RESUMO
In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.
Assuntos
Cricetulus , NAD , Niacinamida , Células CHO , Animais , NAD/metabolismo , Niacinamida/metabolismo , Niacinamida/análogos & derivados , Meios de Cultura/química , Meios de Cultura/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Niacina/metabolismo , Compostos de Piridínio/metabolismo , Cricetinae , Técnicas de Cultura de Células/métodos , Anticorpos Monoclonais/metabolismo , Glucose/metabolismoRESUMO
We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular , Bibliotecas de Moléculas Pequenas , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proliferação de Células/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Azepinas/farmacologia , Azepinas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Indolizinas/química , Indolizinas/farmacologia , Linhagem Celular Tumoral , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ligantes , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/síntese química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas que Contêm Bromodomínio , Óxidos N-Cíclicos , Compostos de PiridínioRESUMO
Friedreich's ataxia (FRDA) is a progressive disorder caused by insufficient expression of frataxin, which plays a critical role in assembly of iron-sulfur centers in mitochondria. Individuals are cognitively normal but display a loss of motor coordination and cardiac abnormalities. Many ultimately develop heart failure. Administration of nicotinamide adenine dinucleotide-positive (NAD+) precursors has shown promise in human mitochondrial myopathy and rodent models of heart failure, including mice lacking frataxin in cardiomyocytes. We studied mice with systemic knockdown of frataxin (shFxn), which display motor deficits and early mortality with cardiac hypertrophy. Hearts in these mice do not "fail" per se but become hyperdynamic with small chamber sizes. Data from an ongoing natural history study indicate that hyperdynamic hearts are observed in young individuals with FRDA, suggesting that the mouse model could reflect early pathology. Administering nicotinamide mononucleotide or riboside to shFxn mice increases survival, modestly improves cardiac hypertrophy, and limits increases in ejection fraction. Mechanistically, most of the transcriptional and metabolic changes induced by frataxin knockdown are insensitive to NAD+ precursor administration, but glutathione levels are increased, suggesting improved antioxidant capacity. Overall, our findings indicate that NAD+ precursors are modestly cardioprotective in this model of FRDA and warrant further investigation.
Assuntos
Modelos Animais de Doenças , Frataxina , Ataxia de Friedreich , Proteínas de Ligação ao Ferro , NAD , Animais , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Humanos , NAD/metabolismo , Fenótipo , Masculino , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Mononucleotídeo de Nicotinamida/farmacologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Compostos de Piridínio , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
BACKGROUND/AIM: Cholangiocarcinoma (CCA) is a highly aggressive disease. Most of CCA patients are diagnosed in an advanced stage of the disease, when it is unresectable and there is chemoresistance, resulting in poor prognosis. However, effective therapeutic regimens and molecular targets for CCA remain poor. Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression. Aberrant CDK activation is a hallmark of cancer. Dinaciclib is a small molecule inhibitor of multiple CDKs, currently under clinical evaluation for treating advanced malignancies. The efficacy of anti-tumor activity of dinaciclib against chemotherapy resistant CCA cells was examined in vitro and in vivo. MATERIALS AND METHODS: In this study, the effect of dinaciclib on growth and cell cycle in CCA cell lines were determined using the MTT assay and cell cycle analysis. The anti-tumor activity of dinaciclib was investigated in CCA-inoculated mice. In addition, the chemosensitizing effect of dinaciclib was investigated in gemcitabine-treated CCA cell lines. RESULTS: Dinaciclib significantly suppressed cell proliferation, induced G1/S phase cell cycle arrest and apoptosis of CCA cell lines. It significantly suppressed the growth of CCA cells in xenograft mouse models. We also found that dinaciclib significantly inhibited the growth of gemcitabine-resistant CCA cell lines (KKU-213A-GemR and KKU-100-GemR). Furthermore, dinaciclib significantly enhanced the anti-tumor activity of gemcitabine in CCA cell lines. CONCLUSION: Dinaciclib has the potential to be an effective therapeutic agent to control tumor cell growth of both parental and gemcitabine-resistant CCA cells.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Proliferação de Células , Colangiocarcinoma , Óxidos N-Cíclicos , Indolizinas , Compostos de Piridínio , Ensaios Antitumorais Modelo de Xenoenxerto , Indolizinas/farmacologia , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Animais , Óxidos N-Cíclicos/farmacologia , Humanos , Compostos de Piridínio/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gencitabina , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ciclo Celular/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
Two ionic liquids (ILs) with amphiphilic properties composed of 1-butyl-3-methylimidazolium dioctylsulfosuccinate (bmim-AOT) and 1-hexyl-3-methylimidazolium dioctylsulfosuccinate (hmim-AOT) form unilamellar vesicles spontaneously simply by dissolving the IL-like surfactant in water. These novel vesicles were characterized using two different and highly sensitive fluorescent probes: 6-propionyl-2-(dimethylaminonaphthalene) (PRODAN) and trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (HC). These fluorescent probes provide information about the physicochemical properties of the bilayer, such as micropolarity, microviscosity, and electron-donor capacity. In addition, the biocompatibility of these vesicles with the blood medium was evaluated, and their toxicity was determined using Dictyostelium discoideum amoebas. First, using PRODAN and HC, it was found that the bilayer composition and the chemical structure of the ions at the interface produced differences between both amphiphiles, making the vesicles different. Thus, the bilayer of hmim-AOT vesicles is less polar, more rigid, and has a lower electron-donor capacity than those made by bmim-AOT. Finally, the results obtained from the hemolysis studies and the growth behavior of unicellular amoebas, particularly utilizing the D. discoideum assay, showed that both vesicular systems do not produce toxic effects up to a concentration of 0.02 mg/mL. This elegant assay, devoid of animal usage, highlights the potential of these newly organized systems for the delivery of drugs and bioactive molecules of different polarities.
Assuntos
Líquidos Iônicos , Tensoativos , Lipossomas Unilamelares , Líquidos Iônicos/química , Tensoativos/química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Nanomedicina , Corantes Fluorescentes/química , Compostos de Piridínio/química , Imidazóis/química , Bicamadas Lipídicas/químicaRESUMO
Purpose: Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG. Methods: Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed. Results: NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo. Conclusions: Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.
Assuntos
Modelos Animais de Doenças , Matriz Extracelular , Glaucoma , Glucocorticoides , Pressão Intraocular , Camundongos Endogâmicos C57BL , Mitocôndrias , Niacinamida , Compostos de Piridínio , Malha Trabecular , Animais , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Piridínio/farmacologia , Glucocorticoides/toxicidade , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Camundongos , Glaucoma/metabolismo , Glaucoma/tratamento farmacológico , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Humanos , Malha Trabecular/metabolismo , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologia , Células Cultivadas , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Espécies Reativas de Oxigênio/metabolismo , Dexametasona/farmacologia , MasculinoRESUMO
Mitophagy is a mechanism that maintains mitochondrial integrity and homeostasis and is thought to promote longevity and reduce the risk of age-related neurodegenerative diseases, including Alzheimer's disease (AD). Here, we investigate the abundance of mitochondrial reactive oxygen species (ROS), mitochondrial function, and mitophagy in primary fibroblasts from patients with sporadic AD (sAD) and normal healthy controls. The results show increased levels of mitochondrial ROS, changes in mitochondrial morphology, altered bioenergetic properties, and defects in autophagy, mitophagy, and lysosome-mediated degradation pathways in sAD fibroblasts relative to control fibroblasts. Interestingly, lysosome abundance and the staining of lysosomal markers remained high, while the capacity of lysosome-dependent degradation was lower in sAD fibroblasts than in controls fibroblasts. Nicotinamide riboside supplementation decreased mitochondrial ROS, while capacity for lysosomal degradation remained unchanged in sAD fibroblasts relative to healthy control fibroblasts. These findings provide insight into molecular mechanisms involving the dysregulation of lysosome and autophagy/mitophagy pathways that may contribute significantly to clinical signs and pathological features of sAD.
Assuntos
Doença de Alzheimer , Autofagia , Fibroblastos , Lisossomos , Mitocôndrias , Mitofagia , Espécies Reativas de Oxigênio , Humanos , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Lisossomos/metabolismo , Lisossomos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Células Cultivadas , Estudos de Casos e Controles , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de PiridínioRESUMO
Nicotinamide riboside (NR), a NAD+ precursor, has received attention due to several health benefits it has induced in experimental models. Studies in cultured cells, animals, and humans consistently show increased NAD+ availability after NR supplementation, which is considered the only mode of NR action that leads to health benefits. In the present study, we show that a persistently low NR concentration (1 µM) in the growth medium of BEAS-2B human cells, grown in a monolayer, induces energy stress, which precedes a cellular NAD+ increase after 192 h. NR concentrations greater than 1 µM under the specified conditions were cytotoxic in the 2D cell culture model, while all concentrations tested in the 3D cell culture model (BEAS-2B cell spheroids exposed to 1, 5, 10, and 50 µM NR) induced apoptosis. Shotgun proteomics revealed that NR modulated the abundance of proteins, agreeing with the observed effects on cellular energy metabolism and cell growth or survival. Energy stress may activate pathways that lead to health benefits such as cancer prevention. Accordingly, the premalignant 1198 cell line was more sensitive to NR cytotoxicity than the phenotypically normal parent BEAS-2B cell line. The role of a mild energy stress induced by low concentrations of NR in its beneficial effects deserves further investigation. On the other hand, strategies to increase the bioavailability of NR require attention to toxic effects that may arise.
Assuntos
Metabolismo Energético , Niacinamida , Compostos de Piridínio , Humanos , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Piridínio/farmacologia , Metabolismo Energético/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Reprogramação MetabólicaRESUMO
Nicotinamide adenine dinucleotide (NAD +) plays a pivotal role in numerous cellular functions. Reduced NAD + levels are postulated to be associated with cancer. As interest in understanding NAD + dynamics in cancer patients with therapeutic applications in mind grows, there remains a shortage of comprehensive data. This study delves into NAD + dynamics in patients undergoing surgery for different digestive system cancers. This prospective study enrolled 99 patients with eight different cancers. Fasting blood samples were obtained during the perioperative period. The concentrations of NAD + , nicotinamide mononucleotide (NMN), and nicotinamide riboside were analyzed using tandem mass spectrometry. After erythrocyte volume adjustment, NAD + remained relatively stable after surgery. Meanwhile, NMN decreased the day after surgery and displayed a recovery trend. Interestingly, liver and pancreatic cancer patients exhibited poor postoperative NMN recovery, suggesting a potential cancer type-specific influence on NAD + metabolism. This study illuminated the behavior of NAD + in surgically treated cancer patients. We identified which cancer types have particularly low levels and at what point depletion occurs during the perioperative period. These insights suggest the need for personalized NAD + supplementation strategies, calibrated to individual patient needs and treatment timelines. Clinical trial registration jRCT1020210066.
Assuntos
NAD , Niacinamida , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , NAD/metabolismo , Neoplasias/cirurgia , Neoplasias/metabolismo , Niacinamida/uso terapêutico , Niacinamida/metabolismo , Niacinamida/análogos & derivados , Mononucleotídeo de Nicotinamida/metabolismo , Estudos Prospectivos , Compostos de Piridínio , Espectrometria de Massas em TandemRESUMO
HIV disease remains prevalent in the United States and is particularly prevalent in sub-Saharan Africa. Recent investigations revealed that mitochondrial dysfunction in kidney contributes to HIV-associated nephropathy (HIVAN) in Tg26 transgenic mice. We hypothesized that nicotinamide adenine dinucleotide (NAD) deficiency contributes to energetic dysfunction and progressive tubular injury. We investigated metabolomic mechanisms of HIVAN tubulopathy. Tg26 and wild-type (WT) mice were treated with the farnesoid X receptor (FXR) agonist INT-747 or nicotinamide riboside (NR) from 6 to 12 wk of age. Multiomic approaches were used to characterize kidney tissue transcriptomes and metabolomes. Treatment with INT-747 or NR ameliorated kidney tubular injury, as shown by serum creatinine, the tubular injury marker urinary neutrophil-associated lipocalin, and tubular morphometry. Integrated analysis of metabolomic and transcriptomic measurements showed that NAD levels and production were globally downregulated in Tg26 mouse kidneys, especially nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway. Furthermore, NAD-dependent deacetylase sirtuin3 activity and mitochondrial oxidative phosphorylation activity were lower in ex vivo proximal tubules from Tg26 mouse kidneys compared with those of WT mice. Restoration of NAD levels in the kidney improved these abnormalities. These data suggest that NAD deficiency might be a treatable target for HIVAN.NEW & NOTEWORTHY The study describes a novel investigation that identified nicotinamide adenine dinucleotide (NAD) deficiency in a widely used HIV-associated nephropathy (HIVAN) transgenic mouse model. We show that INT-747, a farnesoid X receptor agonist, and nicotinamide riboside (NR), a precursor of nicotinamide, each ameliorated HIVAN tubulopathy. Multiomic analysis of mouse kidneys revealed that NAD deficiency was an upstream metabolomic mechanism contributing to HIVAN tubulopathy.
Assuntos
Nefropatia Associada a AIDS , Camundongos Transgênicos , NAD , Niacinamida , Compostos de Piridínio , Sirtuína 3 , Animais , NAD/metabolismo , Nefropatia Associada a AIDS/metabolismo , Nefropatia Associada a AIDS/genética , Nefropatia Associada a AIDS/patologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Piridínio/farmacologia , Sirtuína 3/metabolismo , Sirtuína 3/genética , Sirtuína 3/deficiência , Modelos Animais de Doenças , Nicotinamida Fosforribosiltransferase/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Progressão da Doença , Metabolômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/deficiência , Rim/metabolismo , Rim/patologia , Rim/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Citocinas/metabolismoRESUMO
The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.
Assuntos
Niacinamida , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Relação Estrutura-Atividade , Compostos de Piridínio/metabolismo , Compostos de Piridínio/química , N-Glicosil Hidrolases/metabolismo , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/química , Uridina/metabolismo , Uridina/análogos & derivados , Uridina/química , Especificidade por Substrato , Humanos , Modelos MolecularesRESUMO
Ionic liquids, i.e., organic salts with a low melting point, can be used as gas chromatographic liquid stationary phases. These stationary phases have some advantages such as peculiar selectivity, high polarity, and thermostability. Many previous works are devoted to such stationary phases. However, there are still no large enough retention data sets of structurally diverse compounds for them. Consequently, there are very few works devoted to quantitative structure-retention relationships (QSRR) for ionic liquid-based stationary phases. This work is aimed at closing this gap. Three ionic liquids with substituted pyridinium cations are considered. We provide large enough data sets (123-158 compounds) that can be used in further works devoted to QSRR and related methods. We provide a QSRR study using this data set and demonstrate the following. The retention index for a polyethylene glycol stationary phase (denoted as RI_PEG), predicted using another model, can be used as a molecular descriptor. This descriptor significantly improves the accuracy of the QSRR model. Both deep learning-based and linear models were considered for RI_PEG prediction. The ability to predict the retention indices for ionic liquid-based stationary phases with high accuracy is demonstrated. Particular attention is paid to the reproducibility and reliability of the QSRR study. It was demonstrated that adding/removing several compounds, small perturbations of the data set can considerably affect the results such as descriptor importance and model accuracy. These facts have to be considered in order to avoid misleading conclusions. For the QSRR research, we developed a software tool with a graphical user interface, which we called CHERESHNYA. It is intended to select molecular descriptors and construct linear equations connecting molecular descriptors with gas chromatographic retention indices for any stationary phase. The software allows the user to generate several hundred molecular descriptors (one-dimensional and two-dimensional). Among them, predicted retention indices for popular stationary phases such as polydimethylsiloxane and polyethylene glycol are used as molecular descriptors. Various methods for selecting (and assessing the importance of) molecular descriptors have been implemented, in particular the Boruta algorithm, partial least squares, genetic algorithms, L1-regularized regression (LASSO) and others. The software is free, open-source and available online: https://github.com/mtshn/chereshnya.
Assuntos
Líquidos Iônicos , Compostos de Piridínio , Software , Líquidos Iônicos/química , Cromatografia Gasosa/métodos , Compostos de Piridínio/química , Reprodutibilidade dos Testes , Relação Quantitativa Estrutura-Atividade , Modelos Lineares , Polietilenoglicóis/químicaRESUMO
The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.
Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Reativadores da Colinesterase , Músculo Esquelético , Organofosfatos , Oximas , Animais , Oximas/farmacologia , Oximas/química , Ratos , Acetilcolinesterase/metabolismo , Acetilcolinesterase/sangue , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/toxicidade , Organofosfatos/toxicidade , Masculino , Reativadores da Colinesterase/farmacologia , Reativadores da Colinesterase/química , Compostos de Piridínio/farmacologia , Ratos Sprague-DawleyRESUMO
Enzymes play a fundamental role in cellular metabolism. A wide range of enzymes require the presence of complementary coenzymes and cofactors to function properly. While coenzymes are believed to have been part of the last universal ancestor (LUCA) or have been present even earlier, the syntheses of crucial coenzymes like the redox-active coenzymes flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide (NAD+) remain challenging. Here, we present a pathway to NAD+ under prebiotic conditions starting with ammonia, cyanoacetaldehyde, prop-2-ynal and sugar-forming precursors, yielding inâ situ the nicotinamide riboside. Regioselective phosphorylation and water stable light activated adenosine monophosphate derivatives allow for topographically and irradiation-controlled formation of NAD+. Our findings indicate that NAD+, a coenzyme vital to life, can be formed non-enzymatically from simple organic feedstock molecules via photocatalytic activation under prebiotically plausible early Earth conditions in a continuous process under aqueous conditions.
Assuntos
NAD , NAD/química , NAD/metabolismo , Amônia/química , Niacinamida/química , Niacinamida/análogos & derivados , Fosforilação , Prebióticos , Monofosfato de Adenosina/química , Catálise , Acetaldeído/química , Oxirredução , Água/química , Compostos de Piridínio/química , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
People with lower extremity peripheral artery disease (PAD) have increased oxidative stress, impaired mitochondrial activity, and poor walking performance. NAD+ reduces oxidative stress and is an essential cofactor for mitochondrial respiration. Oral nicotinamide riboside (NR) increases bioavailability of NAD+ in humans. Among 90 people with PAD, this randomized double-blind clinical trial assessed whether 6-months of NR, with and without resveratrol, improves 6-min walk distance, compared to placebo, at 6-month follow-up. At 6-month follow-up, compared to placebo, NR significantly improved 6-min walk (+7.0 vs. -10.6 meters, between group difference: +17.6 (90% CI: + 1.8,+∞). Among participants who took at least 75% of study pills, compared to placebo, NR improved 6-min walk by 31.0 meters and NR + resveratrol improved 6-min walk by 26.9 meters. In this work, NR meaningfully improved 6-min walk, and resveratrol did not add benefit to NR alone in PAD. A larger clinical trial to confirm these findings is needed.
Assuntos
Niacinamida , Doença Arterial Periférica , Compostos de Piridínio , Resveratrol , Humanos , Doença Arterial Periférica/tratamento farmacológico , Niacinamida/análogos & derivados , Niacinamida/uso terapêutico , Masculino , Feminino , Idoso , Método Duplo-Cego , Resveratrol/uso terapêutico , Resveratrol/farmacologia , Pessoa de Meia-Idade , Caminhada , Resultado do Tratamento , Estresse Oxidativo/efeitos dos fármacosRESUMO
The extraction of 99TcO4- from radioactive effluents is extremely crucial for the purposes of nuclear disposal and environmental remediation. Herein, utilizing a facile and low-cost synthesis method, we report a pyridinium-based cationic polymer network, CPP-Cl, with impressive adsorption performance and ultrafast adsorption kinetics towards ReO4-. The structure featuring highly density of charged pyridinium units was synthesized, making it an effective adsorbent for capturing ReO4-. The material showed fast ReO4- adsorption kinetics reaching adsorption equilibrium within 30 s, an excellent capture capability of 1069.7 mg/g, and exceptional separation efficiency of 94.3% for removing 1000 ppm ReO4-. Furthermore, it possessed excellent reusability in multiple sorption/desorption trials and good uptake capacity within a widely ranging pH values. It is noteworthy that the extraction efficiency of CPP-Cl for ReO4- from simulated nuclear waste can be up to 94.2%. The favorable performance of the material in multiple tests revealed that CPP-Cl has tremendous potential as a high-efficiency sorbent for capturing 99TcO4-/ReO4- in complex nuclear associated environmental systems.
Assuntos
Polímeros , Adsorção , Polímeros/química , Porosidade , Cinética , Compostos de Piridínio/química , Recuperação e Remediação Ambiental/métodosRESUMO
Coumarin is a natural product known for its diverse biological activities. While its antifungal properties in agricultural chemistry have been extensively studied, there is limited research on its antibacterial potential. In this study, we developed several novel coumarin derivatives by combining coumarin with pyridinium salt through molecular hybridization and chemical synthesis. Our findings reveal that most of these derivatives exhibit promising antibacterial activity. Among them, derivative A25 has been identified as the most effective compound based on three-dimensional quantitative structure-activity relationships. It demonstrates significant in vitro and in vivo activity against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), and Xanthomonas campestris pv. citri (Xac), outperforming the commercially available thiediazole copper. Initial investigations into its mechanism of action suggest that A25 disrupts the cell membranes of Xoc and Xoo, thereby inhibiting bacterial growth. Additionally, A25 enhances the activity of defense enzymes in rice and modulates the expression of proteins related to the pyruvate metabolism pathway. This dual action contributes to rice's resistance against bacterial infestation. We anticipate that this study will serve as a foundation for the development of coumarin-based bactericides.
Assuntos
Antibacterianos , Cumarínicos , Testes de Sensibilidade Microbiana , Oryza , Xanthomonas , Cumarínicos/farmacologia , Cumarínicos/síntese química , Cumarínicos/química , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Xanthomonas/efeitos dos fármacos , Oryza/microbiologia , Compostos de Piridínio/farmacologia , Compostos de Piridínio/química , Compostos de Piridínio/síntese química , Xanthomonas campestris/efeitos dos fármacos , Desenho de Fármacos , Sais/farmacologia , Sais/química , Relação Estrutura-AtividadeRESUMO
This study demonstrates the potential of gelatin nanoparticles as a nanodelivery system for antagonists of nicotinic acetylcholine receptors (nAChRs) to improve chemotherapy efficacy and reduce off-target effects. Too often, chemotherapy for lung cancer does not lead to satisfactory results. Therefore, new approaches directed at multiple pharmacological targets in cancer therapy are being developed. Following the activation of nAChRs (e.g. by nicotine), cancer cells begin to proliferate and become more resistant to chemotherapy-induced apoptosis. This work shows that the 3-alkylpyridinium salt, APS7, a synthetic analog of a toxin from the marine sponge Haliclona (Rhizoneira) sarai, acts as an nAChR antagonist that inhibits the pro-proliferative and anti-apoptotic effects of nicotine on A549 human lung adenocarcinoma cells. In this study, gelatin-based nanoparticles filled with APS7 (APS7-GNPs) were prepared and their effects on A549 cells were compared with that of free APS7. Both APS7 and APS7-GNPs inhibited Ca2+ influx and increased the efficacy of cisplatin chemotherapy in nicotine-stimulated A549 cells. However, significant benefits from APS7-GNPs were observed - a stronger reduction in the proliferation of A549 lung cancer cells and a much higher selectivity in cytotoxicity towards cancer cells compared with non-tumorigenic lung epithelial BEAS-2B cells.
Assuntos
Proliferação de Células , Gelatina , Neoplasias Pulmonares , Nanopartículas , Humanos , Gelatina/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Células A549 , Nanopartículas/química , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Compostos de Piridínio/química , Cisplatino/farmacologia , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/química , Linhagem Celular TumoralRESUMO
Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.
Assuntos
Colesterol , Microscopia de Fluorescência , Espectrometria de Fluorescência , Colesterol/química , Corantes Fluorescentes/química , Fosfatidilcolinas/química , Lipossomos/química , Compostos de Piridínio/química , Fluidez de Membrana , Fosfatidilgliceróis/químicaRESUMO
The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.