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1.
Chem Commun (Camb) ; 60(22): 3023-3026, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38356394

RESUMO

The design and synthesis of leu-enkephalin analogs by replacing the glycine residues with N-(2-thioethyl)glycines and opening the cyclisation potential is presented. The cyclization (stapling) was achieved using bifunctional reagents (hexafluorobenzene and trithiocyanuric acid derivatives). The CD conformational studies of the stapled analogs suggest that the peptides adopt the type I ß-turn conformation, which is in agreement with the theoretical analysis. The analog containing a trithiocyanuric acid derivative with a benzyl substituent shows potent analgesic activity.


Assuntos
Encefalina Leucina , Glicina , Encefalina Leucina/farmacologia , Reagentes de Ligações Cruzadas , Conformação Molecular , Analgésicos/farmacologia , Analgésicos/química
2.
Mol Pharm ; 21(2): 688-703, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38243899

RESUMO

Persistent pain is a significant healthcare problem with limited treatment options. The high incidence of comorbid chronic pain and depression significantly reduces life quality and complicates the treatment of both conditions. Antidepressants are less effective for pain and depression than for depression alone and they induce severe side effects. Opioids are highly efficacious analgesics, but rapid development of tolerance, dependence, and debilitating side effects limit their efficacy and safe use. Leucine-enkephalin (Leu-ENK), the endogenous delta opioid receptor agonist, controls pain and mood and produces potent analgesia with reduced adverse effects compared to conventional opioids. High proteolytic instability, however, makes Leu-ENK ineffective after systemic administration and limits its clinical usefulness. KK-103, a Leu-ENK prodrug, was developed to overcome these limitations of Leu-ENK via markedly increased plasma stability in mice. We showed rapid and substantially increased systemic adsorption and blood plasma exposure of KK-103 compared to Leu-ENK. We also observed brain uptake of radiolabeled KK-103 after systemic administration, indicating a central effect of KK-103. We then established KK-103's prolonged antinociceptive efficacy in the ramped hot plate and formalin test. In both models, KK-103 produced a comparable dose to the maximum antinociceptive-effect relationship. The pain-alleviating effect of KK-103 primarily resulted from activating the delta opioid receptor after the likely conversion of KK-103 to Leu-ENK in vivo. Finally, KK-103 produced an antidepressant-like activity comparable to the antidepressant desipramine, but with minimal gastrointestinal inhibition and no incidence of sedation.


Assuntos
Encefalina Leucina , Pró-Fármacos , Camundongos , Animais , Receptores Opioides delta/uso terapêutico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Dor/tratamento farmacológico , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico
3.
Artigo em Inglês | MEDLINE | ID: mdl-37263377

RESUMO

The enkephalins are known to regulate many physiological functions, including reproduction in vertebrates. However, the role of leucine-enkephalin (L-ENK) in the ovarian recrudescence activity of reptiles is not known. In the present study, we studied the influence of L-ENK on seasonal and FSH-induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical gecko Hemidactylus frenatus. In the first experiment, treatment with 5 and 25 µg L-ENK resulted in a dose-dependent inhibitory effect on the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and ovary, as indicated by a significantly decreased percent area of GnRH-immunoreactive (GnRH-ir) fibres in the median eminence and pars distalis of the pituitary gland, concomitant with complete absence of stage V (late vitellogenic) follicles in the ovary compared to those of experimental controls. In the second experiment, administration of FSH to lizards in the regression phase stimulated the recruitment of stage IV and V (vitellogenic) follicles in contrast to their absence in initial controls or treatment controls. However, similar treatment of FSH in combination with 25 µg L-ENK did not result in the development of stage IV or V follicles. Together, these results suggest for the first time that treatment with 5 and 25 µg L-ENK exerts a dose-dependent inhibitory effect on the hypothalamic GnRH release into the median eminence and pituitary gland, leading to the blockade of ovarian recrudescence. These results also suggest a possible direct inhibitory effect of L-ENK at the level of the ovary in the gecko.


Assuntos
Lagartos , Ovário , Feminino , Animais , Leucina , Folículo Ovariano , Encefalina Leucina/farmacologia , Peptídeos Opioides/farmacologia , Estações do Ano , Hormônio Liberador de Gonadotropina/farmacologia , Gonadotropinas/farmacologia , Hormônio Foliculoestimulante , Lagartos/fisiologia
4.
Molecules ; 28(4)2023 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-36838964

RESUMO

Dipeptidyl peptidase III (DPP III), a zinc exopeptidase, is involved in the final steps of intercellular protein degradation and has a marked affinity for opioid peptides such as enkephalins and endomorphins. Recently, we characterized a number of neuropeptides as potential substrates and inhibitors of human DPP III and provided an explanation for their differential behavior. These studies prompted us to investigate the influence of the conserved R399 and R669 on neuropeptides binding to DPP III. Measuring kinetic parameters in inhibitory assays, we found that mutation of R669 to Ala or Met significantly reduced the inhibitory properties of the slow substrates tynorphin and valorphin, whereas the effects on binding of the good substrates Arg2-2NA and Leu-enkephalin were small. Molecular dynamics simulations of wild-type (WT) and mutant DPP III complexes with Leu-enkephalin, tynorphin, valorphin, and Arg2-2NA in conjunction with calculations of binding free energies revealed that the lower inhibitory potency of slow substrates in the R669A mutant can be explained by the lower binding affinity of tynorphin and the higher propensity of valorphin to hydrolyze in the mutant than in WT. The R399A mutation was shown to affect the binding and/or hydrolysis of both good and slow substrates, with the effects on Leu-enkephalin being the most pronounced.


Assuntos
Encefalina Leucina , Encefalinas , Humanos , Domínio Catalítico , Mutação
5.
J Med Chem ; 65(18): 12367-12385, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36099150

RESUMO

Chronic pain and depression are both widely prevalent comorbid medical conditions. While efficient, µ-opioid receptor-based medications are associated with life-threatening side effects, including respiratory depression, dependence, and addiction. The δ-opioid receptor is a promising alternative biological target for chronic pain and depression due to its significantly reduced on-target side effects compared to the µ-opioid receptor. A previous study identified two δ-opioid receptor positive allosteric modulators. Herein, we report the design of five series of compounds targeting previously unexplored regions of the originally described SAR. Analogs were assessed for their ability to potentiate the agonist response of Leu-enkephalin. Of the 30 analogs, compound 6g displayed trends toward enhancing the ERK1/2 phosphorylation signaling compared to cAMP inhibition, while compound 11c exhibited a trend in shifting the signaling bias toward cAMP inhibition. Both 6g and 11c emerged as promising tool compounds toward the design of prospective therapeutics requiring specific downstream signaling attributes.


Assuntos
Dor Crônica , Depressão , Receptores Opioides delta , Antidepressivos/química , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Dor Crônica/tratamento farmacológico , Depressão/tratamento farmacológico , Encefalina Leucina/farmacologia , Humanos , Receptores Opioides mu/agonistas , Xantenos/síntese química , Xantenos/farmacologia
6.
Rapid Commun Mass Spectrom ; 36(20): e9373, 2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-35933590

RESUMO

RATIONALE: The COVID-19 pandemic demonstrated the importance of high-throughput analysis for public health. Given the importance of surface viral proteins for interactions with healthy tissue, they are targets of interest for mass spectrometry-based analysis. For that reason, the possibility of detecting and quantifying peptides using a high-throughput technique, laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS), was explored. METHODS: Two peptides used as models for small peptides (leu-enkephalin and endomorphin-2) and four tryptic peptides (GVYYPDK, NIDGYFK, IADYNYK, and QIAPGQTGK) specific to the SARS-CoV-2 Spike protein were employed. Target peptides were analyzed individually in the positive mode by LDTD-QqQMS. Peptides were quantified by internal calibration using selected reaction monitoring transitions in pure solvents and in samples spiked with 20 µg mL-1 of a bovine serum albumin tryptic digest to represent real analysis conditions. RESULTS: Low-energy fragment ions (b and y ions) as well as high-energy fragment ions (c and x ions) and some of their corresponding water or ammonia losses were detected in the full mass spectra. Only for the smallest peptides, leu-enkephalin and endomorphin-2, were [M + H]+ ions observed. Product ion spectra confirmed that, with the experimental conditions used in the present study, LDTD transfers a considerable amount of energy to the target peptides. Quantitative analysis showed that it was possible to quantify peptides using LDTD-QqQMS with acceptable calibration curve linearity (R2 > 0.99), precision (RSD < 18.2%), and trueness (bias < 8.3%). CONCLUSIONS: This study demonstrated for the first time that linear peptides can be qualitatively and quantitatively analyzed using LDTD-QqQMS. Limits of quantification and dynamic ranges are still inadequate for clinical applications, but other applications where higher levels of proteins must be detected could be possible with LDTD. Given the high-throughput capabilities of LDTD-QqQMS (>15 000 samples in less than 43 h), more studies are needed to improve the sensitivity for peptide analysis of this technique.


Assuntos
COVID-19 , Espectrometria de Massas em Tandem , Encefalina Leucina , Humanos , Íons , Lasers , Pandemias , Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Espectrometria de Massas em Tandem/métodos
7.
J Am Soc Mass Spectrom ; 33(9): 1626-1635, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-35895596

RESUMO

Sustained off-resonance irradiation-cross-sectional areas by Fourier transform ion cyclotron resonance mass spectrometry (SORI-CRAFTI) is an FTICR-MS strategy to collisionally activate precursor ions and then measure their ion-neutral collision cross sections, as well as those of selected products, at the same time. We benchmarked SORI-CRAFTI using protonated leucine-enkephalin, to excellent agreement (typically within 1-2%) with previous studies performed via collision-induced dissociation-ion mobility (CID-IMS). SORI-CRAFTI was then applied to alkali metal-cationized leucine-enkephalin and compared with CID-IMS via precursor/product cross-section ratios. Qualitative agreement between SORI-CRAFTI and CID-IMS was excellent (again, usually within 1-2%); however, neither SORI-CRAFTI nor CID-IMS could determine if metalated leucine-enkephalin was present in its canonical or zwitterionic form. When SORI-CRAFTI was used on [2.2.2]-cryptand+Cs+, SORI activation resulted in a 5% decrease in collision cross section, consistent with migration of the externally bound Cs+ into the cryptand's cavity and similar to the cross section observed when electrospraying from an isopropanol-rich solvent. Thus, SORI-CRAFTI is useful for studying gas-phase ion chemistry of small- to medium-sized molecules and host-guest systems.


Assuntos
Éteres de Coroa , Encefalina Leucina , Íons/química , Leucina , Espectrometria de Massas/métodos
8.
Bull Exp Biol Med ; 173(2): 188-192, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35737163

RESUMO

Hypobaric hypoxia (pO2 65 mm Hg, duration 4 h) induced a significant increase in the number of cardiomyocytes expressing р53, beclin-1, endothelial NO synthase and accumulation and degranulation of mast cells in the epicardium in hearts of prepubertal female rats (age 45-47 days); the number of cardiomyocytes with nucleoli decreased, while the number of single-nucleolar cardiomyocytes increased after this exposure. Five-fold administration of non-opiate analogue of leu-enkephalin (NALE peptide: Phe-D-Ala-Gly-Phe-Leu-Arg; 100 µg/kg) during the neonatal period reduced the severity of the post-hypoxic changes in the heart. Neonatal administration of NALE (100 µg/kg) against the background of NO synthase blockade with L-NAME (50 mg/kg) did not abolish the cardioprotective effects of the peptide. A similar correction of posthypoxic changes in the heart was observed after neonatal administration of original peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly; 100 µg/kg). Thus, NO synthase-NO system and C-terminal amino acid Arg in the molecule of non-opiate analogue of leu-enkephalin are not required for the cardioprotective effects of peptides. Non-opiate leu-enkephalin analogs, peptides NALE and G, can be considered as promising substances for increasing heart resistance to hypoxia during later age periods.


Assuntos
Encefalina Leucina , Hipóxia , Encefalina Leucina/farmacologia , Feminino , Coração , Homeostase , Humanos , Hipóxia/tratamento farmacológico , Ratos
9.
J Formos Med Assoc ; 121(11): 2248-2256, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35570051

RESUMO

PURPOSE: To evaluate the perioperative outcome of laparoendoscopic two-site myomectomy (LETS-M). METHODS: The medical records of 204 women receiving LETS-M in a tertiary referral center, including 183 surgeries performed by the experienced surgeon and 21 surgeries performed by 3 well-supervised trainees were retrospectively reviewed. RESULTS: The age of the participants was 39.3 ± 6.4 years. The mean diameter of the largest myoma and the mean number of myomas were 8.5 ± 2.2 cm and 1.7 ± 1.1, respectively. Thirty-one (15%) operations removed more than 2 myomas larger than 5 cm in diameter. The mean weight of the myomas was 281.1 ± 183.1 g. The operation time was 97.6 ± 40.2 min, and the intraoperative blood loss was 99.3 ± 115.2 mL. There were 3 (1%) cases of excessive blood loss (more than 500 mL) and 2 (1%) of postoperative hematoma. The only significant difference between the experienced surgeon and trainees was the operation time (92.3 ± 32.2 min vs. 141.2 ± 54 min, p < .001), while the myoma number, myoma diameter, myoma weight, and intraoperative blood loss were not significantly different. The operation time did not differ among different myoma locations. In multivariate analysis, virginity, myoma number, more than 2 large myomas, and myoma size were independent variables for longer operation times. No patient experienced any major complications. CONCLUSION: LETS-M using conventional laparoscopic equipment is a minimally invasive surgical method that is safe, effective, and easy to learn for managing uterine myoma. It is useful to achieve a favorable perioperative outcome with acceptable operation time.


Assuntos
Laparoscopia , Mioma , Miomectomia Uterina , Neoplasias Uterinas , Adulto , Perda Sanguínea Cirúrgica , Encefalina Leucina/análogos & derivados , Feminino , Humanos , Laparoscopia/métodos , Pessoa de Meia-Idade , Mioma/cirurgia , Estudos Retrospectivos , Miomectomia Uterina/métodos , Neoplasias Uterinas/cirurgia
10.
Bull Exp Biol Med ; 173(1): 1-4, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35622244

RESUMO

In white rats with experimental hypothyroidism, changes in the myeloid compartment of the blood system induced by 6-h immobilization stress and the corrective effect of the analogue of leu-enkephalin (dalargin) on these shifts was analyzed. It was found that in rats with hypothyroidism, stress in the anxiety stage did not cause leukocytosis typical of euthyroid animals, but at the stage of resistance provoked leukopenia at the expense of eosinopenia and neutropenia with depletion of the intramedullary reserve. Dalargin increased white blood cells count, neutrophil count, and the intramedullary depot of these cells.


Assuntos
Encefalina Leucina , Hipotireoidismo , Animais , Leucina Encefalina-2-Alanina , Ratos
11.
Int J Mol Sci ; 23(3)2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35163780

RESUMO

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes peptides of varying sequence and size. Despite attempts to elucidate and narrow down the broad substrate-specificity of DPP III, there is no explanation as to why some of them, such as tynorphin (VVYPW), the truncated form of the endogenous heptapeptide spinorphin, are the slow-reacting substrates of DPP III compared to others, such as Leu-enkephalin. Using quantum molecular mechanics calculations followed by various molecular dynamics techniques, we describe for the first time the entire catalytic cycle of human DPP III, providing theoretical insight into the inhibitory mechanism of tynorphin. The chemical step of peptide bond hydrolysis and the substrate binding to the active site of the enzyme and release of the product were described for DPP III in complex with tynorphin and Leu-enkephalin and their products. We found that tynorphin is cleaved by the same reaction mechanism determined for Leu-enkephalin. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Encefalina Leucina/química , Oligopeptídeos/química , Domínio Catalítico , Encefalina Leucina/farmacologia , Humanos , Hidrólise , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Oligopeptídeos/farmacologia , Domínios Proteicos , Teoria Quântica
12.
Molecules ; 26(23)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34885825

RESUMO

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a 'NAM-agonist' in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.


Assuntos
Receptores Opioides delta/agonistas , Regulação Alostérica/efeitos dos fármacos , Aminoácidos/química , Sítios de Ligação , AMP Cíclico/metabolismo , Encefalina Leucina/química , Encefalina Leucina/farmacologia , Células HEK293 , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismo
13.
Bull Exp Biol Med ; 172(2): 270-275, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34855076

RESUMO

Incubation of primary culture of pulmonary fibroblasts with non-opiate analogue of leuenkephalin (NALE; Phe-D-Ala-Gly-Phe-Leu-Arg, 0.1 µM) reduced generation of superoxide anion-radical (by 20.7%) and decreased the number of p53+ cells (by 40.2%) induced by exposure to H2O2 (60 µM). The cytoprotective effect of NALE was potentiated by NO synthase inhibitor L-NAME (1 mM): the number of p53+ cells decreased by 65.3% and morphometric parameters of the cell nuclei and nucleoli were improved. Incubation of pulmonary fibroblasts culture with peptide G (Phe-D-Ala-Gly-Phe-Leu-Gly, 0.1 µM) also significantly reduced the damaging effect of H2O2: the number of p53+ cells decreased by 73.5%, the area of cell nuclei returned to normal, and generation of superoxide anion-radical decreased by 18.4%. These results indicate that C-terminal amino acid Arg and activation of NO synthase are not involved in the direct cytoprotective effect of NALE.


Assuntos
Arginina/fisiologia , Encefalina Leucina/farmacologia , Óxido Nítrico/fisiologia , Animais , Arginina/farmacologia , Células Cultivadas , Citoproteção/efeitos dos fármacos , Encefalina Leucina/análogos & derivados , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Peróxido de Hidrogênio/farmacologia , Pulmão/citologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar
14.
Dev Comp Immunol ; 121: 104074, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33775662

RESUMO

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma ß-endorphins increase during bovine uterine inflammation. However, the effect of ß-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of ß-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P < 0.05). By using Western blot and qPCR techniques, we found that ß-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P < 0.05). The co-treatment of ß-endorphins and opioid antagonists showed that the anti-inflammatory effect of ß-endorphins could be blocked (P < 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the µ opioid receptor antagonist CTAP (P > 0.05). In conclusion, ß-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.


Assuntos
Endometrite/imunologia , Endométrio/imunologia , Infecção Puerperal/veterinária , Receptores Opioides delta/metabolismo , beta-Endorfina/metabolismo , Criação de Animais Domésticos , Animais , Bovinos , Células Cultivadas , Endometrite/microbiologia , Endométrio/metabolismo , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Células Epiteliais , Escherichia coli/imunologia , Feminino , Inflamação , Lipopolissacarídeos/imunologia , NF-kappa B/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Cultura Primária de Células , Infecção Puerperal/imunologia , Infecção Puerperal/microbiologia , Receptores Opioides delta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
15.
Medicine (Baltimore) ; 100(8): e24581, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33663067

RESUMO

ABSTRACT: Major depressive disorder (MDD) is a common disease with both affective and cognitive disorders. Alterations in metabolic systems of MDD patients have been reported, but the underlying mechanisms still remains unclear. We sought to identify abnormal metabolites in MDD by metabolomics and to explore the association between differential metabolites and neurocognitive dysfunction.Plasma samples from 53 MDD patients and 83 sex-, gender-, BMI-matched healthy controls (HCs) were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was then used to detect metabolites in those samples. Two different algorithms were applied to identify differential metabolites in 2 groups. Of the 136 participants, 35 MDD patients and 48 HCs had completed spatial working memory test. Spearman rank correlation coefficient was applied to explore the relationship between differential metabolites and working memory in these 2 groups.The top 5 metabolites which were found in sparse partial least squares-discriminant analysis (sPLS-DA) model and random forest (RF) model were the same, and significant difference was found in 3 metabolites between MDD and HCs, namely, gamma-glutamyl leucine, leucine-enkephalin, and valeric acid. In addition, MDD patients had higher scores in spatial working memory (SWM) between errors and total errors than HCs. Valeric acid was positively correlated with working memory in MDD group.Gamma-glutamyl leucine, leucine-enkephalin, and valeric acid were preliminarily proven to be decreased in MDD patients. In addition, MDD patients performed worse in working memory than HCs. Dysfunction in working memory of MDD individuals was associated with valeric acid.


Assuntos
Transtorno Depressivo Maior/sangue , Memória de Curto Prazo/fisiologia , Navegação Espacial/fisiologia , Adolescente , Adulto , Fatores Etários , Algoritmos , Índice de Massa Corporal , Cromatografia Líquida , Transtorno Depressivo Maior/fisiopatologia , Dipeptídeos/sangue , Encefalina Leucina/sangue , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Ácidos Pentanoicos/sangue , Escalas de Graduação Psiquiátrica , Fatores Sexuais , Espectrometria de Massas em Tandem , Adulto Jovem
16.
J Am Soc Mass Spectrom ; 32(3): 670-684, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33573373

RESUMO

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 → π*(C═C) 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 → π*(C═N) 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 Proteína
17.
J Mass Spectrom ; 56(1): e4693, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33277714

RESUMO

Quadrupole time-of-flight (QTof) collision-induced dissociation (CID) and Orbitrap higher-energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry-based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q-Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap-specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).


Assuntos
Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Encefalina Leucina/análise , Encefalina Leucina/química , Células HeLa , Humanos , Peptídeos/análise , Peptídeos/química , Fosfopiruvato Hidratase/química , Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas
18.
Mol Pharm ; 17(11): 4163-4179, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32986444

RESUMO

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, •OH, •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/métodos
19.
Anal Chem ; 92(21): 14558-14567, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-32961052

RESUMO

There are many processes that actively alter the concentrations of solutes in the extracellular space. Enzymatic reactions, either by soluble enzymes or membrane-bound ectoenzymes, and uptake or clearance are two such processes. Investigations of ectoenzymatic reactions in vivo is challenging, particularly in the brain. Studies using microdialysis have revealed some qualitative information about what enzymes may be present, but microdialysis is a sampling technique so it is not designed to control conditions such as a substrate concentration outside the probe. Micropush-pull perfusion has been used to determine which nitric oxide synthase enzymes are active in discrete regions of the rat retina. Ectopeptidases are a particularly important class of ectoenzymes. As far as it is known, the extracellular activity of active peptides in the brain is controlled by ectopeptidases. To understand ectopeptidase activity, we developed a physical probe and an accompanying method. The probe has a two-channel source that supplies substrate or substrate plus inhibitor using electroosmotic perfusion (EOP). It also has a microdialysis probe to collect products and unreacted substrate. The method provides quantitative estimates of substrate-to-product conversion and the influence of inhibitors on this process. The quantitative estimates are made possible by including a d-amino acid-containing peptide analog of the substrate in the substrate-containing solution infused. Quantitative analysis of substrate, substrate analog, and products is carried out by quantitative, online capillary liquid chromatography-tandem mass spectrometry. The electroosmotic perfusion-microdialysis probe and associated method were used to determine the effect of the selective inhibitor HFI-419 on insulin-regulated aminopeptidase (EC 3.4.11.3) in the rat neocortex.


Assuntos
Aminopeptidases/metabolismo , Eletro-Osmose/métodos , Encefalina Leucina/metabolismo , Insulina/metabolismo , Lasers , Microdiálise/métodos , Animais , Hidrólise , Neocórtex/metabolismo , Perfusão , Ratos
20.
J Am Soc Mass Spectrom ; 31(1): 117-123, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-32881522

RESUMO

MeV-SIMS is an emerging mass spectrometry imaging method that employs fast, heavy ions to desorb secondary molecules from the analyzed sample. High yields and low fragmentation rates of large molecules, associated with the dominating electronic sputtering process, make it particularly useful in biomedical research, where insight into the distribution of organic molecules is vital. Both yield and fragmentation of desorbed molecules in MeV-SIMS rely on characteristics of the primary ion but may also be impaired by poor instrumental settings. After utilizing secondary ion optics in the linear mass spectrometer at the micro-analytical center of the Jozef Stefan Institute, we demonstrate very efficient detection of secondary ions. As a result, the secondary ion yield, using such settings, solely depends on the species and the characteristics of the primary ion. In order to analyze the yield dependence on the primary ion energy, and the corresponding stopping power within the electronic excitation regime, we used a continuous electron multiplier detector to measure the primary ion current during each measurement of the mass spectra. Secondary ion yield as a function of the primary ion energy and charge is presented as well as fragmentation rates of organic molecules arginine and leu-enkephalin. Other influential instrumental drawbacks are also studied, and their effect on the results is discussed.


Assuntos
Cloro/química , Espectrometria de Massa de Íon Secundário/métodos , Arginina/química , Fracionamento Químico/métodos , Encefalina Leucina/química , Imagem Molecular/métodos , Espectrometria de Massa de Íon Secundário/instrumentação
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