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[PMID]:29199984
[Au] Autor:Law A; Stergioulis A; Halavaty AS; Minasov G; Anderson WF; Kuhn ML
[Ad] Endereço:Department of Chemistry and Biochemistry, San Francisco State University, USA.
[Ti] Título:Structure of the Bacillus anthracis dTDP-L-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose reductase (RfbD).
[So] Source:Acta Crystallogr F Struct Biol Commun;73(Pt 12):644-650, 2017 Dec 01.
[Is] ISSN:2053-230X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Bacillus anthracis is the causative agent of the deadly disease Anthrax. Its use in bioterrorism and its ability to re-emerge have brought renewed interest in this organism. B. anthracis is a Gram-positive bacterium that adds L-rhamnose to its cell-wall polysaccharides using the activated donor dTDP-ß-L-rhamnose. The enzymes involved in the biosynthesis of the activated donor are absent in humans, which make them ideal targets for therapeutic development to combat pathogens. Here, the 2.65 Šresolution crystal structure of the fourth enzyme in the dTDP-ß-L-rhamnose-biosynthetic pathway from B. anthracis, dTDP-4-dehydro-ß-L-rhamnose reductase (RfbD), is presented in complex with NADP . This enzyme catalyzes the reduction of dTDP-4-dehydro-ß-L-rhamnose to dTDP-ß-L-rhamnose. Although the protein was co-crystallized in the presence of Mg , the protein lacks the conserved residues that coordinate Mg .
[Mh] Termos MeSH primário: Bacillus anthracis/enzimologia
Proteínas de Bactérias/química
Desidrogenases de Carboidrato/química
Desidrogenases de Carboidrato/metabolismo
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Proteínas de Bactérias/metabolismo
Desidrogenases de Carboidrato/genética
Cristalografia por Raios X
Magnésio/metabolismo
Modelos Moleculares
NADP/química
NADP/metabolismo
Açúcares de Nucleosídeo Difosfato/metabolismo
Conformação Proteica
Multimerização Proteica
Homologia Estrutural de Proteína
Especificidade por Substrato
Nucleotídeos de Timina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Nucleoside Diphosphate Sugars); 0 (Thymine Nucleotides); 2147-59-3 (thymidine diphosphate rhamnose); 53-59-8 (NADP); EC 1.1.- (Carbohydrate Dehydrogenases); EC 1.1.1.133 (dTDP-4-dehydrorhamnose reductase); I38ZP9992A (Magnesium)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180222
[Lr] Data última revisão:
180222
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171205
[St] Status:MEDLINE
[do] DOI:10.1107/S2053230X17015746


  2 / 2800 MEDLINE  
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[PMID]:28862868
[Au] Autor:Alnajjar KS; Negahbani A; Nakhjiri M; Krylov IS; Kashemirov BA; McKenna CE; Goodman MF; Sweasy JB
[Ad] Endereço:Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
[Ti] Título:DNA Polymerase ß Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ß-γ Bridging Group of the Incoming dNTP.
[So] Source:Biochemistry;56(40):5449-5456, 2017 Oct 10.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PP ) basicity. Construction of linear free energy relationship plots for the dependence of log(k ) on leaving group pK revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.
[Mh] Termos MeSH primário: DNA Polimerase beta/genética
DNA Polimerase beta/metabolismo
Desoxirribonucleotídeos/química
Desoxirribonucleotídeos/metabolismo
Mutação
Neoplasias/genética
[Mh] Termos MeSH secundário: DNA Polimerase beta/química
Cinética
Modelos Moleculares
Neoplasias/enzimologia
Ligação Proteica
Conformação Proteica
Especificidade por Substrato
Nucleotídeos de Timina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Deoxyribonucleotides); 0 (Thymine Nucleotides); EC 2.7.7.- (DNA Polymerase beta); QOP4K539MU (thymidine 5'-triphosphate)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171019
[Lr] Data última revisão:
171019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170902
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00713


  3 / 2800 MEDLINE  
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[PMID]:28676274
[Au] Autor:Lisitskiy VA; Khan H; Popova TV; Chubarov AS; Zakharova OD; Akulov AE; Shevelev OB; Zavjalov EL; Koptyug IV; Moshkin MP; Silnikov VN; Ahmad S; Godovikova TS
[Ad] Endereço:Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev Ave. 8, Novosibirsk 630090, Russia.
[Ti] Título:Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics.
[So] Source:Bioorg Med Chem Lett;27(16):3925-3930, 2017 08 15.
[Is] ISSN:1464-3405
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.
[Mh] Termos MeSH primário: Antineoplásicos/farmacologia
Nucleotídeos/química
Albumina Sérica/química
Nucleotídeos de Timina/farmacologia
[Mh] Termos MeSH secundário: Antineoplásicos/síntese química
Antineoplásicos/química
Linhagem Celular Tumoral
Proliferação Celular/efeitos dos fármacos
Relação Dose-Resposta a Droga
Ensaios de Seleção de Medicamentos Antitumorais
Seres Humanos
Concentração de Íons de Hidrogênio
Estrutura Molecular
Oxirredução
Relação Estrutura-Atividade
Nucleotídeos de Timina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Antineoplastic Agents); 0 (Nucleotides); 0 (Serum Albumin); 0 (Thymine Nucleotides); 345-03-9 (5-trifluoromethyl-2'-deoxyuridine 5'-triphosphate)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:171125
[Lr] Data última revisão:
171125
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170706
[St] Status:MEDLINE


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[PMID]:28665588
[Au] Autor:Dunsirn MM; Thoden JB; Gilbert M; Holden HM
[Ad] Endereço:Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.
[Ti] Título:Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis.
[So] Source:Biochemistry;56(29):3818-3825, 2017 Jul 25.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N -formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 10 M s . In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N -formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.
[Mh] Termos MeSH primário: Proteínas de Bactérias/química
Hidroximetil e Formil Transferases/química
Modelos Moleculares
Mycobacterium tuberculosis/enzimologia
Fatores de Virulência/química
[Mh] Termos MeSH secundário: Proteínas de Bactérias/metabolismo
Catálise
Cristalografia por Raios X
Desoxiaçúcares/química
Desoxiaçúcares/metabolismo
Formiltetra-Hidrofolatos/química
Formiltetra-Hidrofolatos/metabolismo
Hidroximetil e Formil Transferases/metabolismo
Cinética
Mycobacterium tuberculosis/patogenicidade
Nucleotídeos de Timina/química
Nucleotídeos de Timina/metabolismo
Fatores de Virulência/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Deoxy Sugars); 0 (Formyltetrahydrofolates); 0 (Thymine Nucleotides); 0 (Virulence Factors); 0 (dTDP-4-amino-4,6-dideoxy-glucose); EC 2.1.2.- (Hydroxymethyl and Formyl Transferases)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171018
[Lr] Data última revisão:
171018
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170701
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00506


  5 / 2800 MEDLINE  
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[PMID]:28428278
[Au] Autor:Jariwala N; Rajasekaran D; Mendoza RG; Shen XN; Siddiq A; Akiel MA; Robertson CL; Subler MA; Windle JJ; Fisher PB; Sanyal AJ; Sarkar D
[Ad] Endereço:Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia.
[Ti] Título:Oncogenic Role of SND1 in Development and Progression of Hepatocellular Carcinoma.
[So] Source:Cancer Res;77(12):3306-3316, 2017 Jun 15.
[Is] ISSN:1538-7445
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). In this study, we show that hepatocyte-specific SND1 transgenic mice (Alb/SND1 mice) develop spontaneous HCC with partial penetrance and exhibit more highly aggressive HCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibited a relative increase in inflammatory markers and spheroid-generating tumor-initiating cells (TIC). Mechanistic investigations defined roles for Akt and NF-κB signaling pathways in promoting TIC formation in Alb/SND1 mice. In human xenograft models of subcutaneous or orthotopic HCC, administration of the selective SND1 inhibitor 3', 5'-deoxythymidine bisphosphate (pdTp), inhibited tumor formation without effects on body weight or liver function. Our work establishes an oncogenic role for SND1 in promoting TIC formation and highlights pdTp as a highly selective SND1 inhibitor as a candidate therapeutic lead to treat advanced HCC. .
[Mh] Termos MeSH primário: Carcinoma Hepatocelular/patologia
Neoplasias Hepáticas/patologia
Células-Tronco Neoplásicas/patologia
Proteínas Nucleares/metabolismo
Proteínas Oncogênicas/metabolismo
[Mh] Termos MeSH secundário: Animais
Antineoplásicos/farmacologia
Western Blotting
Transformação Celular Neoplásica/genética
Modelos Animais de Doenças
Progressão da Doença
Citometria de Fluxo
Imunofluorescência
Seres Humanos
Imuno-Histoquímica
Camundongos
Camundongos Transgênicos
Reação em Cadeia da Polimerase
Nucleotídeos de Timina/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antineoplastic Agents); 0 (Nuclear Proteins); 0 (Oncogene Proteins); 0 (SND1 protein, human); 0 (Snd1 protein, mouse); 0 (Thymine Nucleotides); 2863-04-9 (thymidine 3',5'-diphosphate)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170914
[Lr] Data última revisão:
170914
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170422
[St] Status:MEDLINE
[do] DOI:10.1158/0008-5472.CAN-17-0298


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[PMID]:28290677
[Au] Autor:Wilson KA; Wetmore SD
[Ad] Endereço:Department of Chemistry and Biochemistry, University of Lethbridge , 4401 University Drive West, Lethbridge, Alberta T1K 3M4, Canada.
[Ti] Título:Molecular Insights into the Translesion Synthesis of Benzyl-Guanine from Molecular Dynamics Simulations: Structural Evidence of Mutagenic and Nonmutagenic Replication.
[So] Source:Biochemistry;56(13):1841-1853, 2017 Apr 04.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA can be damaged by many compounds in our environment, and the resulting damaged DNA is commonly replicated by translesion synthesis (TLS) polymerases. Because the mechanism and efficiency of TLS are affected by the type of DNA damage, obtaining information for a variety of DNA adducts is critical. However, there is no structural information for the insertion of a dNTP opposite an O6-dG adduct, which is a particularly harmful class of DNA lesions. We used molecular dynamics (MD) simulations to investigate structural and energetic parameters that dictate preferred dNTP insertion opposite O6-benzyl-guanine (Bz-dG) by DNA polymerase IV, a prototypical TLS polymerase. Specifically, MD simulations were completed on all possible ternary insertion complexes and ternary -1 base deletion complexes with different Bz-dG conformations. Our data suggests that the purines are unlikely to be inserted opposite anti- or syn-Bz-dG, and dTTP is unlikely to be inserted opposite syn-Bz-dG, because of changes in the active site conformation, including critical hydrogen-bonding interactions and/or reaction-ready parameters compared to natural dG replication. In contrast, a preserved active site conformation suggests that dCTP can be inserted opposite either anti- or syn-Bz-dG and dTTP can be inserted opposite anti-Bz-dG. This is the first structural explanation for the experimentally observed preferential insertion of dCTP and misincorporation of dTTP opposite Bz-dG. Furthermore, we provide atomic level insight into why Bz-dG replication does not lead to deletion mutations, which is in contrast with the replication outcomes of other adducts. These findings provide a basis for understanding the replication of related O6-dG adducts.
[Mh] Termos MeSH primário: Compostos de Benzil/síntese química
Adutos de DNA/química
DNA Polimerase beta/química
Reparo do DNA
Replicação do DNA
Nucleotídeos de Desoxiguanina/química
Proteínas de Escherichia coli/química
Guanina/síntese química
[Mh] Termos MeSH secundário: Domínio Catalítico
Dano ao DNA
DNA Polimerase beta/genética
DNA Polimerase beta/metabolismo
Nucleotídeos de Desoxiadenina/química
Nucleotídeos de Desoxiadenina/metabolismo
Nucleotídeos de Desoxicitosina/química
Nucleotídeos de Desoxicitosina/metabolismo
Nucleotídeos de Desoxiguanina/metabolismo
Escherichia coli/química
Escherichia coli/enzimologia
Escherichia coli/genética
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Guanina/análogos & derivados
Ligações de Hidrogênio
Simulação de Dinâmica Molecular
Mutagênese
Estrutura Secundária de Proteína
Estrutura Terciária de Proteína
Nucleotídeos de Timina/química
Nucleotídeos de Timina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Benzyl Compounds); 0 (DNA Adducts); 0 (Deoxyadenine Nucleotides); 0 (Deoxycytosine Nucleotides); 0 (Deoxyguanine Nucleotides); 0 (Escherichia coli Proteins); 0 (Thymine Nucleotides); 2056-98-6 (2'-deoxycytidine 5'-triphosphate); 5Z93L87A1R (Guanine); 8C2O37Y44Q (deoxyguanosine triphosphate); EC 2.7.7.- (DNA Polymerase beta); EC 2.7.7.- (Dpo4 protein, E coli); K8KCC8SH6N (2'-deoxyadenosine triphosphate); QOP4K539MU (thymidine 5'-triphosphate)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170515
[Lr] Data última revisão:
170515
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170315
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b01247


  7 / 2800 MEDLINE  
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[PMID]:28255091
[Au] Autor:Li A; Ziehr JL; Johnson KA
[Ad] Endereço:From the Institute for Cell and Molecular Biology, Molecular Biosciences Department, University of Texas at Austin, Austin, Texas 78712.
[Ti] Título:A new general method for simultaneous fitting of temperature and concentration dependence of reaction rates yields kinetic and thermodynamic parameters for HIV reverse transcriptase specificity.
[So] Source:J Biol Chem;292(16):6695-6702, 2017 Apr 21.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Recent studies have demonstrated the dominant role of induced fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking, and equilibrium thermodynamic methods are of no avail because the key parameters can only be determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37 °C, and the raw data were fit globally to derive a single set of rate constants at 37 °C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding ( , Δ , Δ , and Δ at 37 °C) and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.
[Mh] Termos MeSH primário: Transcriptase Reversa do HIV/antagonistas & inibidores
Inibidores da Transcriptase Reversa/farmacologia
Nucleotídeos de Timina/farmacologia
Zidovudina/farmacologia
[Mh] Termos MeSH secundário: Simulação por Computador
Cumarínicos/farmacologia
DNA/química
Relação Dose-Resposta a Droga
Infecções por HIV/tratamento farmacológico
Íons
Cinética
Magnésio/química
Simulação de Dinâmica Molecular
Mutação
Nucleotídeos/química
Nucleotídeos/genética
Conformação Proteica
Software
Especificidade por Substrato
Temperatura Ambiente
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Coumarins); 0 (Ions); 0 (Nucleotides); 0 (Reverse Transcriptase Inhibitors); 0 (Thymine Nucleotides); 156571-46-9 (N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide); 4B9XT59T7S (Zidovudine); 9007-49-2 (DNA); EC 2.7.7.49 (HIV Reverse Transcriptase); I38ZP9992A (Magnesium); QOP4K539MU (thymidine 5'-triphosphate)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170621
[Lr] Data última revisão:
170621
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170304
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M116.760827


  8 / 2800 MEDLINE  
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[PMID]:27936595
[Au] Autor:Achuthan V; Singh K; DeStefano JJ
[Ad] Endereço:Cell Biology and Molecular Genetics, University of Maryland , College Park, Maryland 20742, United States.
[Ti] Título:Physiological Mg Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro.
[So] Source:Biochemistry;56(1):33-46, 2017 Jan 10.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg , whereas the free Mg concentration in cells is much lower. Artificially high Mg concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg . At low concentrations of Mg , NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg , whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on k /K ) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC values) more effective at low than at high Mg concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg -dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg conditions yielded results dramatically different from those from measurements using commonly employed high-Mg in vitro conditions. These results also emphasize differences in Mg sensitivity between the translocation inhibitor EFdATP and other NRTIs.
[Mh] Termos MeSH primário: Didesoxinucleotídeos/farmacologia
Transcriptase Reversa do HIV/antagonistas & inibidores
Magnésio/farmacologia
Nucleosídeos/farmacologia
Inibidores da Transcriptase Reversa/farmacologia
[Mh] Termos MeSH secundário: Nucleotídeos de Desoxicitosina/farmacologia
Nucleotídeos de Desoxiguanina/farmacologia
Relação Dose-Resposta a Droga
Interações Medicamentosas
Eletroforese em Gel de Poliacrilamida
Transcriptase Reversa do HIV/genética
Transcriptase Reversa do HIV/metabolismo
Seres Humanos
Cinética
Mutação
Nucleotídeos de Timina/farmacologia
Zalcitabina/farmacologia
Zidovudina/análogos & derivados
Zidovudina/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Deoxycytosine Nucleotides); 0 (Deoxyguanine Nucleotides); 0 (Dideoxynucleotides); 0 (Nucleosides); 0 (Reverse Transcriptase Inhibitors); 0 (Thymine Nucleotides); 4B9XT59T7S (Zidovudine); 66004-77-1 (2',3'-dideoxycytidine 5'-triphosphate); 68726-28-3 (2',3'-dideoxyguanosine 5'-triphosphate); 6L3XT8CB3I (Zalcitabine); 6RGF96R053 (3'-azido-3'-deoxythymidine 5'-triphosphate); EC 2.7.7.- (reverse transcriptase, Human immunodeficiency virus 1); EC 2.7.7.- (reverse transcriptase, Human immunodeficiency virus 2); EC 2.7.7.49 (HIV Reverse Transcriptase); I38ZP9992A (Magnesium)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170602
[Lr] Data última revisão:
170602
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161213
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b00943


  9 / 2800 MEDLINE  
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[PMID]:27557296
[Au] Autor:Chen X; McAllister KJ; Klein B; Bushman LR; Anderson PL
[Ad] Endereço:University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, USA.
[Ti] Título:Development and validation of an LC-MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate.
[So] Source:Biomed Chromatogr;31(3), 2017 Mar.
[Is] ISSN:1099-0801
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy - the separation of mono-, di- and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides - followed by sensitive quantitation using liquid chromatography-tandem mass spectrometry. The validated analytical range was 50-2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells from 40 clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP and 315 (220, 456) for TTP, in femtomoles per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites or nucleos(t)ide analogs, or for other clinical scenarios.
[Mh] Termos MeSH primário: Cromatografia Líquida/métodos
Nucleotídeos de Desoxiadenina/análise
Nucleotídeos de Desoxicitosina/análise
Espectrometria de Massas em Tandem/métodos
Nucleotídeos de Timina/análise
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Deoxyadenine Nucleotides); 0 (Deoxycytosine Nucleotides); 0 (Thymine Nucleotides); 2056-98-6 (2'-deoxycytidine 5'-triphosphate); K8KCC8SH6N (2'-deoxyadenosine triphosphate); QOP4K539MU (thymidine 5'-triphosphate)
[Em] Mês de entrada:1703
[Cu] Atualização por classe:170308
[Lr] Data última revisão:
170308
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160825
[St] Status:MEDLINE
[do] DOI:10.1002/bmc.3820


  10 / 2800 MEDLINE  
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[PMID]:27694439
[Au] Autor:Patra A; Zhang Q; Guengerich FP; Egli M
[Ad] Endereço:From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
[Ti] Título:Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η.
[So] Source:J Biol Chem;291(46):24304-24313, 2016 Nov 11.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:O -Methyl-2'-deoxyguanosine (O -MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O -MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O -MeG and 2"-deoxythymidine-5"-[(α, ß)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O - MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O -MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O -MeG:dC or O -MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η.
[Mh] Termos MeSH primário: DNA Polimerase Dirigida por DNA/química
DNA/química
Nucleotídeos de Desoxicitosina/química
Nucleotídeos de Timina/química
[Mh] Termos MeSH secundário: Proteínas de Bactérias/química
Proteínas de Bactérias/metabolismo
Cristalografia por Raios X
DNA/biossíntese
DNA Polimerase beta/química
DNA Polimerase beta/metabolismo
DNA Polimerase Dirigida por DNA/metabolismo
Nucleotídeos de Desoxicitosina/metabolismo
Seres Humanos
Sulfolobus solfataricus/enzimologia
Nucleotídeos de Timina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Deoxycytosine Nucleotides); 0 (Thymine Nucleotides); 2056-98-6 (2'-deoxycytidine 5'-triphosphate); 9007-49-2 (DNA); EC 2.7.7.- (DNA Polymerase beta); EC 2.7.7.7 (DNA polymerase N, human); EC 2.7.7.7 (DNA-Directed DNA Polymerase); QOP4K539MU (thymidine 5'-triphosphate)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171111
[Lr] Data última revisão:
171111
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161004
[St] Status:MEDLINE



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