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1.
Org Biomol Chem ; 22(15): 2963-2967, 2024 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-38529657

RESUMO

A type of modified nucleotide, deoxynucleotide γ-amidotriphosphates (dNTPγNH2s), exhibited around five times higher stability than dNTPs. These phosphamide nucleotides can be utilized by several DNA polymerases, and the amplification of a 10 kb DNA fragment through the polymerase chain reaction (PCR) can be accomplished even under conditions of high temperature, extended storage, or repeated freeze-thaw cycles. However, the control PCR with standard dNTPs was unsuccessful. These results indicate that dNTPγNH2s have the potential to substitute dNTPs in PCR.


Assuntos
DNA , Dimetoato , DNA Polimerase Dirigida por DNA , Nucleotídeos/genética
2.
Nature ; 554(7692): 387-391, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29414941

RESUMO

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.


Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA/química , DNA/metabolismo , Sítios de Ligação , Proliferação de Células , Ilhas de CpG/genética , Cristalografia por Raios X , DNA/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Nucleic Acids Res ; 47(18): 9495-9501, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504784

RESUMO

We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.


Assuntos
DNA Polimerase I/genética , Replicação do DNA/genética , DNA/biossíntese , Nucleotídeos/genética , DNA/genética , Nucleotídeos de Desoxicitosina/genética , Nucleotídeos de Desoxiguanina/genética , Cinética , Temperatura
4.
Nucleic Acids Res ; 45(20): 11515-11524, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29036687

RESUMO

Nucleotide quality surveillance enzymes play important roles in human health, by detecting damaged molecules in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or adversely affect metabolism. In particular, deamination of adenine moiety in (deoxy)nucleoside triphosphates, resulting in formation of (d)ITP, can be deleterious, leading to DNA damage, mutagenesis and other harmful cellular effects. The 21.5 kDa human enzyme that mitigates this damage by conversion of (d)ITP to monophosphate, ITPA, has been proposed as a possible therapeutic and diagnostic target for multiple diseases. Measuring the activity of this enzyme is useful both in basic research and in clinical applications involving this pathway, but current methods are nonselective and are not applicable to measurement of the enzyme from cells or tissues. Here, we describe the design and synthesis of an ITPA-specific chimeric dinucleotide (DIAL) that replaces the pyrophosphate leaving group of the native substrate with adenosine triphosphate, enabling sensitive detection via luciferase luminescence signaling. The probe is shown to function sensitively and selectively to quantify enzyme activity in vitro, and can be used to measure the activity of ITPA in bacterial, yeast and human cell lysates.


Assuntos
Trifosfato de Adenosina/química , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Inosina Monofosfato/análogos & derivados , Medições Luminescentes/métodos , Pirofosfatases/metabolismo , Extratos Celulares/química , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , Desaminação , Células HeLa , Humanos , Inosina Monofosfato/química , Pirofosfatases/genética , Interferência de RNA , RNA Interferente Pequeno/genética
5.
Nucleic Acids Res ; 45(12): 7276-7284, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28531304

RESUMO

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/genética , Desoxiadenosinas/metabolismo , Desoxicitidina/análogos & derivados , Timidina/análogos & derivados , Sequência de Bases , Sistemas CRISPR-Cas , DNA/metabolismo , Adutos de DNA/genética , Adutos de DNA/metabolismo , Dano ao DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxicitidina/metabolismo , Edição de Genes , Células HEK293 , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Timidina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
J Am Chem Soc ; 140(6): 2105-2114, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29376367

RESUMO

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 µM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 µM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 µM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , DNA Glicosilases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células CACO-2 , DNA Glicosilases/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo
7.
Bioconjug Chem ; 29(5): 1614-1621, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29578692

RESUMO

Nucleotide surveillance enzymes play important roles in human health, by monitoring damaged monomers in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or disrupt nucleotide metabolism. In particular, deamination of cytosine, leading to uracil in DNA and in the nucleotide pool, can be deleterious, causing DNA damage. The enzyme deoxyuridine triphosphatase (dUTPase) is currently under study as a therapeutic and prognostic target for cancer. Measuring the activity of this enzyme is important both in basic research and in clinical applications involving this pathway, but current methods are nonselective, detecting pyrophosphate, which is produced by many enzymes. Here we describe the design and synthesis of a dUTPase enzyme-specific chimeric dinucleotide (DUAL) that replaces the pyrophosphate leaving group of the native substrate with ATP, enabling sensitive detection via luciferase luminescence signaling. The DUAL probe functions sensitively and selectively to quantify enzyme activities in vitro and in cell lysates. We further report the first measurements of dUTPase activities in eight different cell lines, which are found to vary by a factor of 7-fold. We expect that the new probe can be of considerable utility in research involving this clinically significant enzyme.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Substâncias Luminescentes/química , Nucleotídeos/química , Pirofosfatases/análise , Uridina Trifosfato/análogos & derivados , Linhagem Celular Tumoral , Ensaios Enzimáticos/métodos , Células HEK293 , Humanos , Medições Luminescentes/métodos , Especificidade por Substrato
8.
J Am Chem Soc ; 138(29): 9005-8, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27413803

RESUMO

The enzyme MTH1 cleanses the cellular nucleotide pool of oxidatively damaged 8-oxo-dGTP, preventing mutagenesis by this nucleotide. The enzyme is considered a promising therapeutic target; however, methods to measure its activity are indirect and laborious and have low sensitivity. Here we describe a novel ATP-linked chimeric nucleotide (ARGO) that enables luminescence signaling of the enzymatic reaction, greatly simplifying the measurement of MTH1 activity. We show that the reporting system can be used to identify inhibitors of MTH1, and we use it to quantify enzyme activity in eight cell lines and in colorectal tumor tissue. The ARGO reporter is likely to have considerable utility in the study of the biology of MTH1 and potentially in analyzing patient samples during clinical testing.


Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias Colorretais/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Medições Luminescentes/métodos , Terapia de Alvo Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Humanos
9.
Angew Chem Int Ed Engl ; 55(6): 2087-91, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26836342

RESUMO

A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP-releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA/biossíntese , Luciferases/metabolismo , Nucleotídeos/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/química , DNA/química , Luciferases/química , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA/química , RNA/metabolismo
10.
J Am Chem Soc ; 136(33): 11582-5, 2014 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-25073028

RESUMO

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells. Moreover, 5-hmrC was present at appreciable levels (∼1 per 5000 5-methylcytidine) in RNA of mammalian cells and tissues. Our results suggest the involvement of this oxidation in RNA biology.


Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Citosina/biossíntese , Citosina/química , Citosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Dioxigenases/química , Dioxigenases/deficiência , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Oxigenases de Função Mista , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , RNA/química
11.
Anal Chem ; 86(9): 4550-8, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24689502

RESUMO

Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate ((S)GTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of (S)GTP to a GTP-binding protein, Rac1. However, it remains unclear whether (S)GTP can also bind to other cellular proteins. Here, we introduced an orthogonal approach, encompassing nucleotide-affinity profiling and nucleotide-binding competition assays, to characterize comprehensively (S)GTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of (S)GTP and GTP affinity probes, we identified 165 (S)GTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward (S)GTP and GTP, which allowed for the revelation of the relative binding affinities of the two nucleotides toward the nucleotide-binding motif sequence of proteins. Our results suggest that (S)GTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that (S)GTP binds to several cyclin-dependent kinases (CDKs), which may perturb the CDK-mediated phosphorylation and cell cycle progression. Together, this represents the first comprehensive characterization of (S)GTP-binding property for the entire human proteome. We reason that a similar strategy can be generally employed for the future characterization of the interaction of other modified nucleotides with the global proteome.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Marcadores de Afinidade , Ligação Competitiva , Proteínas de Ligação ao GTP/química , Humanos , Proteoma
12.
Chem Res Toxicol ; 27(7): 1304-9, 2014 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-24979327

RESUMO

Recently 5-hydroxymethyl-2'-deoxycytidine (5hmdC), 5-formyl-2'-deoxycytidine (5fdC), and 5-carboxyl-2'-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2'-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells. Here, we examined the effects of the three modified nucleosides on the efficiency and accuracy of DNA replication in HEK293T human kidney epithelial cells. Our results demonstrated that a single, site-specifically incorporated 5fdC or 5cadC conferred modest drops, by approximately 30%, in replication bypass efficiency without inducing detectable mutations in human cells, whereas replicative bypass of 5hmdC is both accurate and efficient. The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/farmacologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Células HEK293 , Humanos , Oligodesoxirribonucleotídeos , Oxirredução , Proteínas Proto-Oncogênicas/metabolismo
13.
Bioorg Med Chem Lett ; 24(5): 1307-9, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24513047

RESUMO

An activity screening between 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide (NAD) analogs and malic enzyme (ME) mutants identified some mutants capable of taking NAD analogs as the cofactor. One particular pair, ME-L310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. The new system gained about 1200-fold cofactor specificity shift from NAD toward B-8 in terms of oxidative decarboxylation of l-malate. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathways.


Assuntos
Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , NAD/análogos & derivados , Triazóis/química , Biocatálise , Descarboxilação , Cinética , Malato Desidrogenase/química , Malatos/química , Malatos/metabolismo , Mutação , NAD/metabolismo , Oxirredução , Ligação Proteica
14.
Dalton Trans ; 53(11): 5020-5033, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38294042

RESUMO

Due to the radioactivity of uranium, the discharged nuclear wastewater not only causes certain damage to the ecology, but also causes certain harm to human life and health. Adsorption is considered to be one of the most effective ways to remove uranium. In this paper, a kind of MoS2 adsorbent was prepared by the solid phase synthesis method and functionalized with NiCo-LDH. The raw materials of MoS2 are cheap and easy to obtain, and the preparation conditions are simple, and large quantities can be obtained without limitations. MoS2 functionalized with NiCo-LDH provides more adsorption sites for the adsorbent and at the same time improves the hydrophilicity of the adsorbent, so that the active sites can fully combine with uranyl ions. The maximum adsorption capacity of the Langmuir isothermal adsorption model is 492.83 mg g-1. The selective adsorption capacity of uranium can reach 76.12% in the multi-ion coexistence system. By analyzing the adsorption mechanism with FT-IR and XRD, it is believed that on the one hand, UO22+ forms a covalent bond with Mo in MoS2 and coordinates with S on the surface of MoS2. On the other hand, UO22+ enters the NiCo-LDH layer for ion exchange with NO3- and coordinates with -OH on the surface of NiCo-LDH. The successful preparation of the MoS2/NiCo-LDH composite provides a certain application prospect for the uranium adsorption field.

15.
Dalton Trans ; 52(29): 10136-10144, 2023 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-37431306

RESUMO

The toxicity and radioactivity of uranium (U)-containing wastewater pose a serious threat to the environment of humans, animals, and plants. It is necessary to remove U from contaminated wastewater. With high adsorption capacity and fast adsorption rate, a composite CNT-P/HAP, which comprises carbon nanotubes (CNT) modified with polyethyleneimine (PEI), was functionalized further by hydroxyapatite (HAP) using the hydrothermal method. Adsorption experiments indicated that the optimal performance for CNT-P/HAP was 1330.64 mg g-1 of adsorption capacity and 40 min of adsorption equilibrium at a pH of 3. In addition, the adsorption capacity of CNT-P/HAP was over 2 times that of HAP at a pH of 7. The synergistic effect in both synthesis and adsorption gave CNT-P/HAP an excellent adsorption capacity for U. The XRD and FT-IR analysis indicated that the adsorption mechanism of CNT-P/HAP for U is decided by the pH of the solution. CNT-P/HAP could be used in multiple conditions to remediate U-containing wastewater.

16.
Adv Healthc Mater ; 12(25): e2300503, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37306493

RESUMO

Photodynamic therapy (PDT) uses photosensitizers to convert oxygen (O2 ) to reactive oxygen species (ROS) under irradiation to induce DNA damage and kill cancer cells. However, the effect of PDT is usually alleviated by apoptosis resistance mechanism of tumor living cells. MTH1 enzyme is known to be such an apoptosis-resistance enzyme which is over expressed as a scavenger to repair the damaged DNA. In this work, a hypoxia-activated nanosystem FTPA, which can be degraded to release the encapsulated PDT photosensitizer 4-DCF-MPYM and an inhibitor TH588 is proposed. The inhibitor TH588 can inhibit the DNA repair process by reducing the activity of MTH1 enzyme, and achieve the purpose of amplifying the therapeutic effect of PDT. This work demonstrates that a precise and augmented tumor PDT is achieved by integration of hypoxia-activation and inhibition resistance of tumor cells to apoptosis.


Assuntos
Fotoquimioterapia , Humanos , Linhagem Celular Tumoral , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Oxigênio , Hipóxia/tratamento farmacológico , Apoptose
17.
ACS Chem Biol ; 17(8): 2074-2087, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35830623

RESUMO

Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 µM and 410 ± 60% at 5 µM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Neoplasias , Monoéster Fosfórico Hidrolases/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Carcinogênese , DNA , Dano ao DNA , Humanos , Nucleotídeos , Estresse Oxidativo
18.
J Am Chem Soc ; 133(51): 20857-62, 2011 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-22098020

RESUMO

Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.


Assuntos
Bactérias/enzimologia , Flucitosina/metabolismo , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Niacinamida/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Bactérias/química , Bactérias/genética , Descarboxilação , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Flucitosina/química , Lactobacillus helveticus/química , Lactobacillus helveticus/enzimologia , Lactobacillus helveticus/genética , Malato Desidrogenase/química , Malato Desidrogenase/genética , Dados de Sequência Molecular , Mutação , Niacinamida/química , Nucleotídeos/química , Nucleotídeos/metabolismo , Oxirredução , Oxirredutases/química , Oxirredutases/genética
19.
Bioorg Med Chem Lett ; 21(6): 1667-9, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21324682

RESUMO

Eleven triazole moiety-containing nucleotide analogs were synthesized starting form tetra-O-acetylribose in 55-63% total yields. The synthesis involved two key steps, the lipase-mediated selective deacylation of 1-azido-2,3,5-tri-O-acetyl-ß-D-ribofuranoside and the Huisgen 1,3-dipolar cycloaddition between terminal alkynes and the 1-azido ribofuranoside derivative. These analogs showed inhibitory effects against a recombinant Escherichia coli NAD-dependent malic enzyme.


Assuntos
Inibidores Enzimáticos/síntese química , Malato Desidrogenase/antagonistas & inibidores , Nucleotídeos/síntese química , Triazóis/química , Escherichia coli/efeitos dos fármacos
20.
RSC Adv ; 11(39): 24027-24031, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35479041

RESUMO

Determination of aflatoxin B1 (AFB1) is still a big issue in food safety. In this paper, we developed a luminescence AFB1 detection method combined with ATP-releasing nucleotides (ARNs) and AFB1 aptamer. Firstly, using a new coupling method, we synthesized two ARNs (dTP4A and dGP4A) in a yield of 67% and 58%, respectively. The newly prepared ARNs show a much lower background. Then, we developed a new isothermal polymerase amplification method. In this method, two DNA hairpins were used to substitute the circle DNA template in rolling circle amplification. Using this amplification method and combined with AFB1 aptamer, a new AFB1 detection method is developed. A detection limit as low as 0.3 pM is achieved. This method is simple and efficient, and will have a great potential to be used for food safety and public health.

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