the lovastatin site in LFA-1. Previously we showed that the volatile anesthetics isoflurane and sevoflurane bound to this site. Taken together, the lovastatin site is an example of the common binding sites for anesthetics currently used clinically.

Beyond the Detergent Effect: a Binding Site for Sodium Dodecyl Sulfate (SDS) in Mammalian Apoferritin

Although sodium dodecyl sulfate (SDS) is widely used as an anionic detergent, it can also exert specific pharmacological effects that are independent of the surfactant properties of the molecule. However, structural details of how proteins recognize SDS are scarce. Here, it is demonstrated that SDS binds specifically to a naturally occurring four-helix bundle protein: horse apoferritin. The X-ray crystal structure of the apoferritin-SDS complex was determined at a resolution of 1.9 Å and revealed that the SDS binds in an internal cavity that has previously been shown to recognize various general anesthetics. A dissociation constant of 24 ± 9 µM at 293 K was determined by isothermal titration calorimetry. SDS binds in this cavity by bending its alkyl tail into a horseshoe shape; the charged SDS head group lies in the opening of the cavity at the protein surface. This crystal structure provides insights into the protein-SDS interactions that give rise to binding and may prove useful in the design of novel SDS-like ligands for some proteins.

Molecular mechanism of anesthetic-induced depression of myocardial contraction

Isoflurane and propofol are known to depress cardiac contraction, but the molecular mechanisms involved are not known. In this study, we determined whether decreasing myofilament Ca2+ responsiveness underlies anesthesia‐induced depression of contraction and uncovered the molecular targets of isoflurane and propofol. Force and intracellular Ca2+ ([Ca2+]i) were measured in rat trabeculae superfused with Krebs‐Henseleit solution, with or without propofol or isoflurane. Photoaffinity labeling of myofilament proteins with meta‐Azi‐propofol (AziPm) and Azi‐isoflurane (Azi‐iso) and molecular docking were also used. Both propofol and isoflurane dose dependently depressed force from low doses (propofol, 27 ± 6 μM; isoflurane, 1.0 ± 0.1%) to moderate doses (propofol, 87 ± 4 μM; isoflurane, 3.0 ± 0.25%), without significant alteration [Ca2+]i. During steady‐state activations in both intact and skinned preparations, propofol and isoflurane depressed maximum Ca2+‐activated force and increased the [Ca2+]i required for 50% of activation. Myofibrils photolabeled with AziPm and Azi‐iso identified myosin, actin, and myosin light chain as targets of the anesthetics. Several adducted residues in those proteins were located in con‐formationally sensitive regions that underlie contractile function. Thus, propofol and isoflurane decrease force development by directly depressing myofilament Ca2+ responsiveness and have binding sites in key regions for contraction in both actin and myosin.—Meng, T., Bu, W., Ren, X., Chen, X., Yu, J., Eckenhoff, R. G., Gao, W. D. Molecular mechanism of anesthetic‐induced depression of myocardial contraction. FASEB J. 30, 2915‐2925 (2016). www.fasebj.org