Active Uptake and Trafficking of Nucleoside Triphosphates <i>In Vivo</i>

Schreier, Verena N.; Loehr, Morten O.; Lattmann, Evelyn; Luedtke, Nathan W.

doi:10.1021/acschembio.2c00153

Cited by 14 publications

(14 citation statements)

References 55 publications

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“…In the literature, native triphosphate nucleosides exhibit poor cell permeability [24] . Recently, Hocek et al.…”

Section: Resultsmentioning

confidence: 99%

“…In the literature, native triphosphate nucleosides exhibit poor cell permeability. [24] Recently, Hocek et al have reported the transfection of modified dNTP using a unique transporter, synthetic nucleoside triphosphate transporter (SNTT). [25] However, nucleosides and related molecules are prodrugs for various diseases including antiviral, anticancer, and antibiotic drugs.…”

Section: Chembiochemmentioning

confidence: 99%

See 1 more Smart Citation

Tropolone‐Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization

2023

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Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.

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“…In the literature, native triphosphate nucleosides exhibit poor cell permeability [24] . Recently, Hocek et al.…”

Section: Resultsmentioning

confidence: 99%

Section: Chembiochemmentioning

confidence: 99%

Tropolone‐Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization

2023

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“…In addition to phenotypic classifications possible from fluorescently tagged proteins or anatomical regions such as the gut, body wall, pharynx or neurons, the three-fluorescent channel capability of LSC would, in principle, enable colocalization and multiplexed assay formats ( Auld et al, 2006 ). For example, metabolic incorporation of TAMRA-dATP into genomic DNA or ‘cell painting’ could facilitate a range of phenotypic profiles in C. elegans ( Gustafsdottir et al, 2013 ; Schreier et al, 2022 ). Although we conducted the current study with LSC, using automated microscopy as a primary or LSC-coupled output is certainly feasible ( Gosai et al, 2010 ).…”

Section: Discussionmentioning

confidence: 99%

In vivoquantitative high-throughput screening for drug discovery and comparative toxicology

Dranchak

Oliphant

Queme

et al. 2023

Disease Models &Amp; Mechanisms

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Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk, and increasingly for academic probe discovery. Phenotypic HTS assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. C. elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser scanning cytometry is described. GFP-expressing organisms revealing phenotype-modifying structure-activity relationships guide subsequent life-stage and proteomic analyses. E. coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in 7-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations having a physiological impact. We anticipate this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.

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“…In addition to phenotypic classifications possible from fluorescently tagged proteins or anatomical regions such as the gut, body wall, phalanx, or neurons, the three fluorescent channel capability of LSC would in principle enable colocalization and multiplexed assay formats (Auld et al, 2006). For example, metabolic incorporation of TAMRA-dATP into genomic DNA or “cell painting” could facilitate a range of phenotypic profiles in C. elegans (Gustafsdottir et al, 2013; Schreier et al, 2022). While we conducted the current study with LSC, using automated microscopy as a primary or LSI-coupled output is certainly feasible (Gosai et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

Inglese

Dranchak

Oliphant

et al. 2022

Preprint

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Quantitative high-throughput screening (qHTS) evaluates the pharmacology of drug and investigational agent libraries for potential therapeutic uses, toxicological risk assessment, and increasingly for academic chemical tool discovery. Phenotypic HTS assays aim to interrogate molecular pathways and networks, often relying on cell culture systems, historically with less emphasis on multicellular organisms. C. elegans has served as a powerful eukaryotic model organism for human biology and disease by virtue of genetic conservation and experimental tractability. Here we describe a paradigm to enable C. elegans in qHTS using 384-well microtiter plate laser scanning cytometry. GFP-expressing organisms are used to reveal phenotype-modifying structure-activity relationships to guide subsequent life stages and proteomic analysis. E. coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over 7-days spanning two life cycles to mitigate complications from bacterial overgrowth. We demonstrate the method with a library composed of anti-infective agents, or molecules of general toxicological concern. Each was tested in 7-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations having a physiological impact. We anticipate a broader application of this qHTS-coupled proteomics approach will enable the analysis of C. elegans orthologous transgenic phenotypes of human pathologies to facilitate drug and probe profiling from high-impact chemical libraries for a range of therapeutic indications and study of potential toxicological signatures.Graphic Abstract

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Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

Cited by 14 publications

References 55 publications

Tropolone‐Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization

Tropolone‐Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization

In vivoquantitative high-throughput screening for drug discovery and comparative toxicology

In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

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