Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

Ligasová, Anna; Liboska, Radek; Friedecký, David; Mičová, Kateřina; Adam, Tomáš; Oždian, Tomáš; Rosenberg, Ivan; Koberna, Karel

doi:10.1098/rsob.150172

Cited by 20 publications

(26 citation statements)

References 40 publications

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“…To reduce or abrogate potential issues of growth inhibition with EdU and EdC toward T. cruzi, the incubation time with these probes was reduced to 2 and 4 hours, which corresponded to no observed inhibitory activity against T. cruzi intracellular amastigotes. In these studies, EdC also exhibited toxicity toward T. cruzi, perhaps as deamination of EdC monophosphate leads to the production of EdU [34]. Due to the amount of F-ara-EdU needed to show incorporation following 48 hours incubation, this compound was not investigated over a shorter exposure period.…”

Section: Discussionmentioning

confidence: 99%

“…Due to the amount of F-ara-EdU needed to show incorporation following 48 hours incubation, this compound was not investigated over a shorter exposure period. The incubation time was reduced for EdU as a DNA probe to detect replication in mammalian cells in a similar effort to reduce the toxic effects of this compound [34]. The percentage level of incorporation of EdU into parasites following 2 hours exposure was increased in comparison to a report utilising cancer cell lines [23].…”

Section: Discussionmentioning

confidence: 99%

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Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

Sykes¹,

Hilko²,

Kung³

et al. 2020

PLoS Negl Trop Dis

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Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2 0 -deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 μM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

Sykes¹,

Hilko²,

Kung³

et al. 2020

PLoS Negl Trop Dis

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“…One week before the experiment, the culture medium was exchanged for a HAT-free medium. For more details see the study by Ligasová et al [108].…”

Section: Cell Culturesmentioning

confidence: 99%

“…The sample preparation of the cells treated with EdU or EdC is described previously in the study by Ligasová et al [108]. Briefly, cells cultivated in a medium with or without nucleoside analogue for a definite time were immediately vacuum-filtered.…”

Section: Sample Preparationmentioning

confidence: 99%

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Mass Spectrometry for the Sensitive Analysis of Intracellular Nucleotides and Analogues

Mičová¹,

Friedecký²,

Adam³

2017

Mass Spectrometry

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Nowadays, mass spectrometry is very important and widely applied tool in nucleotide analysis. As a result of technological advances in sample preparation, separation and mass spectrometry detection, the developed methods allow sensitive and selective measurement of polar compounds occurring in low levels in various biological matrices. This enables more potential uses in clinical field. Direct methods require no special sample pre-treatment before analysis in contrast to indirect methods, where fractionation, dephosphorylation and purification are needed. The use of ion-pairing agent, ion exchange chromatography with pH gradient, porous graphitic carbon columns and HILIC in liquid chromatography represents the most common methods of nucleotide analysis. High separation efficiency is also achieved with the use of CE with MS detection. Analysis of nucleotides was also described by the means of MALDI-TOF, but poor reproducibility and lack of applications make a limitation for this approach. The chapter summarizes different techniques and approaches for determination of endogenous nucleotides and its analogues in various clinical applications. or deoxyribose) through a glycosidic bond. Nucleotides are formed by phosphorylation of nucleoside molecule. They represent a fundamental building block of the structure of nucleic acids formed by the polymerase-mediated synthesis of DNA and RNA from deoxynucleotides and nucleotides, respectively. All deoxynucleotides are synthesized from the corresponding nucleotides. Nucleotides play another important role in enzyme activation and metabolism. They are incorporated into important cofactors of enzymatic reactions such as transfer of various groups by coenzyme A (e.g. acetyl-CoA and succinyl-CoA) in many metabolic processes, NAD, NADP and FAD as coenzymes of oxidoreductases catalyses redox reactions in the cell. ATP and other nucleoside triphosphates are engaged as source energy providers. In the form of coenzymes, they transfer phosphate residues or nucleotide constituent and participate in the activation (e.g. uridine diphosphate glucose for pasting glucose units to polymeric saccharides or cytidine diphosphate-choline for phospholipid synthesis). Cyclic nucleotides act as secondary messengers in many signal transduction pathways. The most important nucleotide signal molecule is cyclic adenosine monophosphate (cAMP), which is an activator of protein kinases and participates in many metabolic pathways by transferring the effect of hormones into cells. Analogical molecule-cyclic guanosine monophosphate (cGMP)-also acts as a secondary messenger in the phototransduction process.Nucleotide metabolism is one of the main therapeutic cellular targets. Synthetic analogues of these compounds have been widely applied in anti-cancer, anti-viral and immunosuppressive therapies [1][2][3][4][5][6]. Nucleoside analogues (e.g. cytarabine, gemcitabine and zidovudine) are modified at the base or sugar moiety. This 'prodrug' form requires intracellular activation by phosphorylation to mono, di or...

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CyTOF Mass Cytometry for Click Proliferation Assays

Toševski

Ulashchik

Trovato³

et al. 2017

CP Cytometry

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Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

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Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

Cited by 20 publications

References 40 publications

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

Mass Spectrometry for the Sensitive Analysis of Intracellular Nucleotides and Analogues

CyTOF Mass Cytometry for Click Proliferation Assays

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