RESUMO
Sedatives/anesthetics are important medical tools to facilitate medical care and increase patients' comfort. Increasingly, there is recognition that sedatives/anesthetics can modulate immune functions. Toll-like receptors (TLRs) are major pattern recognition receptors involved in the recognition of microbial components. TLR7 recognizes single-strand RNA virus such as influenza and SARS-CoV2 viruses and initiates interferon (IFN) responses. IFN production triggered by TLR7 stimulation is a critical anti-viral response. For example, patients with TLR7 variants including loss-of- function variants were associated with severe COVID-19. Taken together, it is important to determine if sedatives/anesthetics mitigate TLR7 function. We have previously showed that TLR7-mediated activation was not affected by volatile anesthetics. However, we found that propofol attenuated TLR7 activation among intravenous sedatives in the reporter assay. TLR7 agonist R837 stimulation increased TNF-α, IL-1ß, IL-6, IL-10, and IFN-ß mRNA levels in bone marrow-derived dendritic cells, while these levels were attenuated by propofol. Our murine lung slice experiments showed that propofol attenuated IFN production. R837 increased IFN-ß expression in the lungs, and propofol attenuated IFN-ß expression in an in vivo model of R837 intranasal instillation. We also found that propofol directly bound to and hindered its association of TLR7 with MyD88. Our analysis using fropofol, propofol derivative showed that the hydroxyl group in propofol was important for propofol-TLR7 interaction.
Assuntos
COVID-19 , Propofol , Animais , Células Dendríticas , Humanos , Hipnóticos e Sedativos/farmacologia , Imiquimode , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Camundongos , Propofol/análogos & derivados , Propofol/farmacologia , RNA Viral/metabolismo , SARS-CoV-2 , Receptor 7 Toll-LikeRESUMO
Toll like receptors (TLRs) are critical receptors to respond to danger signals, and their functions are relevant in the perioperative period. We previously reported that volatile anesthetics directly bound to TLR2 and TLR4 and attenuated their functions. Given that TLR9 can respond to mitochondrial DNA, a danger signal that is released upon tissue injury, we examined the role of anesthetics on TLR9 function. Our reporter assay showed that volatile anesthetics isoflurane and sevoflurane increased the activation of TLR9, while propofol attenuated it. TLR9 activation occurs via its dimerization. The dimerization is facilitated by unmethylated cytosine-phosphate-guanine (CpG) DNA as well as DNA containing cytosine at the second position from 5'-end (5'-xCx DNA). Our structural analysis using photoactivable anesthetics and rigid docking simulation showed that isoflurane and sevoflurane bound to both TLR9 dimer interface and 5'-xCx DNA binding site. Propofol bound to the TLR9 antagonist binding site. This is the first illustration that anesthetics can affect the binding of nucleic acids to their receptor. This study sets the foundation for the effect of anesthetics on TLR9 and will pave the way for future studies to determine the significance of such interactions in the clinical setting.
Assuntos
Anestésicos Inalatórios/farmacologia , Isoflurano/farmacologia , Sevoflurano/farmacologia , Receptor Toll-Like 9/química , Anestésicos Inalatórios/química , Animais , Sítios de Ligação , Células HEK293 , Cavalos , Humanos , Isoflurano/química , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Sevoflurano/química , Receptor Toll-Like 9/metabolismoRESUMO
Propofol is a clinically important intravenous anesthetic. We previously reported that it directly inhibited 5-lipoxygenase (5-LOX), a key enzyme for leukotriene biosynthesis. Because the hydroxyl group in propofol (propofol 1-hydroxyl) is critical for its anesthetic effect, we examined if its presence would be inevitable for 5-lipoxygenase recognition. Fropofol is developed by substituting the hydroxy group in propofol with fluorine. We found that propofol 1-hydroxyl was important for 5-lipoxygenase recognition, but it was not absolutely necessary. Azi-fropofol bound to 5-LOX at one of the two propofol binding sites of 5-LOX (pocket around Phe-187), suggesting that propofol 1-hydroxyl is important for 5-LOX inhibition at the other propofol binding site (pocket around Val-431). Interestingly, 5-hydroperoxyeicosatetraenoic acid (5-HpETE) production was significantly increased by stimulation with calcium ionophore A23187 in HEK293 cells expressing 5-LOX, suggesting that the fropofol binding site is important for the conversion from 5-HpETE to leukotriene A4. We also indicated that propofol 1-hydroxyl might have contributed to interaction with wider targets among our body.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Propofol/química , Propofol/metabolismo , Anestésicos Intravenosos/química , Anestésicos Intravenosos/metabolismo , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/genética , Ácido Araquidônico/sangue , Sítios de Ligação , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Células HEK293 , Humanos , Leucotrieno B4/metabolismo , Leucotrienos/metabolismo , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/metabolismo , Simulação de Acoplamento Molecular , Mutagênese , Propofol/farmacologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
General anesthesia has been the requisite component of surgical procedures for over 150 yr. Although immunomodulatory effects of volatile anesthetics have been growingly appreciated, the molecular mechanism has not been understood. In septic mice, the commonly used volatile anesthetic isoflurane attenuated the production of 5-lipoxygenase products and IL-10 and reduced CD11b and intercellular adhesion molecule-1 expression on neutrophils, suggesting the attenuation of TLR4 signaling. We confirmed the attenuation of TLR4 signaling in vitro and their direct binding to TLR4-myeloid differentiation-2 (MD-2) complex by photolabeling experiments. The binding sites of volatile anesthetics isoflurane and sevoflurane were located near critical residues for TLR4-MD-2 complex formation and TLR4-MD-2-LPS dimerization. Additionally, TLR4 activation was not attenuated by intravenous anesthetics, except for a high concentration of propofol. Considering the important role of TLR4 system in the perioperative settings, these findings suggest the possibility that anesthetic choice may modulate the outcome in patients or surgical cases in which TLR4 activation is expected.-Okuno, T., Koutsogiannaki, S., Hou, L., Bu, W., Ohto, U., Eckenhoff, R. G., Yokomizo, T., Yuki, K. Volatile anesthetics isoflurane and sevoflurane directly target and attenuate Toll-like receptor 4 system.
Assuntos
Anestésicos Inalatórios/farmacologia , Isoflurano/farmacologia , Sevoflurano/farmacologia , Receptor 4 Toll-Like/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Receptor 4 Toll-Like/químicaRESUMO
BACKGROUND: The misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication. METHODS: We determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for ß-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison. RESULTS: The affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the ß-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the ß-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand. CONCLUSIONS: In conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the ß-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.
Assuntos
Analgésicos Opioides/farmacologia , Butorfanol/farmacologia , Neurônios/efeitos dos fármacos , Receptores Opioides kappa/agonistas , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Analgésicos Opioides/toxicidade , Animais , Butorfanol/química , Butorfanol/metabolismo , Butorfanol/toxicidade , Linhagem Celular Tumoral , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Neurônios/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , beta-Arrestinas/metabolismoRESUMO
Supranormal contractile properties are frequently associated with cardiac diseases. Anesthetic agents, including propofol, can depress myocardial contraction. We tested the hypothesis that fropofol, a propofol derivative, reduces force development in cardiac muscles via inhibition of cross-bridge cycling and may therefore have therapeutic potential. Force and intracellular Ca2+ concentration ([Ca2+]i) transients of rat trabecular muscles were determined. Myofilament ATPase, actin-activated myosin ATPase, and velocity of actin filaments propelled by myosin were also measured. Fropofol dose dependently decreased force without altering [Ca2+]i in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca2+-activated force ( Fmax) and increased the [Ca2+]i required for 50% of Fmax (Ca50) at steady state without affecting the Hill coefficient in both intact and skinned cardiac fibers. The drug also depressed cardiac myofibrillar and actin-activated myosin ATPase activity. In vitro actin sliding velocity was significantly reduced when fropofol was introduced during rigor binding of cross-bridges. The data suggest that the depressing effects of fropofol on cardiac contractility are likely to be related to direct targeting of actomyosin interactions. From a clinical standpoint, these findings are particularly significant, given that fropofol is a nonanesthetic small molecule that decreases myocardial contractility specifically and thus may be useful in the treatment of hypercontractile cardiac disorders.-Ren, X., Schmidt, W., Huang, Y., Lu, H., Liu, W., Bu, W., Eckenhoff, R., Cammarato, A., Gao, W. D. Fropofol decreases force development in cardiac muscle.
Assuntos
Anestésicos/farmacologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Propofol/farmacologia , Actinas/metabolismo , Actomiosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Contração Muscular/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miosinas/metabolismo , RatosRESUMO
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.
Assuntos
Flúor/química , Íons/química , Sódio/química , Canais de Sódio Disparados por Voltagem/química , Sítios de Ligação , Fenômenos Biofísicos , Ativação do Canal Iônico/genética , Isoflurano/química , Cinética , Espectroscopia de Ressonância Magnética , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Propofol is an intravenous anesthetic that produces its anesthetic effect, largely via the GABAA receptor in the CNS, and also reduces the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil respiratory burst. Because fMLP-stimulated neutrophils produce leukotriene (LT)B4, we examined the effect of propofol on LTB4 production in vivo and in vitro Cecal ligation and puncture surgery was performed in mice, with or without exposure to propofol. Propofol attenuated the production of 5-lipoxygenase (5-LOX)-related arachidonic acid (AA) derivatives in the peritoneal fluid. Also, in the in vitro experiments on fMLP-stimulated neutrophils and 5-LOX-transfected human embryonic kidney cells, propofol attenuated the production of 5-LOX-related AA derivatives. Based on these results, we hypothesized that propofol would directly affect 5-LOX function. Using meta-azi-propofol (AziPm), we photolabeled stable 5-LOX protein, which had been used to solve the X-ray crystallographic structure of 5-LOX, and examined the binding site(s) of propofol on 5-LOX. Two propofol binding pockets were identified near the active site of 5-LOX. Alanine scanning mutagenesis was performed for the residues of 5-LOX in the vicinity of propofol, and we evaluated the functional role of these pockets in LTB4 production. We demonstrated that these pockets were functionally important for 5-LOX activity. These two pockets can be used to explore a novel 5-LOX inhibitor in the future.-Okuno, T., Koutsogiannaki, S., Ohba, M., Chamberlain, M., Bu, W., Lin, F.-Y., Eckenhoff, R. G., Yokomizo T., Yuki, K. Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.
Assuntos
Anestésicos Intravenosos/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Propofol/farmacologia , Animais , Araquidonato 5-Lipoxigenase/química , Araquidonato 5-Lipoxigenase/genética , Ácidos Araquidônicos/metabolismo , Sítios de Ligação , Células Cultivadas , Células HEK293 , Humanos , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ligação ProteicaRESUMO
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 Ca(2+) responsiveness underlies anesthesia-induced depression of contraction and uncovered the molecular targets of isoflurane and propofol. Force and intracellular Ca(2+) ([Ca(2+)]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 [Ca(2+)]i During steady-state activations in both intact and skinned preparations, propofol and isoflurane depressed maximum Ca(2+)-activated force and increased the [Ca(2+)]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 conformationally sensitive regions that underlie contractile function. Thus, propofol and isoflurane decrease force development by directly depressing myofilament Ca(2+) 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.
Assuntos
Anestésicos Inalatórios/farmacologia , Hipnóticos e Sedativos/farmacologia , Isoflurano/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Propofol/farmacologia , Anestésicos Inalatórios/química , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Corantes , Humanos , Hipnóticos e Sedativos/química , Isoflurano/química , Modelos Moleculares , Miosinas/química , Miosinas/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Propofol/química , Ligação Proteica , Conformação ProteicaRESUMO
BACKGROUND: Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. METHODS: We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1ß3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. RESULTS: Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. CONCLUSIONS: The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.
Assuntos
Anestésicos/metabolismo , Anestésicos/farmacologia , Canais Iônicos de Abertura Ativada por Ligante/antagonistas & inibidores , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Anestésicos/química , Animais , Sítios de Ligação/fisiologia , Dickeya chrysanthemi , Feminino , Canais Iônicos de Abertura Ativada por Ligante/química , Estrutura Secundária de Proteína , Xenopus laevisRESUMO
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 γ-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.
Assuntos
Anestésicos/química , Anestésicos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Animais , Calorimetria , Feminino , Flunitrazepam/metabolismo , Larva , Camundongos , Camundongos Endogâmicos C57BL , Fenóis/química , Fenóis/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Reflexo/efeitos dos fármacos , XenopusRESUMO
As the primary Ca 2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca 2+ release, sarcomere tension and heat production. Restoration of Ca 2+ into the SR also consumes ATP, generating a further untenable metabolic load. When anesthetizing patients with known MH mutations, the non-triggering intravenous general anesthetic propofol is commonly substituted for triggering anesthetics. Evidence of direct binding of anesthetic agents to RyR1 or its binding partners is scant, and the atomic-level interactions of propofol with RyR1 are entirely unknown. Here, we show that propofol decreases RyR1 opening in heavy SR vesicles and planar lipid bilayers, and that it inhibits activator-induced Ca 2+ release from SR in human skeletal muscle. In addition to confirming direct binding, photoaffinity labeling using m- azipropofol (AziP m ) revealed several putative propofol binding sites on RyR1. Prediction of binding affinity by molecular dynamics simulation suggests that propofol binds at least one of these sites at clinical concentrations. These findings invite the hypothesis that in addition to propofol not triggering MH, it may also be protective against MH by inhibiting induced Ca 2+ flux through RyR1.
RESUMO
BACKGROUND: While a number of anesthetics has been shown potentially associated with neurotoxicity in the developing brain, dexmedetomidine, a drug that was rather recently introduced into the perioperative setting, is considered beneficial from neurological wellbeing. However, the underlying mechanism of how dexmedetomidine affects brain health remains to be determined. Based on our recent study, we hypothesized that dexmedetomidine would directly bind to and inhibit Toll-like receptor 4 (TLR4), a critical receptor largely expressed in microglia and responsible for neurological insult. METHODS: We used TLR4 reporter assays to test if dexmedetomidine attenuates TLR4 activation. Furthermore, a direct binding of dexmedetomidine on TLR4 was tested using photoactivatable medetomidine. Lastly, the effect of dexmedetomidine on ketamine (anesthetic)-induced neurotoxicity was tested in rat pups (P7). RESULTS: We showed that dexmedetomidine attenuated TLR4 activation using reporter assay (IC50 = 5.8 µg/mL). Photoactivatable dexmedetomidine delineated its direct binding sites on TLR4. We also showed that dexmedetomidine attenuated microglia activation both in vitro and in vivo. DISCUSSION: We proposed a novel mechanism of dexmedetomidine-mediated neuroprotection.
RESUMO
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 isoflurane's 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 ßI domain. Mutagenesis of the α'1 helix showed that isoflurane binding sites at the ßI 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 ßI domains allosteric to the ICAM-1 binding site, and that isoflurane binding stabilizes LFA-1 in the closed conformation.
Assuntos
Isoflurano/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Sítios de Ligação , Células HEK293 , Humanos , Isoflurano/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Ácido Araquidônico/metabolismo , Ácidos Graxos/metabolismo , Ferritinas/metabolismo , Ferro/metabolismo , Apoferritinas/química , Sítios de Ligação , Calorimetria , Caprilatos/metabolismo , Cristalografia por Raios X , Ferritinas/química , Modelos MolecularesRESUMO
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] αL I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT αL 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 αL 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.
Assuntos
Anestésicos Inalatórios/metabolismo , Anestésicos Intravenosos/metabolismo , Isoflurano/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Éteres Metílicos/metabolismo , Propofol/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Ligantes , Lovastatina/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Ligação Proteica/fisiologia , SevofluranoRESUMO
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.
Assuntos
Apoferritinas/química , Dodecilsulfato de Sódio/metabolismo , Tensoativos/metabolismo , Animais , Apoferritinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cavalos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Ketamine is an anesthetic, analgesic, and antidepressant whose secondary metabolite (2R,6R)-hydroxynorketamine (HNK) has N-methyl-d-aspartate-receptor-independent antidepressant activity in a rodent model. In humans, naltrexone attenuates its antidepressant effect, consistent with opioid pathway involvement. No detailed biophysical description is available of opioid receptor binding of ketamine or its metabolites. Using molecular dynamics simulations with free energy perturbation, we characterize the binding site and affinities of ketamine and metabolites in µ and κ opioid receptors, finding a profound effect of the protonation state. G-protein recruitment assays show that HNK is an inverse agonist, attenuated by naltrexone, in these receptors with IC50 values congruous with our simulations. Overall, our findings are consistent with opioid pathway involvement in ketamine function.
Assuntos
Ketamina , Antidepressivos/farmacologia , Depressão , Ketamina/análogos & derivados , Ketamina/farmacologia , Receptores Opioides kappaRESUMO
Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated the prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling (PAL) with a diazirine derivative of propofol (AziPm), electrophysiological methods, and mutagenesis. AziPm inhibits Nav function in a manner that is indistinguishable from that of the parent compound by promoting activation-coupled inactivation. In several replicates (8/9) involving NaChBac and NavMs, we found adducts at residues located at the C-terminal end of the S4 voltage sensor, the S4-S5 linker, and the N-terminal end of the S5 segment. However, the non-inactivating mutant NaChBac-T220A yielded adducts that were different from those found in the wild-type counterpart, which suggested state-dependent changes at the binding site. Then, using molecular dynamics simulations to further elucidate the structural basis of Nav modulation by propofol, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. Supporting the PAL and MD simulation results, we also found that Ala mutations of a subset of adducted residues have distinct effects on gating modulation of NaChBac and NavMs by propofol. The results of this study provide direct insights into the structural basis of the mechanism through which propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.
Assuntos
Anestésicos Gerais , Propofol , Canais de Sódio Disparados por Voltagem , Sítios de Ligação , Canais Iônicos , Propofol/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.