Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemistry
2.1.1. General Synthetic Experimental
2′-C-Ethynyluridine Aryloxy Phosphoramidate
5-((Benzoyloxy)methyl)-3-vinyltetrahydrofuran-2,3,4-triyl tribenzoate 2
5-((Benzoyloxy)Methyl)-2-(2,4-Dioxo-3,4-Hihydropyrimidin-1(2H)-yl)-3-Vinyltetrahydrofuran-3,4-diyl Benzoate 3
1-(3,4-dihydroxy-5-(hydromethyl)-3-vinyltetrahydrofuran2-yl)pyrimidine-2,4(1H,3H)-dione 4
isopropyl(((5-(2,4-dioxo-3,4-dihydropyridimin-1(2H)-yl)-3,4-dihydroxy-3-inyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate 5
(5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl (2-(methylthio)ethyl) (2-(1H-indol-3-yl)ethyl)phosphoramidate (2′-C-methyluridine methylthioethyl tryptamine ProTide)
2.2. Cell Culture
2.3. Viruses
2.4. CellTiter-Glo Activity Assay
2.5. Bright-Field Microscopy
2.6. Plaque Assay
2.7. ZIKV RdRP Expression and Purification
2.8. Single Nucleotide Incorporation Assay
2.9. Structural Alignment
3. Results
3.1. ProTide Library Design
3.2. Activity against ZIKV and Toxicity of Nucleoside Analogue ProTides in Human Neural Stem Cells
3.3. Protection from ZIKV-Induced Cytopathic Effect by 2′-C-methyluridine Aryloxyl Phosphoramidate ProTide and Sofosbuvir in a ZIKV-Sensitive Glioblastoma Stem Cell Line
3.4. Repression of ZIKV Titers by 2′-C-methyluridine Aryloxyl Phosphoramidate ProTide and Sofosbuvir in Neural Stem Cells
3.5. Enzymatic Incorporation of the Active Triphosphate Metabolites of 2′-C-methyluridine and Sofosbuvir Over Time Reveals Higher Levels of Incorporation for the Former Compound
3.6. Molecular Superpositioning of the ZIKV NS5 Active Site: Consequences for 2′-C-Derivatitized Nucleoside Analogues
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Nucleoside Analogue ProTide | EC50 PRVABC59 ZIKV, µM | EC50 H/PAN ZIKV, µM | CC50, µM | SI 1 PRVABC59 ZIKV | SI 1 H/PAN ZIKV |
---|---|---|---|---|---|
2′-C-Methylcytidine aryloxyl phosphoramidate | 48 ± 1 | >50 | >50 | >1 | >1 |
3′-Deoxyadenosine aryloxyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
5′-Fluorocytidine aryloxyl phosphoramidate | >50 | >50 | 10 ± 1 | >0.2 | >0.2 |
3′-O-Methyluridine aryloxyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
2′-O-Methylcytidine aryloxyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
2′-C-methyluridinearyloxyl phosphoramidate | 1 ± 1 | 2 ± 1 | 47 ± 1 | >47 | >23 |
2′-C-Methyladenosine aryloxyl phosphoramidate | 10 ± 1 | 18 ± 1 | 42 ± 1 | >4 | >2 |
3′-Deoxycytidine aryloxyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
3′-Deoxy-3′-Fluoroguanosine aryloxyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
2′-C-Ethenyluridine aryloxyl phosphoramidate | >50 | >50 | 10 ± 1 | >0.1 | >0.1 |
2′-C-Ethynyluridine aryloxyl phosphoramidate | 0.3 ± 1 | 0.8 ± 1 | >50 | >166 | >62 |
2′-C-methyluridine 2-(methylthio)ethyl phosphoramidate | >50 | >50 | >50 | >1 | >1 |
Sofosbuvir | 35 ± 1 | 46 ± 1 | >50 | >1.4 | >1.1 |
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Bernatchez, J.A.; Coste, M.; Beck, S.; Wells, G.A.; Luna, L.A.; Clark, A.E.; Zhu, Z.; Hecht, D.; Rich, J.N.; Sohl, C.D.; et al. Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells. Viruses 2019, 11, 365. https://doi.org/10.3390/v11040365
Bernatchez JA, Coste M, Beck S, Wells GA, Luna LA, Clark AE, Zhu Z, Hecht D, Rich JN, Sohl CD, et al. Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells. Viruses. 2019; 11(4):365. https://doi.org/10.3390/v11040365
Chicago/Turabian StyleBernatchez, Jean A., Michael Coste, Sungjun Beck, Grace A. Wells, Lucas A. Luna, Alex E. Clark, Zhe Zhu, David Hecht, Jeremy N. Rich, Christal D. Sohl, and et al. 2019. "Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells" Viruses 11, no. 4: 365. https://doi.org/10.3390/v11040365