RESUMO
Methionine-enkephalin (Met-Enk) is an endogenous opioid peptide that is involved in various physiological processes including memory. A technological gap in the understanding of Met-Enk's role in memory is the lack of rapid measurement tools to selectively quantify Met-Enk concentrations in situ. Here, we integrate molecularly imprinted polymers (MIPs) with carbon fiber microelectrodes (CFMs) to selectively detect Met-Enk by using fast-scan cyclic voltammetry (FSCV). We report two MIP conditions that yield 2-fold and 5-fold higher selectivity toward Met-Enk than the tyrosine-containing hexapeptide fragment angiotensin II (3-8). We demonstrate that MIP technology can be combined with FSCV at CFMs to create rapid and selective sensors for Met-Enk. This technology is a promising platform for creating selective sensors for other peptides and biomarkers.
Assuntos
Fibra de Carbono , Técnicas Eletroquímicas , Encefalina Metionina , Microeletrodos , Fibra de Carbono/química , Encefalina Metionina/análise , Encefalina Metionina/química , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Impressão Molecular , Polímeros Molecularmente Impressos/química , Carbono/químicaRESUMO
Near-edge X-ray absorption mass spectrometry (NEXAMS) is an action-spectroscopy technique of growing interest for investigations into the spatial and electronic structure of biomolecules. It has been used successfully to give insights into different aspects of the photodissociation of peptides and to probe the conformation of proteins. It is a current question whether the fragmentation pathways are sensitive toward effects of conformational isomerism, tautomerism, and intramolecular interactions in gas-phase peptides. To address this issue, we studied the cationic fragments of cryogenically cooled gas-phase leucine enkephalin ([LeuEnk+H]+) and methionine enkephalin ([MetEnk+H]+) produced upon soft X-ray photon absorption at the carbon, nitrogen, and oxygen K-edges. The interpretation of the experimental ion yield spectra was supported by density-functional theory and restricted-open-shell configuration interaction with singles (DFT/ROCIS) calculations. The analysis revealed several effects that could not be rationalized based on the peptide's amino acid sequences alone. Clear differences between the partial ion yields measured for both peptides upon C 1s â π*(CâC) excitations in the aromatic amino acid side chains give evidence for a sulfur-aromatic interaction between the methionine and phenylalanine side chain of [MetEnk+H]+. Furthermore, a peak associated with N 1s â π*(CâN) transitions, linked to a tautomeric keto-to-enol conversion of peptide bonds, was only present in the photon energy resolved ion yield spectra of [MetEnk+H]+.
Assuntos
Encefalinas/química , Peptídeos/química , Espectroscopia por Absorção de Raios X/métodos , Encefalina Leucina/química , Encefalina Metionina/química , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, â¢OH, â¢CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.
Assuntos
Encefalina Leucina/química , Encefalina Metionina/química , Compostos Férricos/química , Luz/efeitos adversos , Preparações Farmacêuticas/química , Fotólise/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Acetatos/química , Soluções Tampão , Ácidos Carboxílicos/química , Cromatografia Líquida de Alta Pressão/métodos , Ácido Cítrico/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Oxirredução/efeitos da radiação , Fármacos Fotossensibilizantes/química , Ácido Succínico/química , Espectrometria de Massas em Tandem/métodosRESUMO
Numerous studies demonstrate the promise of opioid peptides as analgesics, but poor oral bioavailability has limited their therapeutic development. This study sought to increase the oral bioavailability of opioid peptides by cyclization, using Hantzsch-based macrocyclization strategies to produce two new series of cyclized DAMGO and Leu/Met-enkephalin analogs. Opioid receptor affinity and selectivity for compounds in each series were assessed in vitro with radioligand competition binding assays. Compounds demonstrated modest affinity but high selectivity for the mu, delta, and kappa opioid receptors (MOR, DOR and KOR), while selectivity for mu opioid receptors varied by structure. Antinociceptive activity of each compound was initially screened in vivo following intracerebroventricular (i.c.v.) administration and testing in the mouse 55 °C warm-water tail-withdrawal test. The four most active compounds were then evaluated for dose- and time-dependent antinociception, and opioid receptor selectivity in vivo. Cyclic compounds 1924-10, 1936-1, 1936-7, and 1936-9 produced robust and long- lasting antinociception with ED50 values ranging from 0.32-0.75 nmol following i.c.v. administration mediated primarily by mu- and delta-opioid receptor agonism. Compounds 1924-10, 1936-1 and 1936-9 further displayed significant time-dependent antinociception after oral (10 mg kg-1, p.o.) administration. A higher oral dose (30 mg kg-1. p.o.) of all four cyclic peptides also reduced centrally-mediated respiration, suggesting successful penitration into the CNS. Overall, these data suggest cyclized opioid peptides synthesized by a Hantzsch-based macrocyclization strategy can retain opioid agonist activity to produce potent antinociception in vivo while conveying improved bioavailability following oral administration.
Assuntos
Analgésicos Opioides/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Encefalina Metionina/farmacologia , Receptores Opioides/agonistas , Tiazóis/farmacologia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/química , Animais , Ciclização , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/administração & dosagem , Ala(2)-MePhe(4)-Gly(5)-Encefalina/química , Encefalina Metionina/administração & dosagem , Encefalina Metionina/química , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Taxa Respiratória , Tiazóis/administração & dosagem , Tiazóis/químicaRESUMO
Locating the minimum free energy paths (MFEPs) between two conformational states is among the most important tasks of biomolecular simulations. For example, knowledge of the MFEP is critical for focusing the effort of unbiased simulations that are used for the construction of Markov state models to the biologically relevant regions of the system. Typically, existing path searching methods perform local sampling around the path nodes in a pre-selected collective variable (CV) space to allow a gradual downhill evolution of the path toward the MFEP. Despite the wide application of such a strategy, the gradual path evolution and the non-trivial a priori choice of CVs are also limiting its overall efficiency and automation. Here we demonstrate that non-local perpendicular sampling can be pursued to accelerate the search, provided that all nodes are reordered thereafter via a traveling-salesman scheme. Moreover, path-CVs can be computed on-the-fly and used as a coordinate system, minimizing the necessary prior knowledge about the system. Our traveling-salesman based automated path searching method achieves a 5-8 times speedup over the string method with swarms-of-trajectories for two peptide systems in vacuum and solution, making it a promising method for obtaining initial pathways when investigating functional conformational changes between a pair of structures.
Assuntos
Dipeptídeos/química , Encefalina Metionina/química , Modelos Químicos , Termodinâmica , Cadeias de Markov , Conformação ProteicaRESUMO
Quercetin and resveratrol are polyphenolic compounds, members of the flavonoid and the stilbene family, respectively, both medicinally important as dietary anticancer and antioxidant agents. They are present in a variety of foods-including fruits, vegetables, tea, wine, as well as other dietary supplements-and are responsible for various health benefits. Different quercetin and resveratrol esters of Leu/Met-enkephalin and tetrapeptide Leu-Ser-Lys-Leu (LSKL) were synthesized as model systems for monitoring the influence of the peptides on biological activity of resveratrol and quercetin. General formula of the main peptidyl-quercetin derivatives is 2-[3-(aa)n-4-hydroxyphenyl]-3,5,7-tri-hydroxy-4H-1-benzopyran-4-on, and the general formula of the main peptidyl-resveratrol derivatives is (E)-5-[4-(aa)n)styryl]benzene-1,3-diol. The antioxidant and anticancer activities of prepared compounds were investigated. Significant anticancer activity was obtained for the LSKL-based both quercetin and resveratrol derivatives. All prepared compounds exhibit antioxidant activity, in particular quercetin derivative containing Met-enkephalin.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Neoplasias/dietoterapia , Quercetina/análogos & derivados , Quercetina/farmacologia , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Antioxidantes/síntese química , Antioxidantes/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais , Encefalina Leucina/química , Encefalina Metionina/química , Ésteres/síntese química , Células HCT116 , Humanos , Células MCF-7 , Peptídeos/química , Compostos Fitoquímicos/síntese química , Quercetina/síntese química , Quercetina/uso terapêutico , Resveratrol/síntese química , Resveratrol/uso terapêutico , Solubilidade , Fator de Crescimento Transformador beta/metabolismoRESUMO
Molecular dynamics (MD) simulation software allows probing the equilibrium structural dynamics of a molecule of interest, revealing how a molecule navigates its structure space one structure at a time. To obtain a broader view of dynamics, typically one needs to launch many such simulations, obtaining many trajectories. A summarization of the equilibrium dynamics requires integrating the information in the various trajectories, and Markov State Models (MSM) are increasingly being used for this task. At its core, the task involves organizing the structures accessed in simulation into structural states, and then constructing a transition probability matrix revealing the transitions between states. While now considered a mature technology and widely used to summarize equilibrium dynamics, the underlying computational process in the construction of an MSM ignores energetics even though the transition of a molecule between two nearby structures in an MD trajectory is governed by the corresponding energies. In this paper, we connect theory with simulation and analysis of equilibrium dynamics. A molecule navigates the energy landscape underlying the structure space. The structural states that are identified via off-the-shelf clustering algorithms need to be connected to thermodynamically-stable and semi-stable (macro)states among which transitions can then be quantified. Leveraging recent developments in the analysis of energy landscapes that identify basins in the landscape, we evaluate the hypothesis that basins, directly tied to stable and semi-stable states, lead to better models of dynamics. Our analysis indicates that basins lead to MSMs of better quality and thus can be useful to further advance this widely-used technology for summarization of molecular equilibrium dynamics.
Assuntos
Algoritmos , Encefalina Metionina/química , Cadeias de Markov , Simulação de Dinâmica Molecular , Análise por Conglomerados , Visualização de Dados , Modelos Moleculares , Software , TermodinâmicaRESUMO
Collective variable (CV) or order parameter based enhanced sampling algorithms have achieved great success due to their ability to efficiently explore the rough potential energy landscapes of complex systems. However, the degeneracy of microscopic configurations, originating from the orthogonal space perpendicular to the CVs, is likely to shadow "hidden barriers" and greatly reduce the efficiency of CV-based sampling. Here we demonstrate that systematic machine learning CV, through enhanced sampling, can iteratively lift such degeneracies on the fly. We introduce an active learning scheme that consists of a parametric CV learner based on deep neural network and a CV-based enhanced sampler. Our active enhanced sampling algorithm is capable of identifying the least informative regions based on a historical sample, forming a positive feedback loop between the CV learner and sampler. This approach is able to globally preserve kinetic characteristics by incrementally enhancing both sample completeness and CV quality.
Assuntos
Simulação de Dinâmica Molecular , Algoritmos , Dipeptídeos/química , Encefalina Metionina/química , Aprendizado de Máquina , Redes Neurais de ComputaçãoRESUMO
The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10â¯pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000â¯pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.
Assuntos
Encefalina Leucina/sangue , Encefalina Metionina/sangue , Cromatografia Líquida de Alta Pressão , Encefalina Leucina/química , Encefalina Metionina/química , Humanos , Espectrometria de Massas , Estrutura MolecularRESUMO
Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.
Assuntos
Agonistas Adrenérgicos/metabolismo , Encefalina Metionina/metabolismo , Morfina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores Opioides mu/metabolismo , Acetilcolina/química , Acetilcolina/metabolismo , Agonistas Adrenérgicos/química , Sequência de Aminoácidos , Animais , Encefalina Metionina/química , Histamina/química , Histamina/metabolismo , Humanos , Metionina/química , Metionina/metabolismo , Camundongos , Morfina/química , Ligação Proteica , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Opioides mu/química , Espectrofotometria UltravioletaRESUMO
We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the µ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and ß-funaltrexamine (ß-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP. Antinociception induced by analog 2 through 4 was not reversed by δ opioid receptor (DOP) or κ opioid receptor (KOP) antagonist; antibodies against dynorphin-A (1-17), dynorphin-B (1-13), and Leu5/Met5-enkephalin had no impact on the antinociceptive effects of these analogs. In contrast, antinociceptive effects induced by a spinal injection of the fluorine substituted analog 1 were significantly reversed by KOP antagonism. Furthermore, intrathecal pretreatment with antibodies against dynorphin-B (1-13) attenuated the antinociceptive effect of analog 1. These results indicate that the antinociceptive activity exerted by intrathecally-administered analog 1 is mediated, in part, through KOP with increased release of dynorphin-B (1-13). The chemical modifications used in the present study may serve as a useful tool to gain insight into the mechanisms of endomorphins activity.
Assuntos
Analgésicos Opioides/química , Analgésicos Opioides/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peptídeos Opioides/química , Peptídeos Opioides/farmacologia , Analgesia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/antagonistas & inibidores , Análise de Variância , Animais , Anticorpos/imunologia , Dinorfinas/administração & dosagem , Dinorfinas/antagonistas & inibidores , Dinorfinas/química , Dinorfinas/farmacologia , Encefalina Leucina/administração & dosagem , Encefalina Leucina/antagonistas & inibidores , Encefalina Leucina/química , Encefalina Leucina/farmacologia , Encefalina Metionina/administração & dosagem , Encefalina Metionina/antagonistas & inibidores , Encefalina Metionina/química , Encefalina Metionina/farmacologia , Flúor/química , Injeções Espinhais , Masculino , Camundongos , Naloxona/administração & dosagem , Naloxona/farmacologia , Naltrexona/administração & dosagem , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Oligopeptídeos/administração & dosagem , Oligopeptídeos/antagonistas & inibidores , Peptídeos Opioides/administração & dosagem , Peptídeos Opioides/antagonistas & inibidores , Dor/tratamento farmacológico , Dor/metabolismo , Medição da Dor , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Receptores sigma/antagonistas & inibidoresRESUMO
Hidden Markov models (HMMs) provide a framework to analyze large trajectories of biomolecular simulation datasets. HMMs decompose the conformational space of a biological molecule into finite number of states that interconvert among each other with certain rates. HMMs simplify long timescale trajectories for human comprehension, and allow comparison of simulations with experimental data. In this chapter, we provide an overview of building HMMs for analyzing bimolecular simulation datasets. We demonstrate the procedure for building a Hidden Markov model for Met-enkephalin peptide simulation dataset and compare the timescales of the process.
Assuntos
Simulação por Computador , Encefalina Metionina/química , Cadeias de Markov , Fragmentos de Peptídeos/química , Algoritmos , Biologia Computacional/métodos , Bases de Dados de Proteínas , HumanosRESUMO
Affinities of quaternary ammonium-chitosan conjugates, their thiolated derivatives and corresponding nanostructured aggregates towards the hydrophilic drug [5-methionine]enkephalin were compared by Nuclear Magnetic Resonance (NMR) spectroscopic methods based on proton selective relaxation rate measurements. Nanoaggregates showed enhanced drug affinity in comparison with corresponding polymers, especially in the case of thiolated systems.
Assuntos
Quitosana/química , Encefalina Metionina/química , NanoestruturasRESUMO
We describe an NMR method to generate singlet order in spin pairs from longitudinal spin magnetization and suppress residual background signals. This method can also be used for generating and observing long-lived spin states. A singlet order selection (SOS) filter is proposed, which allows us to find signals of the spin pair of interest buried in a crowded NMR spectrum. Likewise, SOS filtering enables proton NMR measurements in H2O without pulse sequences for solvent suppression. We demonstrate that the method works perfectly for both weakly and strongly coupled spin pairs. Furthermore, it can be combined with standard NMR pulse sequences: in this way, T1- and T2-relaxation times for spin pairs of interest can be measured. The power of the SOS-filter is demonstrated by relaxation studies in biomolecules.
Assuntos
Encefalina Metionina/química , Oligopeptídeos/química , Ubiquitina/química , Concentração de Íons de Hidrogênio , Espectroscopia de Prótons por Ressonância Magnética/normas , Padrões de ReferênciaRESUMO
This study describes a novel approach to polymeric nanocarriers of the therapeutic peptide met-enkephalin based on the aggregation of thermoresponsive polymers. Thermoresponsive bioconjugate poly((di(ethylene glycol) monomethyl ether methacrylate)-ran-(oligo(ethylene glycol) monomethyl ether methacrylate) is synthesized by AGET ATRP using modified met-enkephalin as a macroinitiator. The abrupt heating of bioconjugate water solution leads to the self-assembly of bioconjugate chains and the formation of mesoglobules of controlled sizes. Mesoglobules formed by bioconjugates are stabilized by coating with cross-linked two-layer shell via nucleated radical polymerization of N-isopropylacrylamide using a degradable cross-linker. The targeting peptide RGD, containing the fluorescence marker carboxyfluorescein, is linked to a nanocarrier during the formation of the outer shell layer. In the presence of glutathione, the whole shell is completely degradable and the met-enkephalin conjugate is released. It is anticipated that precisely engineered nanoparticles protecting their cargo will emerge as the next-generation platform for cancer therapy and many other biomedical applications.
Assuntos
Portadores de Fármacos/química , Encefalina Metionina/química , Nanopartículas/química , Oligopeptídeos/química , Polímeros/química , Polimerização , Propriedades de SuperfícieRESUMO
The Met-enkephalin, Tyr-Gly-Gly-Phe-Met, was synthesized by the solution-phase synthesis (SPS) methodology employing -OBzl group as carboxyls' protection, while the t-Boc groups were employed for the N-terminal α-amines' protection for the majority of the amino acids of the pentapeptide sequence. The l-methionine (l-Met) amino acid was used as PTSA.Met-OBzl obtained from the simultaneous protection of the α-amino, and carboxyl group with para-toluene sulfonic acid (PTSA) and as-OBzl ester, respectively in a C-terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single-step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l-methionine and l-phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.
Assuntos
Benzenossulfonatos/química , Encefalina Metionina/síntese química , Metionina/química , Peptídeos/síntese química , Encefalina Metionina/químicaRESUMO
The TaBoo SeArch (TBSA) algorithm [ Harada et al. J. Comput. Chem. 2015 , 36 , 763 - 772 and Harada et al. Chem. Phys. Lett. 2015 , 630 , 68 - 75 ] was recently proposed as an enhanced conformational sampling method for reproducing biologically relevant rare events of a given protein. In TBSA, an inverse histogram of the original distribution, mapped onto a set of reaction coordinates, is constructed from trajectories obtained by multiple short-time molecular dynamics (MD) simulations. Rarely occurring states of a given protein are statistically selected as new initial states based on the inverse histogram, and resampling is performed by restarting the MD simulations from the new initial states to promote the conformational transition. In this process, the definition of the inverse histogram, which characterizes the rarely occurring states, is crucial for the efficiency of TBSA. In this study, we propose a simple modification of the inverse histogram to further accelerate the convergence of TBSA. As demonstrations of the modified TBSA, we applied it to (a) hydrogen bonding rearrangements of Met-enkephalin, (b) large-amplitude domain motions of Glutamine-Binding Protein, and (c) folding processes of the B domain of Staphylococcus aureus Protein A. All demonstrations numerically proved that the modified TBSA reproduced these biologically relevant rare events with nanosecond-order simulation times, although a set of microsecond-order, canonical MD simulations failed to reproduce the rare events, indicating the high efficiency of the modified TBSA.
Assuntos
Algoritmos , Proteínas de Transporte/química , Encefalina Metionina/química , Proteína Estafilocócica A/química , Proteínas de Bactérias/química , Conformação Proteica , Dobramento de Proteína , Staphylococcus aureusRESUMO
Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies indicated that NPYFa induced an early onset, potent, dose-dependent and prolonged antinociception. Additionally, antagonists (MOR, KOR, and DOR) pretreatment studies determined a KOR-mediated antinociception activity of the ligand. Further, in vitro binding studies using the Eu-GTP-γS binding assay on cell lines expressing opioid and NPFF receptors showed binding to both the opioid and neuropeptide FF receptors suggesting a multiple receptor binding character of NPYFa. Moreover, chronic (6 days) treatment with NPYFa exhibited an absence of tolerance development subsequent to its analgesia. The current study proposes NPYFa as a potent, long-acting antinociceptor lacking tolerance development as well as a probe to study opioid analgesia and the associated complex mechanisms of tolerance development.
Assuntos
Analgesia , Analgésicos , Encefalina Metionina , Oligopeptídeos , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Encefalina Metionina/análogos & derivados , Encefalina Metionina/química , Encefalina Metionina/farmacocinética , Encefalina Metionina/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Receptores Opioides/agonistas , Receptores Opioides/metabolismo , Fatores de TempoRESUMO
Recent experimental results about the oxidation of methionine enkephalin by ·OH radicals indicated an intramolecular electron transfer between the C-terminal methionine radical cation and the tyrosine N-terminus too fast to be observed. We have investigated the thermodynamic possibility of this intramolecular electron transfer by calculating the one-electron redox potentials of both residues for several conformations of the peptide, extracted from the experimental data of the Protein Data Bank (1PLW). Using a QM/MM approach, we show that the redox potential of the Met(â¢+)/Met couple is higher than that of the TyrOH(â¢+)/TyrOH one (tyrosine is denoted as TyrOH) for all conformations. The intramolecular electron transfer between both residues (from TyrOH to Met(â¢+)) is thus always thermodynamically allowed. Previously, we had performed topological studies on the intramolecular electron transfer which predicted this charge transfer. A study by cyclic voltammetry pointed out that the wave belonging to methionine is not present when methionine enkephalin is oxidized and only the direct involvement of the tyrosine residue is observed.
Assuntos
Encefalina Metionina/química , Cátions/química , Bases de Dados de Proteínas , Transporte de Elétrons , Elétrons , Concentração de Íons de Hidrogênio , Radical Hidroxila/química , Modelos Químicos , Estrutura Molecular , Oxirredução , Teoria Quântica , TermodinâmicaRESUMO
Nuclear Magnetic Relaxation Dispersion (NMRD) of protons was studied in the pentapeptide Met-enkephalin and the amino acids, which constitute it. Experiments were run by using high-resolution Nuclear Magnetic Resonance (NMR) in combination with fast field-cycling, thus enabling measuring NMRD curves for all individual protons. As in earlier works, Papers I-III, pronounced effects of intramolecular scalar spin-spin interactions, J-couplings, on spin relaxation were found. Notably, at low fields J-couplings tend to equalize the apparent relaxation rates within networks of coupled protons. In Met-enkephalin, in contrast to the free amino acids, there is a sharp increase in the proton T1-relaxation times at high fields due to the changes in the regime of molecular motion. The experimental data are in good agreement with theory. From modelling the relaxation experiments we were able to determine motional correlation times of different residues in Met-enkephalin with atomic resolution. This allows us to draw conclusions about preferential conformation of the pentapeptide in solution, which is also in agreement with data from two-dimensional NMR experiments (rotating frame Overhauser effect spectroscopy). Altogether, our study demonstrates that high-resolution NMR studies of magnetic field-dependent relaxation allow one to probe molecular mobility in biomolecules with atomic resolution.