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1.
Sci Rep ; 10(1): 611, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953472

RESUMO

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.


Assuntos
Química Click/métodos , Cobre/química , Desoxirribonucleotídeos/análise , Nucleotídeos de Desoxiuracil/química , Reação de Cicloadição , Nucleotídeos de Desoxiadenina/análise , Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxicitosina/análise , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxiguanina/análise , Nucleotídeos de Desoxiguanina/química , Desoxirribonucleotídeos/química , Células HCT116 , Células HEK293 , Humanos , Células K562 , Rodaminas/química , Coloração e Rotulagem , Nucleotídeos de Timina/análise , Nucleotídeos de Timina/química
2.
Talanta ; 205: 120120, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450426

RESUMO

Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40 µL). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [M - H]- ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4 °C in ammonium acetate buffer), after 20 uses and more than 750 min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5 min), efficient (conversion efficiency > 55%), precise and repeatable (CV < 3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).


Assuntos
Reatores Biológicos , Enzimas Imobilizadas/química , Núcleosídeo-Fosfato Quinase/química , Organofosfonatos/química , Timidina Monofosfato/química , Nucleotídeos de Timina/química , Humanos , Espectrometria de Massas/métodos , Fosforilação , Estudo de Prova de Conceito , Vírus Vaccinia/enzimologia
3.
Biochemistry ; 58(6): 697-705, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30571104

RESUMO

Proteins forming dimers or larger complexes can be strongly influenced by their effector-binding status. We investigated how the effector-binding event is coupled with interface formation via computer simulations, and we quantified the correlation of two types of contact interactions: between the effector and its binding pocket and between protein monomers. This was achieved by connecting the protein dynamics at the monomeric level with the oligomer interface information. We applied this method to ribonucleotide reductase (RNR), an essential enzyme for de novo DNA synthesis. RNR contains two important allosteric sites, the s-site (specificity site) and the a-site (activity site), which bind different effectors. We studied these different binding states with atomistic simulation and used their coarse-grained contact information to analyze the protein dynamics. The results reveal that the effector-protein dynamics at the s-site and dimer interface formation are positively coupled. We further quantify the resonance level between these two events, which can be applied to other similar systems. At the a-site, different effector-binding states (ATP vs dATP) drastically alter the protein dynamics and affect the activity of the enzyme. On the basis of these results, we propose a new mechanism of how the a-site regulates enzyme activation.


Assuntos
Ribonucleotídeo Redutases/metabolismo , Nucleotídeos de Timina/metabolismo , Regulação Alostérica/fisiologia , Sítio Alostérico , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Multimerização Proteica/fisiologia , Ribonucleotídeo Redutases/química , Nucleotídeos de Timina/química
4.
Anal Bioanal Chem ; 410(21): 5245-5253, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29947896

RESUMO

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. Graphical abstract ᅟ.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Complexos de Coordenação/química , Portadores de Fármacos/química , Estruturas Metalorgânicas/química , Nanopartículas/química , Zidovudina/administração & dosagem , Fármacos Anti-HIV/química , Antimetabólitos/administração & dosagem , Antimetabólitos/química , Didesoxinucleotídeos/administração & dosagem , Didesoxinucleotídeos/química , Difusão Dinâmica da Luz , Fracionamento por Campo e Fluxo , Modelos Moleculares , Tamanho da Partícula , Refratometria , Espectrofotometria Ultravioleta , Nucleotídeos de Timina/administração & dosagem , Nucleotídeos de Timina/química , Zidovudina/análogos & derivados , Zidovudina/química
5.
J Org Chem ; 83(15): 8353-8363, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29952565

RESUMO

Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (PzO)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that PzO recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring PzO as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.


Assuntos
Primers do DNA/genética , Piridazinas/química , Nucleotídeos de Timina/química , Pareamento de Bases , Primers do DNA/metabolismo , Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Eletricidade Estática , Nucleotídeos de Timina/metabolismo
6.
J Am Chem Soc ; 140(18): 5886-5889, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29489347

RESUMO

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.


Assuntos
Anticorpos/química , DNA de Cadeia Simples/química , Metiltransferases/análise , Selênio/química , Nucleotídeos de Timina/química , Anticorpos/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Metiltransferases/metabolismo , Selênio/metabolismo , Nucleotídeos de Timina/metabolismo
7.
Biochemistry ; 57(22): 3130-3133, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29473739

RESUMO

DesII is a radical SAM lyase that catalyzes a deamination reaction during the biosynthesis of desosamine in Streptomyces venezuelae. Competing mechanistic hypotheses for this radical-mediated reaction are differentiated according to whether a 1,2-migration takes place and the timing of proton abstraction following generation of a substrate α-hydroxyalkyl radical intermediate. In this study, the deuterated C4 epimer of the natural substrate, TDP-4-amino-4-deoxy-d-[3-2H]fucose, was prepared and shown to be a substrate for DesII undergoing deamination alone with a specific activity that is only marginally reduced (∼3-fold) with respect to that of deamination of the natural substrate. Furthermore, pH titration of the deamination reaction implicates the presence of a hydron acceptor that facilitates catalysis but does not appear to be necessary. On the basis of these as well as previously reported results, a mechanism involving direct elimination of ammonium with concerted proton transfer to the nucleofuge from the adjacent α-hydroxyalkyl radical is proposed.


Assuntos
Fucose/química , Açúcares de Nucleosídeo Difosfato/química , Amino Açúcares , Compostos de Amônio/metabolismo , Catálise , Desaminação , Fucose/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Oxirredutases/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologia , Nucleotídeos de Timina/química
8.
J Struct Biol ; 202(2): 175-181, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29331609

RESUMO

Many bacteria require l-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-l-rhamnose, which is produced by the dTDP-l-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-α-d-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.


Assuntos
Bacillus anthracis/enzimologia , Hidroliases/química , Açúcares de Nucleosídeo Difosfato/química , Conformação Proteica , Nucleotídeos de Timina/química , Bacillus anthracis/patogenicidade , Vias Biossintéticas/genética , Carboidratos Epimerases/química , Cristalografia por Raios X
9.
Bioorg Med Chem Lett ; 27(16): 3925-3930, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28676274

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 19F 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.


Assuntos
Antineoplásicos/farmacologia , Nucleotídeos/química , Albumina Sérica/química , Nucleotídeos de Timina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Oxirredução , Relação Estrutura-Atividade , Nucleotídeos de Timina/química
10.
Biochemistry ; 56(29): 3818-3825, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28665588

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 N10-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 104 M-1 s-1. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N5-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.


Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Fatores de Virulência/química , 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
11.
Biochemistry ; 56(13): 1841-1853, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28290677

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.


Assuntos
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 , 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 , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
12.
Sci Rep ; 7: 43904, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28272441

RESUMO

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Desoxiadenosinas/metabolismo , Pareamento de Bases , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA Polimerase Dirigida por DNA/química , Desoxiadenosinas/química , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/metabolismo , Humanos , Cinética , Estrutura Quaternária de Proteína , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
13.
Glycobiology ; 27(4): 358-369, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28096310

RESUMO

The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.


Assuntos
Aciltransferases/genética , Campylobacter jejuni/genética , Galactose/genética , Glicosiltransferases/genética , Nucleotidiltransferases/genética , Aciltransferases/química , Aciltransferases/metabolismo , Vias Biossintéticas/genética , Campylobacter jejuni/enzimologia , Cristalografia por Raios X , Escherichia coli/genética , Galactose/química , Galactose/metabolismo , Glucose/química , Glucose/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
14.
J Biol Chem ; 291(46): 24304-24313, 2016 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-27694439

RESUMO

O6-Methyl-2'-deoxyguanosine (O6-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 O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-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 O6- 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 O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-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.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/química , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Timina/química , 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 , Humanos , Sulfolobus solfataricus/enzimologia , Nucleotídeos de Timina/metabolismo
15.
Curr Pharm Biotechnol ; 17(12): 1089-1099, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27633891

RESUMO

Glucose-1-Phosphate Thymidylyltransferase (RmlA) is one of the enzymes in rhamnose biosynthesis pathway, where rhamnose acts as linker of peptidoglycan and arabinogalacton in the cell wall, therefore RmlA is a potential enzyme for the survival of Mycobacterium tuberculosis (Mtb). To go into the depth of the structure for exploring binding regions, homology model of RmlA was built in Prime, Schrodinger v9.2. The model with lowest Discrete Optimized Potential Energy (DOPE) score of -35524.17 kcal/mol and RMSD of 0.1 Å with the template (1H5R_B) was subjected to Molecular Dynamics Simulation (MDS) for 5 ns to achieve its stable folding state. The tertiary structure of the proposed model is composed of α/ß/α sandwich type protein with quasi-Rossmann type folding pattern. The substrate, deoxy Thymidine tri phosphate (dTTP) comprises of triphosphate (R1) and methyl (R2) side chains where, R1 is highly essential for the survival of Mtb. Therefore, nineteen side chain analogues of dTTP were designed by substituting R1 and R2 chain of dTTP using Combi Glide, Schrodinger v9.2 and docked with the target RmlA protein. Out of which two analogues such as, 6-[(2R,3S,5R)-5-[5-(2- aminoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]-3-hydroxyoxolan-2 yl] hexanoic acid (COMP- 11) and 4-(2-{1-[(1S,3S,4S)-3-(5-carboxypentyl)-4-hydroxy-2-methylidenecyclopentyl]-2,4-dioxo- 1,2,3,4-tetrahydropyrimidin-5-yl}ethyl)morpholin-4-ium (COMP-12) showed the highest GLIDE score (-12.55 Kcal/mol and -11.58 Kcal/mol respectively) than that of substrate (-9.725 Kcal/mol). During simulations, hydrogen bonding profile between the two top hits and protein ranges up to 5 strong polar contacts which were much stronger than that of substrate. Similarly, the computational binding free energy of both the analogues was found to be less than -70 Kcal/mol which is much lower than that of substrate (-52.84 Kcal/mol). All these results suggest that these two compounds have more stable interaction than that of substrate inside the solvent condition and can be used as competitive inhibitors.


Assuntos
Mycobacterium tuberculosis/metabolismo , Nucleotidiltransferases/química , Ligantes , Simulação de Dinâmica Molecular , Nucleotídeos de Timina/química
16.
Int J Biol Macromol ; 88: 565-71, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27044348

RESUMO

Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.


Assuntos
Brugia Malayi/enzimologia , Filariose Linfática/enzimologia , Núcleosídeo-Fosfato Quinase/química , Proteínas Recombinantes/química , Animais , Brugia Malayi/patogenicidade , Domínio Catalítico , Dimerização , Filariose Linfática/tratamento farmacológico , Filariose Linfática/parasitologia , Humanos , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Nucleotídeos de Timina/química
17.
Angew Chem Int Ed Engl ; 55(17): 5255-8, 2016 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-27008042

RESUMO

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV/efeitos dos fármacos , Nucleosídeos/farmacologia , Polifosfatos/farmacologia , Pró-Fármacos/farmacologia , Nucleotídeos de Timina/farmacologia , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Permeabilidade da Membrana Celular , Esterases/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Nucleosídeos/química , Nucleosídeos/metabolismo , Nucleosídeos/farmacocinética , Polifosfatos/química , Polifosfatos/metabolismo , Polifosfatos/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Suínos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
18.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 3): 224-33, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26919527

RESUMO

Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme.


Assuntos
Proteínas de Bactérias/química , Salmonella typhimurium/enzimologia , Timidina Fosforilase/química , Nucleotídeos de Timina/química , Uridina/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Ligantes , Ligação Proteica , Especificidade por Substrato
19.
Nucleic Acids Res ; 44(5): 2310-22, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26850643

RESUMO

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.


Assuntos
Farmacorresistência Viral Múltipla/genética , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Clonagem Molecular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/química , Zidovudina/farmacologia
20.
Anal Biochem ; 498: 53-8, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26778528

RESUMO

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.


Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Hidroliases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Antituberculosos/química , Bioensaio , Inibidores Enzimáticos/química , Glucose/análogos & derivados , Glucose/química , Glucose/farmacologia , Hidroliases/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia
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