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1.
J Environ Pathol Toxicol Oncol ; 40(3): 25-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34587402

RESUMO

This study is intended to explore the anticancer, antiproliferative, and chemopreventive action of troxerutin (TX) in human non-small-cell lung cancer cell (A549) using BALB/c nude mice. 2 × 106 A549 cells were subcutaneously injected into mice, along with 10 µM and 20 µM/kg body weight of TX orally for 19 days. On the last day, tumor weight and volume were assessed. Stress marker enzymes such as Aryl hydrocarbon hydroxylase (AHH), lactate dehydrogenase (LDH), 5'Nucleotidase (5'ND), and γ-glutamyltranspeptidase (γ-GT) were estimated in the lung tissues. Cytotoxicity of TX was assessed using MTT assay. Expression of carcinoembryonic antigen (CEA) and inflammatory cytokines were also analyzed. Histopathological examination of tissue sections and immunohistochemical examination of proliferating cell nuclear antigen (PCNA) were also performed. mRNA expression of p53, p21, cyclin D1, P13k, Akt, and mTOR were analyzed using RT-PCR. TX administered orally in a dose-dependent manner markedly reverted the level of stress marker enzymes to a significant extent. TX also exhibited significant protection against lung cancer cells, as evidenced by cytotoxicity assay and histopathological studies. It was also found to reduce the expression of PCNA, cyclin D1, P13k, Akt, and mTOR, but increase the expression of p53 and p21. TX has also been shown to reduce cancer cell inflammation, as was evidenced by reduced expression of inflammatory cytokines. Thus TX could be used as an effective chemopreventive and anticancer agent in treating cancer.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Hidroxietilrutosídeo/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/efeitos dos fármacos , Enzimas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxietilrutosídeo/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Soft Matter ; 17(38): 8585-8589, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34553206

RESUMO

Chemical and shape recognition are the main assembling mechanisms of complex bio-structures. A refined assessment of relevant thermodynamic parameters includes the consideration of a variety of contributions from different molecular motions and electronic interactions. An additional refinement includes the analysis of recognition involving multiple distant partners. This perspective note highlights some of the above issues in the cases of fibrillogenesis, and enzyme-substrate and antigen-antibody associations. Translational motions are found to be particularly relevant to the fibrillogenesis process. The assembly of enzyme-substrate complexes is discussed in terms of a dynamic equilibrium process.


Assuntos
Enzimas , Modelos Moleculares , Modelos Estruturais , Eletrônica , Enzimas/metabolismo , Conformação Molecular
3.
BMC Plant Biol ; 21(1): 361, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34364372

RESUMO

BACKGROUND: Priming of seed prior chilling is regarded as one of the methods to promote seeds germination, whole plant growth, and yield components. The application of biostimulants was reported as beneficial for protecting many plants from biotic or abiotic stresses. Their value was as important to be involved in improving the growth parameters of plants. Also, they were practiced in the regulation of various metabolic pathways to enhance acclimation and tolerance in coriander against chilling stress. To our knowledge, little is deciphered about the molecular mechanisms underpinning the ameliorative impact of biostimulants in the context of understanding the link and overlap between improved morphological characters, induced metabolic processes, and upregulated gene expression. In this study, the ameliorative effect(s) of potassium silicate, HA, and gamma radiation on acclimation of coriander to tolerate chilling stress was evaluated by integrating the data of growth, yield, physiological and molecular aspects. RESULTS: Plant growth, yield components, and metabolic activities were generally diminished in chilling-stressed coriander plants. On the other hand, levels of ABA and soluble sugars were increased. Alleviation treatment by humic acid, followed by silicate and gamma irradiation, has notably promoted plant growth parameters and yield components in chilling-stressed coriander plants. This improvement was concomitant with a significant increase in phytohormones, photosynthetic pigments, carbohydrate contents, antioxidants defense system, and induction of large subunit of RuBisCO enzyme production. The assembly of Toc complex subunits was maintained, and even their expression was stimulated (especially Toc75 and Toc 34) upon alleviation of the chilling stress by applied biostimulators. Collectively, humic acid was the best the element to alleviate the adverse effects of chilling stress on growth and productivity of coriander. CONCLUSIONS: It could be suggested that the inducing effect of the pretreatments on hormonal balance triggered an increase in IAA + GA3/ABA hormonal ratio. This ratio could be linked and engaged with the protection of cellular metabolic activities from chilling injury against the whole plant life cycle. Therefore, it was speculated that seed priming in humic acid is a powerful technique that can benefit the chilled along with non-chilled plants and sustain the economic importance of coriander plant productivity.


Assuntos
Resposta ao Choque Frio/fisiologia , Coriandrum/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/farmacologia , Sementes/crescimento & desenvolvimento , Aclimatação , Antioxidantes/metabolismo , Metabolismo dos Carboidratos , Carboidratos/análise , Proteínas de Cloroplastos/metabolismo , Resposta ao Choque Frio/efeitos dos fármacos , Resposta ao Choque Frio/efeitos da radiação , Coriandrum/efeitos dos fármacos , Coriandrum/efeitos da radiação , Enzimas/metabolismo , Raios gama , Substâncias Húmicas , Peroxidação de Lipídeos , Pigmentos Biológicos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Compostos de Potássio/química , Compostos de Potássio/farmacologia , Sementes/efeitos dos fármacos , Sementes/efeitos da radiação
4.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
5.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443339

RESUMO

The treatment of environmental pollution by microorganisms and their enzymes is an innovative and socially acceptable alternative to traditional remediation approaches. Microbial biodegradation is often characterized with high efficiency as this process is catalyzed via degrading enzymes. Various naturally isolated microorganisms were demonstrated to have considerable ability to mitigate many environmental pollutants without external intervention. However, only a small fraction of these strains are studied in detail to reveal the mechanisms at the enzyme level, which strictly limited the enhancement of the degradation efficiency. Accordingly, this review will comprehensively summarize the function of various degrading enzymes with an emphasis on catalytic mechanisms. We also inspect the expanded applications of these pollutant-degrading enzymes in industrial processes. An in-depth understanding of the catalytic mechanism of enzymes will be beneficial for exploring and exploiting more degrading enzyme resources and thus ameliorate concerns associated with the ineffective biodegradation of recalcitrant and xenobiotic contaminants with the help of gene-editing technology and synthetic biology.


Assuntos
Biocatálise , Poluentes Ambientais/metabolismo , Enzimas/metabolismo , Poluentes Ambientais/isolamento & purificação
6.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443447

RESUMO

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.


Assuntos
Antifúngicos/farmacologia , Enzimas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Polissacarídeos/metabolismo , Liofilização , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Peso Molecular , Proteólise/efeitos dos fármacos , Alimentos de Soja , Espectrofotometria Ultravioleta , Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
7.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443458

RESUMO

Adverse effects associated with synthetic drugs in diabetes therapy has prompted the search for novel natural lead compounds with little or no side effects. Effects of phenolic compounds from Carpobrotus edulis on carbohydrate-metabolizing enzymes through in vitro and in silico methods were assessed. Based on the half-maximal inhibitory concentrations (IC50), the phenolic extract of the plant had significant (p < 0.05) in vitro inhibitory effect on the specific activity of alpha-amylase (0.51 mg/mL), alpha-glucosidase (0.062 mg/mL) and aldose reductase (0.75 mg/mL), compared with the reference standards (0.55, 0.72 and 7.05 mg/mL, respectively). Molecular interactions established between the 11 phenolic compounds identifiable from the HPLC chromatogram of the extract and active site residues of the enzymes revealed higher binding affinity and more structural compactness with procyanidin (-69.834 ± 6.574 kcal/mol) and 1,3-dicaffeoxyl quinic acid (-42.630 ± 4.076 kcal/mol) as potential inhibitors of alpha-amylase and alpha-glucosidase, respectively, while isorhamnetin-3-O-rutinoside (-45.398 ± 4.568 kcal/mol) and luteolin-7-O-beta-d-glucoside (-45.102 ± 4.024 kcal/mol) for aldose reductase relative to respective reference standards. Put together, the findings are suggestive of the compounds as potential constituents of C. edulis phenolic extract responsible for the significant hypoglycemic effect in vitro; hence, they could be exploited in the development of novel therapeutic agents for type-2 diabetes and its retinopathy complication.


Assuntos
Aizoaceae/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Simulação de Dinâmica Molecular , Fenóis/análise , Fenóis/uso terapêutico , Animais , Cromatografia Líquida de Alta Pressão , Enzimas/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Simulação de Acoplamento Molecular , Ratos , Suínos , Termodinâmica
8.
Molecules ; 26(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34361657

RESUMO

The current review aims to summarise the biodiversity and biosynthesis of novel secondary metabolites compounds, of the phylum Actinobacteria and the diverse range of secondary metabolites produced that vary depending on its ecological environments they inhabit. Actinobacteria creates a wide range of bioactive substances that can be of great value to public health and the pharmaceutical industry. The literature analysis process for this review was conducted using the VOSviewer software tool to visualise the bibliometric networks of the most relevant databases from the Scopus database in the period between 2010 and 22 March 2021. Screening and exploring the available literature relating to the extreme environments and ecosystems that Actinobacteria inhabit aims to identify new strains of this major microorganism class, producing unique novel bioactive compounds. The knowledge gained from these studies is intended to encourage scientists in the natural product discovery field to identify and characterise novel strains containing various bioactive gene clusters with potential clinical applications. It is evident that Actinobacteria adapted to survive in extreme environments represent an important source of a wide range of bioactive compounds. Actinobacteria have a large number of secondary metabolite biosynthetic gene clusters. They can synthesise thousands of subordinate metabolites with different biological actions such as anti-bacterial, anti-parasitic, anti-fungal, anti-virus, anti-cancer and growth-promoting compounds. These are highly significant economically due to their potential applications in the food, nutrition and health industries and thus support our communities' well-being.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Produtos Biológicos , Enzimas/isolamento & purificação , Enzimas/metabolismo , Metabolismo Secundário
9.
Biomolecules ; 11(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34356667

RESUMO

During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Enzimas/metabolismo , Haloferax mediterranei/metabolismo , Coenzimas/metabolismo , Simulação por Computador , Desnitrificação , Enzimas/química , Haloferax mediterranei/enzimologia , Modelos Moleculares , Nitrato Redutase/química , Nitrato Redutase/metabolismo , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Sinais Direcionadores de Proteínas , Alinhamento de Sequência
10.
Biomolecules ; 11(7)2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34356649

RESUMO

Curcumin is a known anti-adipogenic agent for alleviating obesity and related disorders. Comprehensive comparisons of the anti-adipogenic activity of curcumin with other curcuminoids is minimal. This study compared adipogenesis inhibition with curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), and their underlying mechanisms. We differentiated 3T3-L1 cells in the presence of curcuminoids, to determine lipid accumulation and triglyceride (TG) production. The expression of adipogenic transcription factors and lipogenic proteins was analyzed by Western blot. A significant reduction in Oil red O (ORO) staining was observed in the cells treated with curcuminoids at 20 µM. Inhibition was increased in the order of curcumin < DMC < BDMC. A similar trend was observed in the detection of intracellular TG. Curcuminoids suppressed differentiation by downregulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), leading to the downregulation of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). AMP-activated protein kinase α (AMPKα) phosphorylation was also activated by BDMC. Curcuminoids reduced the release of proinflammatory cytokines and leptin in 3T3-L1 cells in a dose-dependent manner, with BDMC showing the greatest potency. BDMC at 20 µM significantly decreased leptin by 72% compared with differentiated controls. Molecular docking computation indicated that curcuminoids, despite having structural similarity, had different interaction positions to PPARγ, C/EBPα, and ACC. The docking profiles suggested a possible interaction of curcuminoids with C/EBPα and ACC, to directly inhibit their expression.


Assuntos
Adipogenia/efeitos dos fármacos , Diarileptanoides/química , Diarileptanoides/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/fisiologia , Adipocinas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , Curcumina/análise , Curcumina/farmacologia , Enzimas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , PPAR gama/química , PPAR gama/metabolismo , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Triglicerídeos/metabolismo
11.
Zoology (Jena) ; 147: 125945, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34217027

RESUMO

The ∼15.000 decapod crustaceans that are mostly omnivorous have evolved a structurally and functionally complex digestive system. They have highly effective cuticular chewing and filtering structures in the stomach, which are regularly renewed by moulting. Decapods produce a broad range of digestive enzymes including chitinases, cellulases, and collagenases with unique properties. These enzymes are synthesized in the F-cells of the hepatopancreas and are encoded in the genome as pre-pro-proteins. In contrast to mammals, they are stored in a mature form in the lumen of the stomach to await the next meal, and therefore, the enzymes are particularly stable. The fat emulsifiers are fatty acyl-dipeptides rather than bile salts. After mechanical and chemical processing of the food in the cardiac stomach, the chyme is filtered by two unique filter systems of different mesh-size. The filtrate is then transferred to the hepatopancreas where the nutrients are absorbed by the R-cells, mostly via carriers, resembling nutrient absorption in the small intestine of mammals. The absorbed nutrients are used to fuel the metabolism of the hepatopancreas, are supplied to other organs, and are stored in the R-cells as glycogen and lipid reserves. Export lipids are secreted from the R-cells into the haemolymph as high density lipoproteins that mainly consist of phospholipids. In contrast to mammals, the midgut tube and hindgut contribute only little to food processing and nutrient absorption. The oesophagus, stomach and hindgut are well innervated but the hepatopancreas lacks nerves. Hormone cells are abundant in the midgut and hepatopancreas epithelia. Microorganisms are often present in the intestine of decapods, but they are apparently not essential for digestion and nutrition.


Assuntos
Decápodes/fisiologia , Digestão/fisiologia , Enzimas/metabolismo , Comportamento Alimentar/fisiologia , Mamíferos/fisiologia , Animais
12.
Methods Mol Biol ; 2342: 3-27, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272689

RESUMO

This chapter will provide a general introduction to the kinetics of enzyme-catalyzed reactions, including a general discussion of catalysts, reaction rates, and binding constants. This section will be followed by a discussion of various types of enzyme kinetics observed in drug metabolism reactions. A large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the Vmax value. However, in other cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 (CYPs) have large active sites that enable binding of multiple molecules (Yano et al., J Biol Chem 279:38091-38094, 2004; Wester et al., J Biol Chem 279:35630-35637, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies.


Assuntos
Enzimas/metabolismo , Algoritmos , Animais , Catálise , Humanos , Cinética
13.
Methods Mol Biol ; 2342: 147-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272694

RESUMO

Differential equations are used to describe time-dependent changes in enzyme kinetics and pharmacokinetics. Analytical and numerical methods can be used to solve differential equations. This chapter describes the use of numerical methods in solving differential equations and its applications in characterizing the complexities observed in enzyme kinetics. A discussion is included on the use of numerical methods to overcome limitations of explicit equations in the analysis of metabolism kinetics, reversible inhibition kinetics, and inactivation kinetics. The chapter describes the advantages of using numerical methods when Michaelis-Menten assumptions do not hold.


Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Algoritmos , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Enzimas/química , Humanos , Cinética , Modelos Teóricos
14.
Methods Mol Biol ; 2342: 419-440, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272703

RESUMO

Intracellular drug metabolism involves transport, bioactivation, conjugation, and other biochemical steps. The dynamics of these steps are each dependent on a number of other cellular factors that can ultimately lead to unexpected behavior. In this review, we discuss the confounding processes and coupled reactions within bioactivation networks that require a systems-level perspective in order to fully understand the time-varying behavior. When converting known in vitro characteristics of drug-enzyme interactions into descriptions of cellular systems, features such as substrate availability, cell-to-cell variability, and intracellular redox state, deserve special focus. Two examples are provided. First, a model of hydrogen peroxide clearance during chemotherapy treatment serves as a basis to discuss an example of sensitivity analysis. Second, an example of doxorubicin bioactivation is used for discussing points of consideration when constructing and analyzing network models of drug metabolism.


Assuntos
Doxorrubicina/farmacocinética , Enzimas/metabolismo , Peróxido de Hidrogênio/farmacocinética , Biologia de Sistemas/métodos , Vias de Eliminação de Fármacos , Tratamento Farmacológico , Enzimas/química , Humanos , Cinética , Oxirredução
15.
Methods Mol Biol ; 2342: 443-479, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272704

RESUMO

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.


Assuntos
Enzimas/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos/citologia , Microssomos Hepáticos/enzimologia , Técnicas de Cultura de Células , Células Cultivadas , Enzimas/genética , Glucuronosiltransferase/genética , Hepatócitos/metabolismo , Humanos , Cinética , Variantes Farmacogenômicos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
Methods Mol Biol ; 2342: 633-642, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272708

RESUMO

This chapter deals with practical considerations on key issues such as choosing an enzyme source, determining linear conditions, and choosing appropriate substrate and organic solvent concentrations. Practical solutions for working with limited resources and carrying out inhibition experiments are also addressed. Thus, after reading this chapter, the novice reader should have a better idea of how to design, develop, and interpret basic experiments using drug metabolism enzymes.


Assuntos
Enzimas/metabolismo , Hepatócitos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Hepatócitos/enzimologia , Humanos , Cinética , Lisossomos/enzimologia , Projetos de Pesquisa
17.
Methods Mol Biol ; 2342: 643-652, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272709

RESUMO

Characterization of enzyme kinetics in an experiment is dependent on measurement of a change in concentration of either the substrate (loss of parent) or the product (formation of metabolite). Modern analytical techniques such as ultrahigh pressure liquid chromatography, high resolution mass spectrometry, etc. have allowed accurate characterization of minute changes in concentration. Therefore, complex kinetic data such as a sigmoidal phase at low substrate concentrations or terminal half-life in a PK curve can be evaluated by stretching the limits of analytical quantification. This chapter presents some elementary dos and don'ts and provides insight into some of the underlying principles for utilizing the best possible analytical techniques when investigating enzyme kinetics. The objective of this case study is to answer the following questions: (a) Why is it necessary to determine lower and upper limits of quantification (LLOQ and ULOQ, respectively) of a bioanalytical assay, specifically for enzyme kinetic assays? How do you utilize LLOQ and ULOQ to correctly interpret your kinetic data? (b) Why should one use a linear fit and not a quadratic fit for standard curves? (c) Is quantification of an analyte possible without a reference standard? Can one assume equal signal intensities regardless of analytical technique (MS, UV)? (d) In the absence of reference standards, can you still determine kinetic constants? (e) With the need to keep substrate depletion at less than 20% for linearity assumptions, does bioanalytical variability matter? (f) What buffer do you use for your enzyme systems? How do you choose your buffer ? Does choice of bioanalytical methods (LC, MS) dictate your choice of buffer ?


Assuntos
Enzimas/metabolismo , Preparações Farmacêuticas/metabolismo , Algoritmos , Cromatografia Líquida , Humanos , Cinética , Limite de Detecção , Farmacocinética , Padrões de Referência , Projetos de Pesquisa , Espectrometria de Massas em Tandem
18.
Methods Mol Biol ; 2342: 551-593, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272706

RESUMO

Almost 50% of prescription drugs lack age-appropriate dosing guidelines and therefore are used "off-label." Only ~10% drugs prescribed to neonates and infants have been studied for safety or efficacy. Immaturity of drug metabolism in children is often associated with drug toxicity. This chapter summarizes data on the ontogeny of major human metabolizing enzymes involved in oxidation, reduction, hydrolysis, and conjugation of drugs. The ontogeny data of individual drug-metabolizing enzymes are important for accurate prediction of drug pharmacokinetics and toxicity in children. This information is critical for designing clinical studies to appropriately test pharmacological hypotheses and develop safer pediatric drugs, and to replace the long-standing practice of body weight- or surface area-normalized drug dosing. The application of ontogeny data in physiologically based pharmacokinetic model and regulatory submission are discussed.


Assuntos
Enzimas/metabolismo , Medicamentos sob Prescrição/farmacocinética , Cálculos da Dosagem de Medicamento , Humanos , Lactente , Recém-Nascido , Medicamentos sob Prescrição/administração & dosagem , Medicamentos sob Prescrição/toxicidade
19.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2256-2271, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327893

RESUMO

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.


Assuntos
Biotecnologia , Enzimas , Biocatálise , Catálise , Composição de Medicamentos , Enzimas/metabolismo
20.
Nat Commun ; 12(1): 4461, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294694

RESUMO

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.


Assuntos
Cristalografia por Raios X/métodos , Enzimas/química , Enzimas/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Galinhas , Cristalografia por Raios X/instrumentação , Desenho de Equipamento , Modelos Moleculares , Muramidase/química , Muramidase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo
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