RESUMO
Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.
Assuntos
Compostos Azo/farmacologia , Inibidores Enzimáticos/farmacologia , Triptofano Sintase/antagonistas & inibidores , Inibidores Enzimáticos/efeitos da radiaçãoRESUMO
Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from biology to materials science. In chemical biology, induction of biological processes via photoactivation is a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable protecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethylamine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has a bathochromic shift in the one-photon absorption maximum from λmax = 315 nm with the unfunctionalized NDBF scaffold to λmax = 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm. To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting, caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live cells using 850-900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experiments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm. Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments in chemical biology and materials science.
Assuntos
Benzofuranos/química , Inibidores Enzimáticos/farmacologia , Compostos de Sulfidrila/farmacologia , Animais , Benzofuranos/síntese química , Benzofuranos/efeitos da radiação , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/efeitos da radiação , Farnesiltranstransferase/antagonistas & inibidores , Raios Infravermelhos , Células Madin Darby de Rim Canino , Fotólise/efeitos da radiação , Fótons , Compostos de Sulfidrila/química , Compostos de Sulfidrila/efeitos da radiaçãoRESUMO
Human aldose reductase (hAR, AKR1B1) has been explored as drug target since the 1980s for its implication in diabetic complications. An activated form of hAR was found in cells from diabetic patients, showing a reduced sensitivity to inhibitors in clinical trials, which may prevent its pharmacological use. Here we report the conversion of native hAR to its activated form by X-ray irradiation simulating oxidative stress conditions. Upon irradiation, the enzyme activity increases moderately and the potency of several hAR inhibitors decay before global protein radiation damage appears. The catalytic behavior of activated hAR is also reproduced as the KM increases dramatically while the kcat is not much affected. Consistently, the catalytic tetrad is not showing any modification. The only catalytically-relevant structural difference observed is the conversion of residue Cys298 to serine and alanine. A mechanism involving electron capture is suggested for the hAR activation. We propose that hAR inhibitors should not be designed against the native protein but against the activated form as obtained from X-ray irradiation. Furthermore, since the reactive species produced under irradiation conditions are the same as those produced under oxidative stress, the described irradiation method can be applied to other relevant proteins under oxidative stress environments.
Assuntos
Aldeído Redutase/genética , Inibidores Enzimáticos/farmacologia , Estresse Oxidativo/efeitos da radiação , Alanina/genética , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/efeitos da radiação , Catálise/efeitos dos fármacos , Catálise/efeitos da radiação , Microambiente Celular/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Inibidores Enzimáticos/efeitos da radiação , Humanos , Oxirredução , Estresse Oxidativo/genética , Serina/genética , Raios XRESUMO
Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe resynthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
Assuntos
Marcadores de Afinidade/química , Ácido Aspártico Endopeptidases/química , Diazometano/análogos & derivados , Gelatinases/química , Glutamato Carboxipeptidase II/química , Proteínas de Membrana/química , Proteômica/métodos , Serina Endopeptidases/química , Marcadores de Afinidade/síntese química , Marcadores de Afinidade/efeitos da radiação , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Biotina/química , Linhagem Celular Tumoral , Diazometano/síntese química , Diazometano/efeitos da radiação , Endopeptidases , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/efeitos da radiação , Fluoresceínas/química , Corantes Fluorescentes/química , Gelatinases/antagonistas & inibidores , Glutamato Carboxipeptidase II/antagonistas & inibidores , Humanos , Espectrometria de Massas/métodos , Proteínas de Membrana/antagonistas & inibidores , Microscopia Confocal/métodos , Ácidos Polimetacrílicos/química , Raios UltravioletaRESUMO
Microcystin-LR (MCLR) is the most common cyanotoxin in contaminated aquatic systems. MCLR inhibits protein phosphatases 1 and 2A, leading to liver damage and tumor formation. MCLR is relatively stable owing to its cyclic structures. The combined UV/H2O2 technology can degrade MCLR efficiently. The second-order rate constant of the reaction between MCLR and hydroxyl radical (·OH) is 2.79(±0.23)×1010 M-1 s-1 based on the competition kinetics model using nitrobenzene as reference compound. The probable degradation pathway was analyzed through liquid chromatography mass spectrometry. Results suggested that the major destruction pathways of MCLR were initiated by ·OH attack on the benzene ring and diene of the Adda side chain. The corresponding aldehyde or ketone peptide residues were formed through further oxidation. Another minor destruction pathway involved ·OH attack on the methoxy group of the Adda side chain, followed by complete removal of the methoxy group. The combined UV/H2O2 system is a promising technology for MCLR removal in contaminated aquatic systems.
Assuntos
Inibidores Enzimáticos/química , Peróxido de Hidrogênio/farmacologia , Radical Hidroxila/química , Microcistinas/química , Raios Ultravioleta , Cromatografia Líquida , Inibidores Enzimáticos/efeitos da radiação , Cinética , Toxinas Marinhas , Espectrometria de Massas , Microcistinas/efeitos dos fármacos , Microcistinas/efeitos da radiação , Oxidantes/farmacologia , Oxirredução , Purificação da Água/métodosRESUMO
High-resolution structural data of protein inhibitor complexes are the key to rational drug design. Synchrotron radiation allows for atomic resolutions but is frequently accompanied by radiation damage to protein complexes. In this study a human aldose reductase mutant complexed with a bromine-substituted inhibitor was determined to atomic resolution [Protein Data Bank (PDB) code 3onc]. Though the radiation dose was moderate, a selective disruption of a bromine-inhibitor bond during the experiment was observed while the protein appears unaffected. A covalent bond to bromine is cleaved and the displaced atom is not scattered throughout the crystal but can most likely be assigned as a bromide to an additional difference electron density peak observed in the structure. The bromide relocates to an adjacent unoccupied site where promising interactions to protein residues stabilize its position. These findings were verified by a second similar structure determined with considerably higher radiation dose (PDB code 3onb).
Assuntos
Acetatos/efeitos da radiação , Aldeído Redutase/efeitos da radiação , Bromo/efeitos da radiação , Inibidores Enzimáticos/efeitos da radiação , Fenoxiacetatos/efeitos da radiação , Tioamidas/efeitos da radiação , Acetatos/química , Aldeído Redutase/química , Aldeído Redutase/genética , Bromo/química , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Fenoxiacetatos/química , Síncrotrons , Tioamidas/químicaRESUMO
ZRBA1 is a molecule termed 'combi-molecule' designed to induce DNA-alkylating lesions and to block epidermal growth factor receptor (EGFR) tyrosine kinase. Owing to its ability to downregulate the EGFR tyrosine kinase-mediated antiapoptotic signaling and DNA repair proteins, we inferred that it could significantly sensitize cells to ionizing radiation. Using the MDA-MB-468 human breast cancer cell line in which ZRBA1 has already been reported to induce significant EGFR/DNA-targeting potency, the results showed that: (i) concurrent administration of ZRBA1 and 4 Gy radiation led to a significant decrease in cell viability, (ii) the greater efficacy of the combination was sequential, being limited to conditions wherein the drug was administered concurrently with radiation or before radiation, and (iii) the efficacy enhancement of the combination was further confirmed by clonogenic assays from which a dose enhancement factor of 1.34 could be observed at survival fraction of 0.01. Flow cytometric analysis showed significant enhancement of cell cycle arrest in G2/M (P<0.046, irradiated cells vs. cells treated with ZRBA1 and radiation) and increased apoptosis when ZRBA1 was combined with radiation. Likewise, significant levels of double-strand breaks were observed for the combination, as determined by neutral comet assay (P<0.045, irradiated cells vs. cells treated with ZRBA1 and radiation). These results in toto suggest that the superior efficacy of the ZRBA1 plus radiation combination may be secondary to the ability of ZRBA1 to arrest the cells in G2/M, a cell cycle phase in which tumor cells are sensitive to radiation. Furthermore, the increased levels of DNA damage, combined with the concomitant downregulation of EGFR-mediated signaling by ZRBA1, may account for the significant levels of cell killing induced by the combination.
Assuntos
Quinazolinas/farmacologia , Radiossensibilizantes/farmacologia , Triazenos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/efeitos da radiação , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/efeitos da radiação , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/efeitos da radiação , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Microscopia de Fluorescência , Quinazolinas/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/efeitos da radiação , Radiossensibilizantes/metabolismo , Radiossensibilizantes/farmacocinética , Radiossensibilizantes/efeitos da radiação , Triazenos/metabolismo , Triazenos/farmacocinética , Triazenos/efeitos da radiação , Ensaio Tumoral de Célula-Tronco , Raios XAssuntos
Anidrase Carbônica I/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/efeitos da radiação , Sulfonamidas/farmacologia , Sulfonamidas/efeitos da radiação , Tiofenos/farmacologia , Tiofenos/efeitos da radiação , Anidrase Carbônica I/química , Domínio Catalítico , Inibidores Enzimáticos/química , Humanos , Luz , Fotoquímica , Conformação Proteica , Sulfonamidas/química , Tiofenos/químicaRESUMO
The potent O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline has been modified by buffer- or acylation-induced imine-to-enamine conversion and then electrophile or radical addition (Xn = D3, F, N3, OH, SMe, COCF3, CF3). Several functionalized GlcNAc-thiazolines show highly selective inhibition of OGA vs human hexosaminidase and thus have promise as tools for targeted investigations of OGA, an enzyme linked to diabetes and neurodegeneration. A new radical addition/fragmentation reaction of the N-(trifluoroacetyl)enamine has been discovered.
Assuntos
Inibidores Enzimáticos/síntese química , Glucosamina/análogos & derivados , Tiazóis/síntese química , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/efeitos da radiação , Radicais Livres/síntese química , Radicais Livres/farmacologia , Radicais Livres/efeitos da radiação , Glucosamina/síntese química , Glucosamina/farmacologia , Glucosamina/efeitos da radiação , Hexosaminidases/antagonistas & inibidores , Humanos , Isomerismo , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Tiazóis/farmacologia , Tiazóis/efeitos da radiação , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
Microcystins are a family of toxins produced by cyanobacteria found throughout the world in marine and freshwater environments. The most commonly encountered form of microcystin is microcystin-LR (MC-LR). Humans are exposed to MC-LR by drinking contaminated water. The toxin accumulates rapidly in the liver where it exerts most of its damage. Treatment of water containing MC-LR by ultrasonic irradiation leads to the breakdown of the toxin. Both the parent toxin and the treated toxin reaction products (TTRP) were evaluated for toxic effects in mice. Animals were exposed to purified MC-LR or an equivalent dose of the TTRP and sacrificed after 4 h or 24 h. Serum was collected and assayed for lactate dehydrogenase (LDH) activity as an indicator of hepatotoxicity. LDH activity was detected in the serum of MC-LR exposed mice indicative of liver damage, but not in control mice. Only a fraction of that activity was detectable in mice exposed to TTRP. Liver RNA was used for microarray analysis and real-time PCR. Individual animals varied in their overall genomic response to the toxin; however, only 20 genes showed consistent changes in expression. These include chaperones which may be part of a generalized stress response; cytochrome P450 which may be involved in metabolizing the toxin; and lipid dystrophy genes such as lipin-2, uridine phosphorylase and a homolog to tribbles, a stress-inducible gene involved in cell death. Of the genes that responded to the MC-LR, none showed significant changes in expression profile in response to TTRP. Taken together, the data indicate that ultrasonic irradiation of MC-LR effectively reduces hepatotoxicity in mice and therefore may be a useful method for detoxification of drinking water.
Assuntos
Perfilação da Expressão Gênica , Microcistinas/toxicidade , Toxicogenética/métodos , Ultrassom , Animais , Argininossuccinato Sintase/genética , Sistema Enzimático do Citocromo P-450/genética , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos da radiação , Inibidores Enzimáticos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Peptídeos e Proteínas de Sinalização Intracelular/genética , L-Lactato Desidrogenase/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos BALB C , Microcistinas/administração & dosagem , Microcistinas/efeitos da radiação , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfatidato Fosfatase , Proteínas Serina-Treonina Quinases/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Uridina Fosforilase/genéticaRESUMO
A simple and efficient synthesis of 2-amino pyrido[2,3-d]pyrimidine derivatives was accomplished via a three-component reaction under microwave irradiation without catalyst. This method had many dramatic advantages such as the short reaction time, high yield, and broad substrate scope, as well as convenient operation. We provide new series of potential biologically active compounds as inhibitors of Cdk4.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/efeitos da radiação , Micro-Ondas , Estrutura Molecular , Piridinas/efeitos da radiação , Pirimidinas/efeitos da radiação , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the most selective GSK-3 inhibitors 7a-d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and target the specific hydrophobic backpocket of the enzyme.
Assuntos
Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinazolinas/farmacologia , Tiazóis/farmacologia , Trifosfato de Adenosina/química , Sítios de Ligação , Cristalografia por Raios X , Quinases Ciclina-Dependentes/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/efeitos da radiação , Ligação de Hidrogênio , Micro-Ondas , Modelos Moleculares , Quinazolinas/química , Quinazolinas/efeitos da radiação , Estereoisomerismo , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/efeitos da radiaçãoRESUMO
Microwave irradiation of substituted hydrazines and beta-ketoesters gives 5-aminopyrazoles in excellent yield, which can be transformed to the corresponding N-carbonyl derivatives by treatment with an isocyanate or chloroformate. Derivatization of 4-nitronaphth-1-ol using predominantly microwave heating methods and reaction with an N-pyrazole carbamate provides a rapid route to the N-pyrazole urea BIRB 796 in high purity, as a potent and selective inhibitor of p38alpha mitogen-activated protein kinase for the study of accelerated ageing in Werner syndrome cells.
Assuntos
Senescência Celular/efeitos dos fármacos , Inibidores Enzimáticos , Micro-Ondas , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Naftalenos , Pirazóis , Linhagem Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/efeitos da radiação , Humanos , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/farmacologia , Naftalenos/efeitos da radiação , Pirazóis/síntese química , Pirazóis/farmacologia , Pirazóis/efeitos da radiação , Estereoisomerismo , Relação Estrutura-Atividade , Síndrome de Werner/enzimologiaRESUMO
The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.
Assuntos
Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/química , Animais , Carbazóis/química , Carbazóis/efeitos da radiação , Linhagem Celular Tumoral , Meios de Cultura , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A1/efeitos da radiação , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/efeitos da radiação , Indóis/química , Indóis/efeitos da radiação , Luz , Neoplasias Hepáticas Experimentais/metabolismo , Fotoquímica , Fármacos Fotossensibilizantes/farmacologia , Dibenzodioxinas Policloradas/farmacologia , Ratos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/efeitos da radiação , Riboflavina/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Teratogênicos/farmacologia , Triptofano/metabolismoRESUMO
The microwave-assisted synthesis of a family of 2,8-substituted thiazoloquinazolinones is described. The preliminary evaluation of the antiproliferative activity and the capacity of these molecules to inhibit CDKs and GSK-3 are reported. A lead compound was identified, constituting a scaffold from which more potent inhibitors could be designed.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinazolinas/síntese química , Quinazolinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/efeitos da radiação , Humanos , Micro-Ondas , Estrutura Molecular , Quinazolinas/efeitos da radiação , Relação Estrutura-AtividadeRESUMO
o,o'-Diphenyleneiodonium cations (DPI) convert relatively harmless radiation-produced electrons into efficient DNA cleaving agents. The cleavage products are unaltered DNA bases, 5-methylenefuranone (5-MF), and a complete set of 3' and 5'-phosphorylated DNA fragments. The production of alkali-labile sites is a minor factor in the process. Based on the production of 5-MF, it is concluded that DNA cleavage by DPI cations involves (but may not be limited to) the C1' chemistry. The loss of 3-aminoDPI (ADPI) cations bound to highly polymerized calf thymus DNA appears to be due to a short-chain reaction with an apparent length of up to 2.1 ADPI cations consumed for each radiation-produced electron. The suggested chain reaction mechanism includes the one-electron oxidation of DNA radicals (including the C1' sugar radical) by ADPI cations bound to the same duplex. The yields of DNA loss in complexes formed by ADPI with short synthetic duplexes indicate that there is more than a 60% probability of DNA damage after one-electron reduction of ADPI.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Elétrons , Inibidores Enzimáticos/efeitos da radiação , Raios gama , Oniocompostos/efeitos da radiação , Animais , Cátions , Bovinos , Radioisótopos de Cobalto , DNA/metabolismo , Inibidores Enzimáticos/metabolismo , Furanos/análise , Cinética , Modelos Químicos , Oniocompostos/metabolismo , Piperidinas/farmacologiaRESUMO
OBJECT: Ionizing radiation is the gold-standard adjuvant treatment for glioblastoma multiforme (GBM), the most aggressive primary brain tumor. The mechanisms underlying neoplastic glial cell growth inhibition after administration of ionizing radiation, however, remain largely unknown. In this report, the authors characterize the response of GBM cells to ionizing radiation and elucidate factors that correlate with the radiosensitivity of these tumors. METHODS: Six human GBM cell lines were subjected to increasing doses of radiation. Each demonstrated a dose-dependent suppression of cell proliferation. In the most radiosensitive cell line, the authors demonstrated a transient increase in the expression of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27, which corresponded with a G1 cell-cycle arrest. In contrast, the most radioresistant cell line demonstrated a decrease in p21 and p27 expression levels, which correlated with a failure to arrest. Apoptosis did not occur in any cell line following irradiation. Instead, autophagic cell changes were observed following administration of radiation, regardless of the relative radiosensitivity of the cell line. CONCLUSIONS: These findings elucidate some of the molecular responses of GBMs to irradiation and suggest novel targets for future therapy.
Assuntos
Autofagia/genética , Autofagia/efeitos da radiação , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Proteínas de Ciclo Celular/efeitos da radiação , Ciclinas/efeitos da radiação , Inibidores Enzimáticos/efeitos da radiação , Fase G1/genética , Fase G1/efeitos da radiação , Glioblastoma/patologia , Glioblastoma/radioterapia , Radiação Ionizante , Proteínas Supressoras de Tumor/efeitos da radiação , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/análise , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/análise , Inibidores Enzimáticos/análise , Glioblastoma/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Organelas/genética , Organelas/efeitos da radiação , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Fatores de Tempo , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/efeitos da radiação , Proteínas Supressoras de Tumor/análiseRESUMO
Flavonol induced pollen germination in petunia is rapid, specific, and achieved at low concentrations of kaempferol or quercetin. To determine the macromolecules that interact with the flavonol signal we have synthesized affinity-tagged kaempferol analogues. The first generation molecules are based on a benzophenone photophore. We find that 2-(3-benzoylphenyl)-3,5,7-trihydroxychromen-4-one (BPKae) antagonizes flavonol-induced pollen germination in a concentration-dependent manner. Further, BPKae acts as an irreversible inhibitor of flavonol 3-O-galactosyltransferase (F3GalTase), the gametophyte-specific enzyme that controls the accumulation of glycosylated flavonols in pollen. The effects of BPKae are mediated by UV-A light treatment. The binding characteristics of BPKae to F3GalTase suggest that it can be used to identify the residues required for flavonol-binding and catalysis.
Assuntos
Benzofenonas/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/metabolismo , Flavonoides/farmacologia , Galactosiltransferases/fisiologia , Germinação/efeitos dos fármacos , Proteínas de Plantas/fisiologia , Pólen/fisiologia , Marcadores de Afinidade , Benzofenonas/síntese química , Benzofenonas/efeitos da radiação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/efeitos da radiação , Flavonoides/síntese química , Flavonoides/efeitos da radiação , Flavonóis , Galactosiltransferases/antagonistas & inibidores , Germinação/fisiologia , Glicosilação , Petunia/enzimologia , Fotoquímica , Proteínas de Plantas/antagonistas & inibidores , Pólen/enzimologia , Solubilidade , Especificidade por Substrato , Raios Ultravioleta , Zea mays/enzimologiaRESUMO
Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.