RESUMO
The DNA as the depository of genetic information is a natural target for chemotherapy. A lot of anticancer and antimicrobial agents derive their biological activity from their selective interaction with DNA in the minor groove and from their ability to interfere with biological processes such as enzyme catalysis, replication and transcription. The discovery of the details of minor groove binding drugs, such as netropsin and distamycin A, oligoamides built of 4-amino-1-methylpyrrole-2-carboxylic acid residues, allowed to develop various DNA sequence-reading molecules, named lexitropsins, capable of interacting with DNA precisely, strongly and with a high specificity, and at the same time exhibiting significant cytotoxic potential. Among such compounds, lexitropsins built of carbocyclic sixmembered aromatic rings occupy a quite prominent place in drug research. This work is an attempt to present current findings in the study of carbocyclic lexitropins, their structures, syntheses and biological investigations such as DNA-binding and antiproliferative activity.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Distamicinas/química , Distamicinas/farmacologia , Desenho de Fármacos , Netropsina/análogos & derivados , Netropsina/farmacologia , Ácidos Carbocíclicos/síntese química , Ácidos Carbocíclicos/química , Ácidos Carbocíclicos/farmacologia , Animais , Antibacterianos/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , Distamicinas/síntese química , Humanos , Neoplasias/tratamento farmacológico , Netropsina/síntese químicaRESUMO
A shell biorefinery would involve fractionation of crustacean shells and incorporation of the components into value-added products, particularly those that contain nitrogen. In a proof-of-concept study that validates this concept, the anticancer alkaloid proximicinâ A has been synthesized from the chitin-derived platform chemical 3-acetamido-5-acetylfuran (3A5AF). This study accentuates the leading role chitin is likely to play in the sustainable production of nitrogen-containing fine chemicals that are not directly attainable from lignocellulose.
Assuntos
Antineoplásicos/metabolismo , Quitina/metabolismo , Netropsina/análogos & derivados , Exoesqueleto/química , Animais , Fracionamento Químico , Quitina/isolamento & purificação , Crustáceos/química , Química Verde , Netropsina/síntese química , Estudo de Prova de ConceitoRESUMO
A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the all new substances prepared in this investigation were fully characterized by NMR ((1)H, (13)C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322 confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.
Assuntos
DNA/metabolismo , Netropsina/análogos & derivados , Netropsina/metabolismo , Netropsina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Netropsina/síntese químicaRESUMO
Strain FIM06054 was isolated from a marine sponge sample collected from the East China Sea and was characterised as a strain of Verrucosispora genus on the basis of its 16S rRNA gene sequence. One new compound, FW054-1 (1), together with a known aminofuran compound proximicin A (2), was isolated from the culture broth of Verrucosispora sp. FIM06054. Their structures were elucidated on the basis of spectral analysis. 1 and 2 showed antiproliferative activity against several human tumour cell lines.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Micromonosporaceae/química , Netropsina/análogos & derivados , Poríferos/química , Ribose/análogos & derivados , Ribose/isolamento & purificação , Ribose/farmacologia , Animais , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Biologia Marinha , Netropsina/química , Netropsina/isolamento & purificação , Netropsina/farmacologia , Ressonância Magnética Nuclear Biomolecular , RNA Ribossômico 16S/genética , Ribose/químicaRESUMO
The proximicins A-C are naturally occurring cytotoxic γ-peptides that contain the unique 4-amino-furan-carboxylic acid. In contrast to the structurally related cytotoxic natural DNA binder netropsin and distamycin, both exhibiting as core building block N-methyl-4-amino-pyrrol-carboxylic acid, no DNA binding was observed for the procimicins. X-ray analysis of crystals of a protected 4-amino-furan-2-carboxylic acid dipeptide revealed a stretched conformation. In contrast, for netropsin and distamycin, sickle-shaped crystal conformations were observed. DFT-calculations elegantly confirm these conformational arrangements. The most stable conformers of the proximicins are linear whereas sickle-shaped conformations are less stable, having higher Gibbs energies. For netropsin, distamycin and the netropsin-proximicin-hybrid a sickle shaped conformation appears energetically favored. The reported results are consistent with the observations that the proximicins A-C do not bind to the DNA and have a different mode of action concerning their cytotoxic activity with respect to netropsin and distamycin.
Assuntos
Antibacterianos/química , Peptídeos/química , Teoria Quântica , Cristalografia por Raios X , Modelos Moleculares , Netropsina/análogos & derivados , Netropsina/química , Conformação ProteicaRESUMO
The criteria currently followed for selecting antitumor compounds include agents that can target apoptosis inhibitor proteins and cancer cell markers. In silico studies are often used to identify suitable antitumor compounds for the cancer targets. The aim of the present study is to evaluate the interactions of some antitumor compounds reported from marine Streptomyces with cancer target proteins. Nine compounds were selected from marine Streptomyces based on previous reports and evaluated for their interactions with cancer target proteins by in silico molecular docking approach. Interactions of the selected ligand with target proteins were studied by PatchDock bioinformatics docking tool. Among the compounds tested marmycin A was interacted very effectively with human epidermal growth factor receptor 2 (HER2) and showed a least binding energy of -472.92 kcal/mol. The compound altemicidin showed a least binding energy of -415.66 kcal/mol with cyclin dependent kinase 4 (CDK4). The ligands resistoflavine and resistomycin also interacted with HER2 and showed the binding energy of -402.10 kcal/mol and -377.78 kcal/mol respectively. Other ligands proximycin A, chandrananimycin C, echinosporin, streptochlorin and streptokordin also showed the binding energy of -341.11 kcal/mol, -313.31 kcal/mol, -305.64 kcal/mol, -291.91 kcal/mol and 222.34 kcal/mol respectively with CDK4 protein. These results of our study suggest that HER2 and CDK4 are better cancer drug targets for therapy.
Assuntos
Antineoplásicos/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Simulação de Acoplamento Molecular/métodos , Receptor ErbB-2/metabolismo , Streptomyces/química , Alcaloides/química , Alcaloides/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Antineoplásicos/análise , Benzopirenos/metabolismo , Biologia Computacional , Humanos , Indóis/metabolismo , Lactonas/metabolismo , Biologia Marinha , Modelos Moleculares , Netropsina/análogos & derivados , Netropsina/metabolismo , Oxazóis/metabolismo , Piridinas/química , Piridinas/metabolismo , Piridonas/metabolismo , Compostos de Enxofre/química , Compostos de Enxofre/metabolismoRESUMO
Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.
Assuntos
Desenho de Fármacos , Oligodesoxirribonucleotídeos/química , Antineoplásicos Fitogênicos/química , Antivirais/química , Sequência de Bases , Benzoxazóis/química , Berberina/química , Bisbenzimidazol/análogos & derivados , Bisbenzimidazol/química , Butiratos , Ligação de Hidrogênio , Imidazóis/química , Ácidos Indolacéticos/química , Ligantes , Simulação de Dinâmica Molecular , Netropsina/análogos & derivados , Netropsina/química , Conformação de Ácido Nucleico , Plasmídeos/química , Vincristina/químicaRESUMO
Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA-damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS-057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI-MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.
Assuntos
DNA/química , Guanina/química , Timina/química , Pareamento Incorreto de Bases , Netropsina/análogos & derivados , Netropsina/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/farmacologia , Netropsina/análogos & derivados , Netropsina/farmacologia , Apoptose/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular Tumoral , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Feminino , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Neoplasias/tratamento farmacológico , Netropsina/síntese química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Verrucosispora maris AB-18-032 is a marine actinomycete that produces atrop-abyssomicin C and proximicin A, both of which have novel structures and modes of action. In order to understand the biosynthesis of these compounds, to identify further biosynthetic potential, and to facilitate rational improvement of secondary metabolite titers, we have sequenced the complete 6.7-Mb genome of Verrucosispora maris AB-18-032.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Micromonosporaceae/genética , Antineoplásicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Micromonosporaceae/isolamento & purificação , Micromonosporaceae/metabolismo , Dados de Sequência Molecular , Netropsina/análogos & derivados , Netropsina/metabolismo , Análise de Sequência de DNA , Microbiologia da ÁguaRESUMO
The protein binding to the origin of replication of the herpes simplex virus type 1 (HSV-1) is DNA helicase encoded by the UL9 gene of the herpes virus. The protein specifically binds to two binding sites in the viral DNA replication origins OriS or OriL. In order to determine the role of the UL9 protein in the initiation of replication and find efficient inhibitors of the UL9 activity, we have synthesized a recombinant UL9 protein expressed in E. coli cells. It was found that the recombinant UL9 protein binds to Boxes I and II in the OriS and possesses the DNA helicase and ATPase activities. In a complex with a fluorescent analog of ATP, two molecules of the ATP analog bind to one protein dimer molecule. It was also found that the UL9 protein in the dimer form can bind simultaneously to two DNA fragments, each containing specific binding sites for the protein. The interaction of the recombinant UL9 protein with the 63-mer double and single-stranded oligonucleotides OriS and OriS* has been investigated, which correspond to the origin of replication of herpes simplex virus. From the titrations of OriS and OriS* by ethidium bromide in the presence and absence of the UL9 protein, the equilibrium affinity constants of the protein binding to OriS and OriS* have been determined. A DNase I footprinting study showed that bis-linked netropsin derivatives exhibit preferences for binding to the AT-cluster in the origin of replication OriS and inhibit the fluctuation opening of AT-base pairs in the AT-cluster. The drugs also prevent the formation of an intermediate conformation of OriS* that involves a disordered tail at the 3'-end and stable Box I-Box III hairpin to which the UL9 helicase selectively binds. The stabilization by bis-netropsins of the AT-rich hairpin at its 3' end can inhibit the helicase activity. It was concluded that the antiviral activity of bis-netropsins may be associated with the inhibitory effects of bis-netropsins on these two stages of the reaction catalyzed by helicase UL9.
Assuntos
Antivirais/química , DNA Helicases/química , DNA Viral/química , Proteínas de Ligação a DNA/química , Herpesvirus Humano 1/enzimologia , Netropsina/análogos & derivados , Proteínas Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Antivirais/uso terapêutico , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Herpes Simples/tratamento farmacológico , Herpes Simples/enzimologia , Herpesvirus Humano 1/genética , Netropsina/química , Netropsina/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação/fisiologia , Proteínas Virais/genéticaRESUMO
We recently demonstrated that poly(ADP-ribose) polymerase (PARP)-1 is involved in angiogenesis and tumour aggressiveness. In this study we have compared the influence of abrogation of PARP-1 expression by stable gene silencing to that of the pharmacological inhibition of cellular PARP activity using PARP-1/-2 inhibitors on the chemosensitivity of tumour cells to the wide spectrum methylating agent temozolomide (TMZ) and to the N3-adenine selective methylating agent {1-methyl-4-[1-methyl-4-(3-methoxysulfonylpropanamido)pyrrole-2-carboxamido]-pyrrole-2-carboxamido}propane (Me-Lex). Silencing of PARP-1 in melanoma or cervical carcinoma lines enhanced in vitro sensitivity to TMZ and Me- Lex, and induced a higher level of cell accumulation at the G2/M phase of cell cycle with respect to controls. GPI 15427, which inhibits both PARP-1 and PARP-2, increased sensitivity to TMZ and Me-Lex both in PARP-1-proficient and - deficient cells. However, it induced different cell cycle modulations depending on PARP-1 expression, provoking a G2/M arrest only in PARP-1 silenced cells. Treatment of PARP-1 silenced cells with TMZ or Me-Lex resulted in a more extensive phosphorylation of Chk-1 and p53 as compared to PARP-1 proficient cells. The combination of the methylating agents with GPI 15427 increased Chk-1 and p53 phosphorylation both in PARP-1 proficient or deficient cells. When mice challenged with PARP-1 silenced melanoma cells were treated with the TMZ and PARP inhibitor combination there was an additional reduction in tumour growth with respect to treatment with TMZ alone. These results suggest the involvement of PARP-2 or other PARPs, in the repair of DNA damage provoked by methylating agents, highlighting the importance of targeting both PARP-1 and PARP-2 for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Netropsina/análogos & derivados , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Sinergismo Farmacológico , Citometria de Fluxo , Fase G2/efeitos dos fármacos , Células HeLa , Humanos , Melanoma Experimental/patologia , Metilação , Camundongos , Netropsina/farmacologia , Poli(ADP-Ribose) Polimerases/genética , TemozolomidaRESUMO
We recently demonstrated that Polzeta and Rev1 contribute to alleviate the lethal effects of Me-lex, which selectively generates 3-methyladenine, by error prone lesion bypass. In order to determine the role of Poleta in the biological fate of Me-lex induced lesions, the RAD30 (Poleta) gene was deleted in the yIG397 parental strain and in its rev3 (Polzeta) derivative, and the strains transformed with plasmid DNA damaged in vitro by Me-lex. While deletion of RAD30 increased the toxicity of Me-lex, the impact on mutagenicity varied depending on the concentration of Me-lex induced DNA damage and the overall TLS capacity of the cells. For the first time the Me-lex induced mutation spectrum in rad30 strain was determined and compared with the spectrum previously determined in WT strain. Overall, the two mutation spectra were not significantly different. The effect on mutation frequency and the features of the Me-lex induced mutation spectra were suggestive of error prone (significant decrease of mutation frequency and significant decrease of AT>TA at a mutation hotspot in rad30 vs RAD30) but also error free (significant increase of AT>GC in rad30 vs RAD30) Poleta dependent bypass of lesions. In summary, our previous results with Polzeta and Rev1 mutants, the present results with Poleta, and the known physical and functional interactions among TLS proteins, lead us to propose that the bypass of Me-lex induced lesions is a multi-DNA polymerases process that is mostly effective when all three yeast TLS polymerases are present.
Assuntos
Adenina/análogos & derivados , Dano ao DNA , DNA Fúngico/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/fisiologia , Mutagênese , Netropsina/análogos & derivados , Saccharomyces cerevisiae/efeitos dos fármacos , Adenina/toxicidade , DNA Fúngico/genética , Deleção de Genes , Mutação/genética , Netropsina/toxicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The proximicins A-C (1-3) are novel naturally occurring gamma-peptides with a hitherto unknown 2,4-disubstituted furan amino acid as a core structure. They show a moderate cytotoxic activity and induce upregulation of cell cycle regulating proteins (p53 and p21) and lead to cell cycle arrest in G0/G1-phase. Hybrid molecules combining structural motifs of the proximicins and of netropsin (4), a structurally related natural product, seem to have similar effects. Herein we describe the synthesis of a netropsin-proximicin-hybrid library and its evaluation regarding cytotoxicity and minor groove binding activity.
Assuntos
Antineoplásicos/síntese química , DNA/metabolismo , Netropsina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Netropsina/síntese química , Netropsina/toxicidade , Proteína Supressora de Tumor p53/metabolismoRESUMO
The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.) (22-33) are described. The molecules consist of an isoxazole that pre-organizes a planar aromatic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb's amide formation technique, using SmCl(3) as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute's (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of the AIMs for G4 over B-DNA is presented.
Assuntos
Antracenos/síntese química , Antracenos/farmacologia , Antineoplásicos/síntese química , Azóis/síntese química , Azóis/farmacologia , Netropsina/análogos & derivados , Antracenos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Azóis/química , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Netropsina/síntese química , Netropsina/química , Netropsina/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
A family of three novel aminofuran antibiotics named as proximicins was isolated from the marine Verrucosispora strain MG-37. Proximicin A was detected in parallel in the marine abyssomicin producer "Verrucosispora maris" AB-18-032. The characteristic structural element of proximicins is 4-amino-furan-2-carboxylic acid, a hitherto unknown gamma-amino acid. Proximicins show a weak antibacterial activity but a strong cytostatic effect to various human tumor cell lines.
Assuntos
Actinobacteria/metabolismo , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Netropsina/análogos & derivados , Actinobacteria/química , Actinobacteria/classificação , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/isolamento & purificação , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Fermentação , Humanos , Testes de Sensibilidade Microbiana , Netropsina/biossíntese , Netropsina/isolamento & purificação , Netropsina/farmacologia , Espectrofotometria UltravioletaAssuntos
Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Netropsina/análogos & derivados , Netropsina/farmacologia , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Actinobacteria/química , Furanos , Estrutura Molecular , Netropsina/química , Netropsina/isolamento & purificaçãoRESUMO
The relative toxicity and mutagenicity of Me-lex, which selectively generates 3-methyladenine (3-MeA), is dependent on the nature of the DNA repair background. Base excision repair (BER)-defective S. cerevisiae strains mag1 and apn1apn2 were both significantly more sensitive to Me-lex toxicity, but only the latter is significantly more prone to Me-lex-induced mutagenesis. To examine the contribution of translesion synthesis (TLS) DNA polymerases in the bypass of Me-lex-induced lesions, the REV3 and REV1 genes were independently deleted in the parental yeast strain and in different DNA repair-deficient derivatives: the nucleotide excision repair (NER)-deficient rad14, and the BER-deficient mag1 or apn1apn2 strains. The strains contained an integrated ADE2 reporter gene under control of the transcription factor p53. A centromeric yeast expression vector containing the wild-type p53 cDNA was treated in vitro with increasing concentrations of Me-lex and transformed into the different yeast strains. The toxicity of Me-lex-induced lesions was evaluated based on the plasmid transformation efficiency compared to the untreated vector, while Me-lex mutagenicity was assessed using the p53 reporter assay. In the present study, we demonstrate that disruption of Polzeta (through deletion of its catalytic subunit coded by REV3) or Rev1 (by REV1 deletion) increased Me-lex lethality and decreased Me-lex mutagenicity in both the NER-defective (rad14) and BER-defective (mag1; apn1apn2) strains. Therefore, Polzeta and Rev1 contribute to resistance of the lethal effects of Me-lex-induced lesions (3-MeA and derived AP sites) by bypassing lesions and fixing some mutations.
Assuntos
Adenina/análogos & derivados , Antimutagênicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Mutagênicos/toxicidade , Netropsina/análogos & derivados , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenina/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Netropsina/toxicidade , Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína Supressora de Tumor p53RESUMO
PURPOSE: Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion. EXPERIMENTAL DESIGN: We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites. RESULTS: (a) The LD(10) for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O(6)-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity. CONCLUSIONS: (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O(6)-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Netropsina/análogos & derivados , Linhagem Celular Tumoral , Adutos de DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Netropsina/farmacologia , Oligonucleotídeos Antissenso/química , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The regulation of gene expression is based on the interaction of DNA with different ligands. A model of adsorption was considered that can be applied to the quantitative analysis of footprinting diagrams for the complexes formed by a ligand with a DNA fragment of known structure. This model allows the probabilities of ligand binding to DNA sites with a known sequence to be calculated and the variance of probabilities of ligand binding with a specified binding site to be estimated. The model was used for quantitative analysis of diagrams of DNAse footprinting for the complexes of the dimeric analogue of the antitumor antibiotic netropsin. Experimental and theoretically calculated profiles of distribution of netropsin bound on DNA are in good agreement with one another.