RESUMO
Tyrosyl-DNA phosphodiesterase 1(TDP1) is a promising target for a new therapy in oncological disease as an adjunct to topoisomerase 1 (TOP1) drugs. In this paper, novel thiazolidin-4-one derivatives with a benzyl and monoterpene substituents were synthesized. Compounds with a monoterpene fragment attached via a phenyloxy linker were active against TDP1 with IC50 values in the 1 ÷ 3 µM range, while direct attachment of monoterpene moiety to the thiazolidin-4-one fragment had no activity. Molecular modelling predicted two plausible binding modes of the active compounds both effectively blocking access to the catalytic site of TDP. At non-toxic concentrations the active ligands potentiated the efficacy of the TOP1 poison topotecan in human cervical cancer HeLa cells, but not in non-cancerous HEK293A cells.
Assuntos
Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases , Esterases/metabolismo , Células HeLa , Humanos , Monoterpenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Relação Estrutura-AtividadeRESUMO
The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC50 values in the 0.3-22.0 µM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects.
Assuntos
Diester Fosfórico Hidrolases , Topotecan , Animais , Camundongos , Humanos , Topotecan/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Nucleosídeos de Purina , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/farmacologia , Esterases/metabolismo , Dano ao DNA , DNA , DNA Topoisomerases Tipo I/metabolismoRESUMO
Inhibiting tyrosyl-DNA phosphodiesterase 1 (TDP1) is a promising strategy for increasing the effectiveness of existing antitumor therapy since it can remove the DNA lesions caused by anticancer drugs, which form covalent complexes with topoisomerase 1 (TOP1). Here, new adamantane-monoterpene conjugates with a 1,2,4-triazole or 1,3,4-thiadiazole linker core were synthesized, where (+)-and (-)-campholenic and (+)-camphor derivatives were used as monoterpene fragments. The campholenic derivatives 14a-14b and 15a-b showed activity against TDP1 at a low micromolar range with IC50 ~5-6 µM, whereas camphor-containing compounds 16 and 17 were ineffective. Surprisingly, all the compounds synthesized demonstrated a clear synergy with topotecan, a TOP1 poison, regardless of their ability to inhibit TDP1. These findings imply that different pathways of enhancing topotecan toxicity other than the inhibition of TDP1 can be realized.
Assuntos
Adamantano , Antineoplásicos , Adamantano/farmacologia , Antineoplásicos/farmacologia , Cânfora , Monoterpenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Topotecan/farmacologiaRESUMO
Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3' phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA-TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4-25.2 µM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.
Assuntos
Benzofuranos/química , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Sulfetos/química , Benzofuranos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/síntese química , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Relação Estrutura-Atividade , Sulfetos/farmacologia , Sulfóxidos/química , Sulfóxidos/farmacologia , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologiaRESUMO
A series of deoxycholic acid (DCA) amides containing benzyl ether groups on the steroid core were tested against the tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) enzymes. In addition, 1,2,4- and 1,3,4-oxadiazole derivatives were synthesized to study the linker influence between a para-bromophenyl moiety and the steroid scaffold. The DCA derivatives demonstrated promising inhibitory activity against TDP1 with IC50 in the submicromolar range. Furthermore, the amides and the 1,3,4-oxadiazole derivatives inhibited the TDP2 enzyme but at substantially higher concentration. Tryptamide 5 and para-bromoanilide 8 derivatives containing benzyloxy substituent at the C-3 position and non-substituted hydroxy group at C-12 on the DCA scaffold inhibited both TDP1 and TDP2 as well as enhanced the cytotoxicity of topotecan in non-toxic concentration in vitro. According to molecular modeling, ligand 5 is anchored into the catalytic pocket of TDP1 by one hydrogen bond to the backbone of Gly458 as well as by π-π stacking between the indolyl rings of the ligand and Tyr590, resulting in excellent activity. It can therefore be concluded that these derivatives contribute to the development of specific TDP1 and TDP2 inhibitors for adjuvant therapy against cancer in combination with topoisomerase poisons.
Assuntos
Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Sítios de Ligação , Linhagem Celular , Fenômenos Químicos , Técnicas de Química Sintética , Ácido Desoxicólico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to C and D rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9-O-sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9-O-sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.
Assuntos
Berberina/análogos & derivados , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Antineoplásicos/química , Berberina/química , Desenho de Fármacos , Células HeLa , Humanos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors having IC50 values in the 0.35-0.57 µM range. The cytotoxicity was determined in the HeLa, HCT-116 and SW837 cancer cell lines; moderate CC50 (µM) values were seen from the mid-teens to no effect at 100 µM. Furthermore, citral derivative 20c, α-pinene-derived compounds 20f, 20g and 25c, and the citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further development as a potential adjunct therapy with Top1 poisons.
Assuntos
Adamantano/farmacologia , Monoterpenos/química , Diester Fosfórico Hidrolases/efeitos dos fármacos , Adamantano/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Humanos , Ligantes , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure-activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Berberina/análogos & derivados , Inibidores de Fosfodiesterase/síntese química , Diester Fosfórico Hidrolases/química , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Berberina/química , Berberina/farmacologia , Sítios de Ligação , Reparo do DNA/efeitos dos fármacos , Combinação de Medicamentos , Desenho de Fármacos , Sinergismo Farmacológico , Células HeLa , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química , Topotecan/químicaRESUMO
Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2',3',5'-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 2',3',5'-Tri-O-benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 µM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 µM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50-100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Diester Fosfórico Hidrolases/metabolismo , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/toxicidade , Células HeLa , Humanos , Nucleosídeos de Pirimidina/toxicidadeRESUMO
Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC50 value of 0.65 µM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k, a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.
Assuntos
Monoterpenos Bicíclicos/química , Desenho de Fármacos , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sistemas CRISPR-Cas , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Inativação de Genes , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/genética , Topotecan/farmacologiaRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.
Assuntos
Carcinoma Krebs 2/tratamento farmacológico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Monoterpenos , Proteínas de Neoplasias , Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases/metabolismo , Animais , Carcinoma Krebs 2/enzimologia , Carcinoma Krebs 2/patologia , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/patologia , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Monoterpenos/síntese química , Monoterpenos/química , Monoterpenos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Relação Estrutura-AtividadeRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Diester Fosfórico Hidrolases , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase ß, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.
Assuntos
Reparo do DNA , Reparo por Excisão , Poli(ADP-Ribose) Polimerase-1 , Humanos , Extratos Celulares , Linhagem Celular , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
Usnic acid (UA) is a secondary metabolite of lichens that exhibits a wide range of biological activities. Previously, we found that UA derivatives are effective inhibitors of tyrosyl-DNA phosphodiesterase 1 (TDP1). It can remove covalent complex DNA-topoisomerase 1 (TOP1) stabilized by the TOP1 inhibitor topotecan, neutralizing the effect of the drugs. TDP1 removes damage at the 3' end of DNA caused by other anticancer agents. Thus, TDP1 is a promising therapeutic target for the development of drug combinations with topotecan, as well as other drugs for cancer treatment. Ten new UA enamino derivatives with variation in the terpene fragment and substituent of the UA backbone were synthesized and tested as TDP1 inhibitors. Four compounds, 11a-d, had IC50 values in the 0.23-0.40 µM range. Molecular modelling showed that 11a-d, with relatively short aliphatic chains, fit to the important binding domains. The intrinsic cytotoxicity of 11a-d was tested on two human cell lines. The compounds had low cytotoxicity with CC50 ≥ 60 µM for both cell lines. 11a and 11c had high inhibition efficacy and low cytotoxicity, and they enhanced topotecan's cytotoxicity in cancerous HeLa cells but reduced it in the non-cancerous HEK293A cells. This "protective" effect from topotecan on non-cancerous cells requires further investigation.
Assuntos
Benzofuranos/química , Monoterpenos/química , Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases/metabolismo , Benzofuranos/farmacologia , Células HEK293 , Humanos , Monoterpenos/farmacologia , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologiaRESUMO
Prominent lesions in DNA are abasic (AP) sites arising spontaneously or as intermediates during base excision repair. An AP site can form a Schiff base intermediate with primary amino groups of proteins. This intermediate can be stabilized by NaBH(4) treatment and, therefore, cross-linking of AP site-containing DNA (AP DNA) can be used as a tool in detecting proteins that interact with AP sites. Using AP DNA, we observed in the extracts derived from several human cell lines a predominant cross-linked product with an apparent molecular mass of 95kDa. The cross-linked protein was identified as the p80 subunit of Ku antigen (Ku80) (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777-1785 [1]). Because the cross-linking of Ku80 to AP sites is efficient and selective, this approach may be useful to estimate the amount of Ku antigen in cell extracts in the presence of other cellular proteins. We compared levels of Ku80 detected by dot-ELISA with Ku80 antibodies to the levels of Ku80 cross-linked to AP DNA in extracts derived from HeLa cells and several melanoma cell lines. The level of Ku80 trapping varied considerably depending on the cell lines and correlated with the amount of Ku80 in the extracts estimated by the immunochemical approach. This approach, unlike western blot or estimation of the Ku content based on mRNA levels, is more suitable for tracking Ku forms active in DNA binding including those having aberrations in Ku80, but retaining an ability to heterodimerize with Ku70, that provides efficient loading of Ku antigen onto DNA ends. As a routine test, borohydride trapping (BHT) is also less time and reagent consuming than blotting and EMSA.
Assuntos
Antígenos Nucleares/análise , Dano ao DNA , Proteínas de Ligação a DNA/análise , Ácido Apurínico , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas , Sondas de DNA , Humanos , Autoantígeno Ku , Melanoma/química , PolinucleotídeosRESUMO
One of the most abundant lesions in DNA is the abasic (AP) sites arising spontaneously or as an intermediate in base excision repair. Certain proteins participating in the processing of these lesions form a Schiff base with the deoxyribose of the AP site. This intermediate can be stabilized by NaBH(4) treatment. By this method, DNA duplexes with AP sites were used to trap proteins in cell extracts. In HeLa cell extract, along with a prevalent trap product with an apparent molecular mass of 95 kDa, less intensive low-molecular-weight products were observed. The major one was identified as the p80-subunit of Ku antigen (Ku). Ku antigen, a DNA binding component of DNA-dependent protein kinase (DNA-PK), participates in double-stranded break repair and is responsible for the resistance of cells to ionizing radiation. The specificity of Ku interaction with AP sites was proven by more efficient competition of DNA duplexes with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with different efficiencies depending on the size and position of strand interruptions opposite to AP sites. Ku antigen as a part of DNA-PK was shown to inhibit AP site cleavage by apurinic/apyrimidinic endonuclease 1.
Assuntos
Antígenos Nucleares/metabolismo , Pareamento Incorreto de Bases , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Antígenos Nucleares/fisiologia , Pareamento Incorreto de Bases/fisiologia , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Células HL-60 , Células HeLa , Humanos , Células K562 , Autoantígeno Ku , Conformação de Ácido Nucleico , Mutação Puntual/fisiologia , Ligação Proteica/efeitos dos fármacosRESUMO
DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.
Assuntos
Extratos Celulares , Reparo do DNA/fisiologia , Poli ADP Ribosilação/fisiologia , Animais , DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Ratos-Toupeira , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Clustered apurinic/apyrimidinic (AP) sites are more cytotoxic than isolated AP lesions because double strand breaks (DSB) can be formed during repair of closely positioned bistranded AP sites. Formation of DSB due to simultaneous cleavage of bistranded AP sites may be regulated by proteins specifically interacting with this complex lesion. A set of AP DNA duplexes containing AP sites in both strands in different mutual orientation (BS-AP DNAs) was used for search in the extracts of human cells proteins specifically recognizing clustered AP sites. A protein, which formed the Schiff-base-dependent covalent products having an apparent molecular mass of 50â¯kDa with the subset of BS-AP DNAs, was identified by mass spectrometry as apurinic/apyrimidinic endonuclease 1 (APE1). The identity of trapped protein was confirmed by Western blot analysis with anti-APE1 antibodies. Purified recombinant human APE1 is also capable of forming the 50â¯kDa-adducts with efficiency of BS-AP DNAs cross-linking to APE1 being dependent on the mutual orientation of AP sites. In spite of formation of the Schiff-base-dependent intermediate, which is prerequisite for the ß-elimination mechanism, APE1 is unable to cleave AP sites. APE1 lacking the first 34â¯amino acids at the N-terminus, unlike wild type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites. The yield of APE1-AP DNA cross-links was found to correlate with the enzyme amount in the extracts estimated by the immunochemical approach; therefore the BS-AP DNA-probes can be useful for comparative analysis of APE1 content in cell extracts.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Bases de Schiff/metabolismo , Sítios de Ligação , DNA/genética , DNA/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Humanos , Espectrometria de Massas , Ligação Proteica , Bases de Schiff/químicaRESUMO
In eukaryotes the stability of genome is provided by functioning of DNA repair systems. One of the main DNA repair pathways in eukaryotes is the base excision repair (BER). This system requires precise regulation for correct functioning. Two members of the PARP family - PARP-1 and PARP-2, which can be activated by DNA damage - are widely considered as regulators of DNA repair processes, including BER. In contrast to PARP-1, the role of PARP-2 in BER has not been extensively studied yet. Since AP site is one of the most frequent type of DNA damage and a key intermediate of BER at the stage preceding formation of DNA breaks, in this paper we focused on the characterization of PARP-2 interaction with AP site-containing DNAs. We demonstrated that PARP-2, like PARP-1, can interact with the intact AP site via Schiff base formation, in spite of crucial difference in the structure of the DNA binding domains of these PARPs. By cross-linking of PARPs to AP DNA, we determined that the N-terminal domains of both PARPs are involved in formation of cross-links with AP DNA. We have also confirmed that DNA binding by PARP-2, in contrast to PARP-1, is not modulated by autoPARylation. PARP-2, like PARP-1, can inhibit the activity of APE1 by binding to AP site, but, in contrast to PARP-1, this inhibitory influence is hardly regulated by PAR synthesis. At the same time, 5'-dRP lyase activity of both PARPs is comparable, although being much weaker than that of Pol ß, which is considered as the main 5'-dRP lyase of the BER process.
Assuntos
DNA/química , Poli(ADP-Ribose) Polimerases/química , Elementos de Resposta , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação ProteicaRESUMO
Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages. Whereas the role of PARP-1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP-2, is less documented. To find out specific DNA targets of PARP-2 we evaluated by EMSA Kd values of PARP-2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes. In addition, we tested these DNA as activators of PARP-1 and PARP-2 in poly(ADP-ribose) synthesis. Like PARP-1, PARP-2 doesn't show correlation between activation efficiency and Kd values for DNA. PARP-2 displayed the highest affinity for flap-containing DNA, but was more efficiently activated by 5'-overhang DNA. Evaluating the influence of PARP-1 and PARP-2 on DNA repair synthesis catalyzed by DNA polymerase ß revealed that both PARPs inhibit DNA polymerase ß activity. However, unlike PARP-1, poly(ADP-ribosyl)ation of PARP-2 does not result in restoration of DNA synthesis efficiency. Similarly, both PARPs proteins inhibited FEN1 activity, but only activation of PARP-1, not PARP-2, could restore FEN1 activity, and only when PARP-2 was not present. Taken together, our data show that PARP-2 can directly regulate BER proteins but also can modulate the influence of PARP-1 on these BER proteins, by decreasing its poly(ADP-ribosyl)ation activity.