- Development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting zika virus
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Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2=-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKVinduced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.
- Bernatchez, Jean A.,Yang, Zunhua,Coste, Michael,Li, Jerry,Beck, Sungjun,Liu, Yan,Clark, Alex E.,Zhu, Zhe,Luna, Lucas A.,Sohl, Christal D.,Purse, Byron W.,Li, Rongshi,Siqueira-Neto, Jair L.
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- Nucleoside analogs with antiviral activity against yellow fever virus
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Yellow fever virus (YFV) is a human Flavivirus reemerging in parts of the world. While a vaccine is available, large outbreaks have recently occurred in Brazil and certain African countries. Development of an effective antiviral against YFV is crucial, as there is no available effective drug against YFV. We have identified several novel nucleoside analogs with potent antiviral activity against YFV with 50% effective concentration (EC50) values between 0.25 and 1 μM with selectivity indices over 100 in culture.
- Zandi, Keivan,Amblard, Franck,Amichai, Sarah,Bassit, Leda,Tao, Sijia,Jiang, Yong,Zhou, Longhu,Russell, Olivia Ollinger,Mengshetti, Seema,Schinazi, Raymond F.
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- Synthesis and biological evaluation of 5′-C-methyl nucleotide prodrugs for treating HCV infections
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Nucleotide prodrugs are of great clinical interest for treating a variety of viral infections due to their ability to target tissues selectively and to deliver relatively high concentrations of the active nucleotide metabolite intracellularly. However, their clinical successes have been limited, oftentimes due to unwanted in vivo metabolic processes that reduce the quantities of nucleoside triphosphate that reach the site of action. In an attempt to circumvent this, we designed novel nucleosides that incorporate a sterically bulky group at the 5′-carbon of the phosphoester prodrug, which we reasoned would reduce the amounts of non-productive P[sbnd]O bond cleavage back to the corresponding nucleoside by nucleotidases. Molecular docking studies with the NS5B HCV polymerase suggested that a nucleotide containing a 5′-methyl group could be accommodated. Therefore, we synthesized mono- and diphosphate prodrugs of 2′,5′-C-dimethyluridine stereoselectively and evaluated their cytotoxicity and anti-HCV activity in the HCV replicon assay. All four prodrugs exhibited anti-HCV activity with IC50 values in the single digit micromolar concentrations, with the 5′(R)-C-methyl prodrug displaying superior potency relative to its 5′(S)-C-methyl counterpart. However, when compared to the unmethylated prodrug, the potency is poorer. The poorer potency of these prodrugs may be due to unfavorable steric interactions of the 5′-C-methyl group in the active sites of the kinases that catalyze the formation of active triphosphate metabolite.
- Dasari, Madhuri,Liotta, Dennis C.,Ma, Peipei,Pelly, Stephen C.,Sharma, Savita K.
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- Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase
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To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2′- and/or 4′-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2′-OH with 2′-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2′-fluorination, the introduction of fluorine at the ribose 4′-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2′-C-ethynyl-4′-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.
- Wang, Gang,Lim, Siew Pheng,Chen, Yen-Liang,Hunziker, Jürg,Rao, Ranga,Gu, Feng,Seh, Cheah Chen,Ghafar, Nahdiyah Abdul,Xu, Haoying,Chan, Katherine,Lin, Xiaodong,Saunders, Oliver L.,Fenaux, Martijn,Zhong, Weidong,Shi, Pei-Yong,Yokokawa, Fumiaki
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p. 2324 - 2327
(2018/05/28)
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- Mechanism-Based Solution to the ProTide Synthesis Problem: Selective Access to Sofosbuvir, Acelarin, and INX-08189
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A general and efficient method for the synthesis of pronucleotide (ProTide) 5'-phosphoramidate monoesters is reported. This method consists of a highly stereoselective 5'-phosphorylation mediated by dimethylaluminum chloride to afford the desired target ProTides in excellent yields without employing 3'-protection strategies. The application of this methodology to the synthesis of a number of pharmaceutically relevant compounds currently marketed or under investigation in clinical research is demonstrated.
- Simmons, Bryon,Liu, Zhuqing,Klapars, Artis,Bellomo, Ana,Silverman, Steven M.
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supporting information
p. 2218 - 2221
(2017/05/12)
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- SYNTHESIS OF PHOSPHORAMIDATES
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A process for preparing a compound of formula (I) or a salt thereof, the process comprising providing a compound of formula (III) wherein PG is an inert electron withdrawing hydroxyl protecting group, reacting the compound of formula (III) with a fluorinating agent, obtaining a compound of formula (II) and deprotecting the compound of formula (II).
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