Inhibitors of the IMPDH Enzyme as Potential Anti-Bovine Viral Diarrhoea Virus Agents

Stuyver, Lieven; Lostia, Stefania; Patterson, Steven; Clark, Jeremy L.; Watanabe, Kyoichi A.; Otto, Michael; Pankiewicz, Krzysztof W.

doi:10.1177/095632020201300602

Cited by 25 publications

(15 citation statements)

References 37 publications

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“…It has also been shown that FdC could be phosphorylated by the HSV-1 viral thymidine kinase (19). However, when FdC was tested against the cytopathogenic BVDV strain NADL in MDBK cells (14), the compound was unable to inhibit the production of viral RNA in the culture supernatant (EC 90 , Ͼ100 M; data not shown) and was not cytotoxic (CC 50 , Ͼ100 M). The inactivity of FdC in the MDBK-cpBVDV model may have resulted from (i) poor initial activation to FdC-monophosphate by the bovine deoxycytidine kinase, (ii) rapid deamination by the bovine cytidine deaminase or deoxycytidine deaminase to the inactive 2Ј-deoxy-2Ј-fluorouridine (FdU), or (iii) an inability of BVDV polymerase to recognize FdCTP as a substrate.…”

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confidence: 96%

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Stuyver

McBrayer

Whitaker

et al. 2004

Antimicrob Agents Chemother

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2-Deoxy-2-fluorocytidine (FdC) is a potent inhibitor of the hepatitis C virus RNA replicon in culture, and FdC-5-triphosphate is an effective inhibitor of the NS5B polymerase. Dynamic profiling of cell growth in an antiviral assay showed that FdC caused cytostasis due to an S-phase arrest. These observations demonstrate that FdC treatment is affecting both a viral target and a cellular target.Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in the United States, with sequelae including fibrosis, cirrhosis, and hepatocellular carcinoma (1). In vivo, HCV replication occurs mainly in the cytoplasm of infected hepatocytes, but it has been difficult to demonstrate replication in vitro. Replicon-based systems have now been developed that sustain efficient replication of HCV RNA in cell culture. Initially, subgenomic replicons that expressed only nonstructural proteins were constructed; however, recent reports described replicons that can express the entire HCV polyprotein (5, 7).In addition to the currently approved standard treatment options for HCV infections that use interferon and ribavirin, several new antiviral agents are in preclinical or clinical development. Similar to the case with human immunodeficiency virus type 1 treatment, multiple drug targets (e.g., protease, helicase, polymerase, and entry) may be needed to limit the emergence of drug-resistant variants. The HCV subgenomic replicon provides an excellent system for evaluating HCV antiviral agents in cell culture (3,5,6,10,16,18). We report here the antiviral activity of 2Ј-deoxy-2Ј-fluorocytidine (FdC) (Fig. 1) measured in the HCV subgenomic replicon system and in the bovine viral diarrhea virus (BVDV)-Madin-Darby bovine kidney (MDBK) cell system. HCV-replicon RNA-containing Huh-7 cells (Clone A cells; Apath, LLC, St. Louis, Mo.) were kept in exponential growth as described previously (16). Antiviral assays were performed in medium without G418. Cells were seeded in a 96-well plate at 1,000 cells per well, and test compounds were added immediately after seeding. After 96 h of incubation, total cellular RNA was isolated (Rneasy 96 kit; Qiagen, Valencia, Ca.), and HCV replicon RNA and an internal control (TaqMan rRNA Control Reagents; Applied Biosystems, Foster City, Ca.) were amplified in a single-step multiplex reverse transcription-PCR protocol. FdC (obtained from the Pharmasset compound library) was tested in a concentration range of 0.1 to 200 M, and a 90% effective concentration (EC 90 ) for reducing the intracellular HCV replicon RNA levels of 5.0 M was found ( Fig. 2A). FdC was found to be more potent than ribavirin (EC 90 , ϳ100 M) and comparable in potency to ␤-D-N 4 -hydroxycytidine (NHC) (EC 90 ϭ 5 M) (16). The cellular toxicity against Huh-7 and HepG2 cells was measured after 96 h of incubation by using the CellTiter 96 AQ ueous One solution cell proliferation assay (Promega, Madison, Wis.), and the concentration resulting in 50% reduction in cell growth (CC 50 ) was found to be greater than 100 M. This resul...

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confidence: 96%

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Stuyver

McBrayer

Whitaker

et al. 2004

Antimicrob Agents Chemother

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“…One of the most important questions in testing antimetabolites for antireplicon (or antiviral) activity is whether induction of the cytostatic and/or cytotoxic condition can be separated from specific antiviral activity, since "dead cells don't make virus." There are many reports in which specific antiviral activity has been ascribed to antimetabolites (1,11,22,23,28), but to date, no systematic study has been conducted to determine their effects on HCV subgenomic replicon. In addition, the antiviral action of antimetabolites such as ribavirin remains controversial (17,18,29).…”

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confidence: 99%

Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites

Stuyver

McBrayer

Tharnish

et al. 2003

J Virol

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Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i) minimal interference with the exponential cell growth, (ii) minimal reduction in cellular host RNA levels, and (iii) reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.Despite the availability of infectious cDNA clones of the hepatitis C virus (HCV), efficient in vitro replication has not been observed (3). After transfection of subgenomic HCV RNA replicons that also express the neomycin phosphotransferase gene selection marker, HCV replication has been reported previously in the human hepatoma cell line Huh-7 (2, 21). Such HCV replicon-harboring cell lines could be cultivated for more than a year without signs of cytopathogenicity (26). High levels of HCV RNAs can be maintained in cells passaged under continuous selection with G418. In addition, high-level replication was reflected in the observed adaptations of the HCV replicon to the host cell (20).A tight coupling of the amount of intracellular HCV RNA and cell growth was observed. High levels of viral RNA were found in exponentially growing cells, but this was followed by a sharp decline in RNA levels when cells reached a confluent state (26,29). This suggests that cellular factors required for RNA replication and/or translation vary in abundance and become limited in resting cells. Several proteins have been suggested, but none of them has as yet been positively identified to be directly responsible (9,15,16,19,25,30). However, there are no reports in which the availability of the cellular nucleoside triphosphate pools were linked to the loss of steadystate level replication of HCV replicon RNA in confluent cells. It is well-known that confluent cells mainly depend on salvage nucleoside biosynthetic pathways, resulting in lowered concentrations of endogenous nucleoside pools. As most antimetabolite agents of the de novo nucleoside biosynthesis are known to chemically deplete nucleoside pools and induce a concentration-dependent cytostasis, evaluating these compounds against HCV replicon can shed light on the relationship between nucleoside pools and replicon dynamics. However, before doing so, a definition of specificity of antiviral ...

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“…Only few examples are reported for the treatment of BVDV such as acridones [21] ribavirin and mycophenolic acid (MPA) [22], and iminosugars [23]. In connection with our strategy in synthesis of new amino acid derivatives [24,25], we report here the synthesis of new peptides bearing naphthalene residue as potential antiviral and antitumor candidates.…”

Section: Introductionmentioning

confidence: 98%

Amino acid derivatives, part 3: New peptide and glycopeptide derivatives conjugated naphthalene. Synthesis, antitumor, anti‐HIV, and BVDV evaluation

Al‐Masoudi

Al-Masoudi

Ali

et al. 2005

Heteroatom Chemistry

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Inhibitors of the IMPDH Enzyme as Potential Anti-Bovine Viral Diarrhoea Virus Agents

Cited by 25 publications

References 37 publications

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Dynamics of Subgenomic Hepatitis C Virus Replicon RNA Levels in Huh-7 Cells after Exposure to Nucleoside Antimetabolites

Amino acid derivatives, part 3: New peptide and glycopeptide derivatives conjugated naphthalene. Synthesis, antitumor, anti‐HIV, and BVDV evaluation

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