RESUMO
Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.
Assuntos
Substâncias para a Guerra Química/toxicidade , Creatina Quinase Forma MM/efeitos dos fármacos , Metionina/metabolismo , Gás de Mostarda/toxicidade , Alquilação/efeitos dos fármacos , Animais , Cromatografia Líquida , Creatina Quinase Forma MM/metabolismo , Cisteína/metabolismo , Coelhos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.
Assuntos
Substâncias para a Guerra Química/toxicidade , Creatina Quinase/metabolismo , Gás de Mostarda/toxicidade , Pele/efeitos dos fármacos , Albuminas/metabolismo , Alquilação/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Cromatografia Líquida , Cisteína/metabolismo , Adutos de DNA/metabolismo , Humanos , Masculino , Ratos , Ratos Wistar , Pele/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
DNA damaging agents have been a cornerstone of cancer therapy for nearly a century. The discovery of many of these chemicals, particularly the alkylating agents, are deeply entwined with the development of poisonous materials originally intended for use in warfare. Over the last decades, their anti-proliferative effects have focused on the specific mechanisms by which they damage DNA, and the factors involved in the repair of such damage. Due to the variety of aberrant adducts created even for the simplest alkylating agents, numerous pathways of repair are engaged as a defense against this damage. More recent work has underscored the role of RNA damage in the cellular response to these agents, although the understanding of their role in relation to established DNA repair pathways is still in its infancy. In this review, we discuss the chemistry of alkylating agents, the numerous ways in which they damage nucleic acids, as well as the specific DNA and RNA repair pathways which are engaged to counter their effects.
Assuntos
Dano ao DNA , DNA/genética , RNA/genética , Alquilantes/toxicidade , Alquilação/efeitos dos fármacos , Animais , DNA/química , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , RNA/químicaRESUMO
Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide-thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys282 of CK and Cys232 of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.
Assuntos
Alquilação/efeitos dos fármacos , Substâncias para a Guerra Química/efeitos adversos , Creatina Quinase/metabolismo , Gás de Mostarda/efeitos adversos , alfa 1-Antitripsina/metabolismo , Creatina Quinase/química , Células HEK293 , Humanos , Modelos Moleculares , alfa 1-Antitripsina/químicaRESUMO
The antioxidant properties of proteins could be enhanced by forming covalent conjugates with polyphenols. In this study, the antioxidant activity of egg white protein (EWP) was improved by conjugating with tea polyphenols (TP) using traditional and ultrasound-assisted alkaline/free radical methods. In addition, the influences of TP conjugation on the antioxidant activities and structural and digestive properties of EWP were comprehensively studied. Compared with the traditional methods, the sonochemistry (40 kHz) approaches significantly increased the efficiency of TP grafting to the EWP (P < 0.05) from 24 h to 1 h. Amino acid analysis showed that in the ultrasound-assisted alkaline method, TP was successfully conjugated to the EWP through proline, glutamic acid, cysteine, and tryptophan residues, whereas proline, cysteine, and tryptophan were involved in the free radical method. However, the number of cross-linking sites was increased significantly after ultrasound-assisted treatments. Moreover, the antioxidant activities of the EWP were significantly improved after covalent conjugation with TP using traditional and ultrasound-assisted alkaline/free radical methods, particularly the ultrasound-assisted approaches. Furthermore, circular dichroism revealed that the ultrasound-assisted approaches had the greatest impact with regard to decreasing the α-helix content and increasing the random coil content, which loosened the protein structure, thereby improving its reactivity and digestibility. Therefore, ultrasound-assisted alkaline/free radical methods were efficient and safe means for the production of EWP-TP conjugates.
Assuntos
Antioxidantes/farmacologia , Proteínas do Ovo/farmacologia , Polifenóis/farmacologia , Sonicação , Chá/química , Alquilação/efeitos dos fármacos , Aminoácidos/análise , Antiácidos/farmacologia , Fenômenos Químicos , Misturas Complexas/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Digestão/efeitos dos fármacos , Tecnologia de Alimentos , Radicais LivresRESUMO
Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.
Assuntos
Apoproteínas/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Alquilação/efeitos dos fármacos , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Monóxido de Carbono/metabolismo , Cisteína/química , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/química , Ensaios Enzimáticos , Iodoacetamida/análogos & derivados , Iodoacetamida/química , Iodoacetamida/farmacologia , Maleimidas/química , Maleimidas/farmacologia , Oxirredução/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
Covalently self-assembled polymer nanocapsules (NCs) based on cucurbit[6]uril have been previously prepared and their applications in payload delivery and bioimaging have been demonstrated, showing significant potentials. However, the preparation of these NCs often requires laborious and tedious multistep reactions, including a low-yield conversion of perhydroxycucurbit[6]uril to perallyloxycucurbit[6]uril, subsequent photopolymerization of perallyloxycucurbit[6]uril with dithiol linkers, and two additional steps of treatment to remove disulfide loops and create cationic sulfoniums. Herein, we report a novel, facile approach leading to cucurbit[6]uril-based polymer NCs via direct alkylation of perhydroxycucurbit[6]uril with a ditopic linker, thereby saving significant time and efforts, which may lead to significant expansion in investigations of these unique materials in various applications, particularly biomedical sciences. As a proof of concept, we have further demonstrated that a photosensitive therapeutic payload, such as chlorin e6, may get encapsulated inside the NCs for improved, targeted photodynamic therapy against cancer cells.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Imidazóis/síntese química , Nanocápsulas/química , Neoplasias/terapia , Fotoquimioterapia , Alquilação/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Clorofilídeos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Nanocápsulas/uso terapêutico , Polímeros/química , Polímeros/farmacologia , Porfirinas/químicaRESUMO
Reduction and alkylation are common processing steps in sample preparation for qualitative and quantitative proteomic analyses. In principle, these steps mitigate the limitations resulting from the presence of disulfide bridges. There has been recurring debate in the proteomics community around their use, with concern over negative impacts that result from overalkylation (off-target, non-thiol sites) or incomplete reduction and/or S-alkylation of cysteine. This chapter integrates findings from a number of studies on different reduction and alkylation strategies, to guide users in experimental design for their optimal use in proteomic workflows.
Assuntos
Cisteína/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica , Alquilantes/farmacologia , Alquilação/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Proteômica/métodos , Substâncias Redutoras/farmacologia , Fluxo de TrabalhoRESUMO
Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Mycobacterium leprae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano/efeitos dos fármacos , Humanos , Hanseníase/microbiologia , Modelos Moleculares , Mycobacterium leprae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Under conditions of oxidative stress, cholesterol aggregates into discrete membrane bilayer domains that precipitate the formation of extracellular crystals, a feature of advanced atheroma in cardiovascular disease. Therapeutic interventions using membrane-directed antioxidants, such as polyphenolic esters, may reduce cholesterol domains and crystal formation. In this study, the effects of rosmarinic acid (RC0) and rosmarinic esters, with alkyl chain lengths ranging from 4 to 16carbons (RC4-RC16), on membrane lipid oxidation and cholesterol domain formation were investigated. METHODS: Model membranes were prepared with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine and cholesterol at different cholesterol-to-phospholipid mole ratios (0.3:1, 0.9:1, and 1.2:1), in the absence or presence of each molecule and exposed to 72â¯h of oxidation. Changes in lipid hydroperoxide (LOOH) and cholesterol domain formation were measured using iodometric and small angle x-ray diffraction approaches, respectively. RESULTS: Rosmarinic acid and its esters had differential effects on LOOH formation based on alkyl chain length. RC8 exhibited the greatest antioxidant effect, reducing LOOH levels by 82%, and inhibited cholesterol domain formation. By contrast, RC0 and RC16 failed to inhibit either LOOH formation or cholesterol domain formation. CONCLUSION: These data indicate that the membrane antioxidant and cholesterol domain inhibition activities of rosmarinic acid esters are dependent, nonlinearly, on alkyl chain length. The mechanism for this effect is attributed to the influence of alkyl chain length on the optimal depth of the polyphenols into the lipid bilayer for trapping free radicals. GENERAL SIGNIFICANCE: These findings provide insight into novel atheroprotective benefits of polyphenol esters that are dependent on their membrane location.
Assuntos
Colesterol/metabolismo , Cinamatos/farmacologia , Depsídeos/farmacologia , Ésteres/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Antioxidantes/farmacologia , Cinamatos/química , Depsídeos/química , Ésteres/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Difração de Raios X , Ácido RosmarínicoRESUMO
Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet329 (-HETE)Phe, LGM(-HETE)F, was detected after pepsin-mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met329 in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.
Assuntos
Alquilação/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Metionina/análise , Gás de Mostarda/toxicidade , Albumina Sérica Humana/química , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.
Assuntos
DNA/química , Mutagênicos/farmacologia , Peptídeos/farmacologia , Policetídeos/farmacologia , Alquilação/efeitos dos fármacos , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Escherichia coli/genética , Humanos , Peptídeos/genéticaRESUMO
It is known that aziridines and nitrogen mustards exert their biological activities, especially in chemotherapy, via DNA alkylation. The studied scaffold, 2-phenyl-1-aziridine, provides a distinct conformation compared to commonly used aziridines, and therefore, leads to a change in high-strained ring reactivity towards biological nucleophiles, such as DNA. The above series of compounds was tested in three breast cell lines: MCF-10, a healthy cell; MCF-7, a hormone responsive cancer cell; and MDA-MB-231, a triple negative breast cancer cell. Both aziridines and their precursors, ß-amino alcohols, showed activity towards these cells, and some of the compounds showed higher selectivity index than cisplatin, the drug used as control. When the type of cell death was investigated, the synthesized compounds demonstrated higher apoptosis and lower necrosis rates than cisplatin, and when the mechanism of action was studied, the compounds were shown to interact with DNA via its minor groove instead of alkylation or intercalation.
Assuntos
Amino Álcoois/farmacologia , Antineoplásicos/farmacologia , Aziridinas/farmacologia , DNA/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Amino Álcoois/química , Antineoplásicos/química , Aziridinas/química , Linhagem Celular , Cisplatino/química , Cisplatino/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next-generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane.
Assuntos
Antineoplásicos Alquilantes/farmacologia , DNA/química , Diazometano/análogos & derivados , RNA/química , Compostos de Trimetilsilil/farmacologia , Alquilação/efeitos dos fármacos , Antineoplásicos Alquilantes/química , Diazometano/química , Diazometano/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/tratamento farmacológico , Compostos de Trimetilsilil/químicaAssuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Ensaios Clínicos como Assunto , Elipticinas/farmacologia , Elipticinas/uso terapêutico , Humanos , Mutação , Neoplasias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Quinuclidinas/farmacologia , Quinuclidinas/uso terapêutico , Relação Estrutura-Atividade , Tiossemicarbazonas/farmacologia , Tiossemicarbazonas/uso terapêutico , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Citocromo P-450 CYP2B1/antagonistas & inibidores , Citocromo P-450 CYP2B6/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Éteres Difenil Halogenados/farmacologia , Alquilação/efeitos dos fármacos , Substituição de Aminoácidos , Compostos de Anilina , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Derivados de Benzeno/farmacologia , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Inibidores das Enzimas do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Citocromos b5/metabolismo , Poluentes Ambientais/metabolismo , Éteres Difenil Halogenados/metabolismo , Humanos , Hidrocarbonetos Halogenados/metabolismo , Concentração Inibidora 50 , Estrutura Molecular , Mutagênese Sítio-Dirigida , NADPH Oxidases/metabolismo , Oxirredução , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.
Assuntos
Anti-Inflamatórios/farmacologia , Organismos Aquáticos/química , Produtos Biológicos/farmacologia , Inflamação/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Antioxidantes/metabolismo , Produtos Biológicos/química , Linhagem Celular , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Feminino , Humanos , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Células MCF-7 , Camundongos , Células RAW 264.7RESUMO
Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.
Assuntos
Enzimas AlkB/antagonistas & inibidores , Antineoplásicos Alquilantes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Glutaminase/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilação/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Glutaminase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Distribuição Aleatória , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ATPase activity of NLRP3 has pivotal role in inflammasome activation and is recognized as a good target for the development of the NLRP3 inflammasome-specific inhibitor. However, signals in the vicinity of the ATPase activity of NLRP3 have not been fully elucidated. Here, we demonstrate NLRP3 inflammasome-specific action of a benzoxathiole derivative, BOT-4-one. BOT-4-one exhibited an inhibition of NLRP3 inflammasome activation, which was attributable to its alkylating capability to NLRP3. In particular, the NLRP3 alkylation by BOT-4-one led to an impaired ATPase activity of NLRP3, thereby obstructing the assembly of the NLRP3 inflammasome. Additionally, we found that NLRP3 alkylators, including BOT-4-one, enhance the ubiquitination level of NLRP3, which might also contribute to the inhibition of NLRP3 inflammasome activation. Finally, BOT-4-one appeared to be superior to other known NLRP3 alkylators in inhibiting the functionality of the NLRP3 inflammasome and its resulting anti-inflammatory activity was confirmed in vivo using a monosodium urate-induced peritonitis mouse model. Collectively, the results suggest that NLRP3 alkylators function by inhibiting ATPase activity and increasing the ubiquitination level of NLRP3, and BOT-4-one could be the type of NLRP3 inhibitor that may be potentially useful for the novel development of a therapeutic agent in controlling NLRP3 inflammasome-related diseases.
Assuntos
Adenosina Trifosfatases/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ubiquitinação/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Feminino , Humanos , Inflamassomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Células THP-1RESUMO
Nitrogen mustards (NMs) are DNA-alkylating compounds that represent the earliest anticancer drugs. However, clinical use of NMs is limited because of their own mutagenic properties. The mechanisms of NM-induced mutagenesis remain unclear. The major product of DNA alkylation by NMs is a cationic NM-N7-dG adduct that can yield the imidazole ring-fragmented lesion, N5-NM-substituted formamidopyrimidine (NM-Fapy-dG). Characterization of this adduct is complicated because it adopts different conformations, including both a canonical ß- and an unnatural α-anomeric configuration. Although formation of NM-Fapy-dG in cellular DNA has been demonstrated, its potential role in NM-induced mutagenesis is unknown. Here, we created site-specifically modified single-stranded vectors for replication in primate (COS7) or Escherichia coli cells. In COS7 cells, NM-Fapy-dG caused targeted mutations, predominantly G â T transversions, with overall frequencies of â¼11-12%. These frequencies were â¼2-fold higher than that induced by 8-oxo-dG adduct. Replication in E. coli was essentially error-free. To elucidate the mechanisms of bypass of NM-Fapy-dG, we performed replication assays in vitro with a high-fidelity DNA polymerase, Saccharomyces cerevisiae polymerase (pol) δ. It was found that pol δ could catalyze high-fidelity synthesis past NM-Fapy-dG, but only on a template subpopulation, presumably containing the ß-anomeric adduct. Consistent with the low mutagenic potential of the ß-anomer in vitro, the mutation frequency was significantly reduced when conditions for vector preparation were modified to favor this configuration. Collectively, these data implicate the α-anomer as a major contributor to NM-Fapy-dG-induced mutagenesis in primate cells.