Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

Abstract

Hepatitis C virus (HCV) is one of the most important Flaviviridae infections in humans and is responsible for the second most common cause of viral hepatitis. Presently, nearly 2% of the U.S. population, and an estimated 170 million people worldwide, are HCV carriers (2). The only approved therapy for chronic hepatitis C is alpha interferon (IFN-␣), either alone or in combination with ribavirin. Anemia is the most common adverse effect associated with ribavirin treatment, and neuropsychiatric adverse effects of IFN-␣ lead to premature cessation of therapy in 10 to 20% of patients (9, 13).As additional treatment options are urgently needed, there is an ongoing search for more potent antiviral compounds with fewer adverse effects. However, the search for improved antiviral agents is hampered by the limited and cumbersome propagation of HCV in vitro (4). Therefore, surrogate models such as the HCV RNA replicon that replicates in the human hepatoma cell line Huh7 have been developed (6,29). Improved versions of these HCV replicons contain adaptive mutations (25), and their use has facilitated the evaluation of candidate anti-HCV drugs.Bovine viral diarrhea virus (BVDV) is one of the best characterized members of the Flaviviridae family and has one of the largest RNA genomes (12.5 kb) in this family (8). This virus has the remarkable property of existing as noncytopathic and cytopathic (cpBVDV) biotypes, with cpBVDV strains showing insertions or viral genome rearrangements at the junction site between nonstructural protein 2 (NS2) and NS3 (32). BVDV may provide a surrogate model for HCV, both for the molecular study of viral proteins (33) and for the evaluation of antiviral compounds (3,7,47).In the search for therapeutic agents, any element that is essential for viral (or replicon) RNA replication may be considered a drug discovery target. Such elements can be either viral proteins (NS2-NS3 protease, NS3-NS4A serine proteinase, NS3 RNA helicase, or RNA-dependent RNA polymerase [3,24,34,36] [18,31,41,43]). Current knowledge of the human genome, combined with array technology and pathogen infection models, will likely lead to more defined host-pathogen-related targets for future drug design (17, 23). Today, however, the most successful classes of antiviral compounds with clinical utility in combat against other human viral pathogens (human immunodeficiency virus type 1 [HIV-1], hepatitis B virus [HBV], herpes simplex virus [HSV], and cytomegalovirus) are the protease, the nonnucleoside analogue, and the nucleoside analogue inhibitors. As the latter class of compounds is crucial in controlling herpesvirus, HIV-1, and HBV infections, it is likely and anticipated that

Combinations of 2'-C-Methylcytidine Analogues with Interferon-α2b and Triple Combination with Ribavirin in the Hepatitis C Virus Replicon System

“…Determination of the 50% effective concentration (EC 50 ) and the 90% effective concentration (EC 90 ) in HCV replicon RNA levels of the compounds were performed as previously described by Stuyver et al [18]. In addition, the 50% cytotoxicity concentration (CC 50 ) of the compounds was also determined by calculating the DCt for rRNA [18].…”

“…Determination of the 50% effective concentration (EC 50 ) and the 90% effective concentration (EC 90 ) in HCV replicon RNA levels of the compounds were performed as previously described by Stuyver et al [18]. In addition, the 50% cytotoxicity concentration (CC 50 ) of the compounds was also determined by calculating the DCt for rRNA [18]. Cell proliferation was also determined by 5-(3-carboxymethoxyphenyl-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl) tetrazolium salt (MTT) reduction test using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer's instructions.…”

Synthesis and Evaluation of 2,6-Modified Purine 2′-C-Methyl Ribonucleosides as Inhibitors of HCV Replication

“…Therefore, in order to overcome the often rate-limiting first phosphorylation step and improve the antiviral activity of our nucleoside analogues, we prepared their corresponding monophosphate McGuigantype prodrugs. 12 The synthesis of phosphoramidates 31−46 was performed following the Uchiyama procedure by reacting the nucleosides 10−12, 14,15,17,18,20,22,23,25,26,28, and 29 with chlorophosphoramidate 30 13 in the presence of N-methylimidazole (Scheme 4). 14,15 It is noteworthy that the use of acetonitrile as a cosolvent improved the solubility of certain nucleosides leading to better overall yields.…”

“…16 Cytotoxicity in Huh7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and cellular rRNA (rRNA). 17 In addition cytotoxicity was determined in primary human peripheral blood mononuclear (PBM) cells, human lymphoblastoid CEM, and African Green monkey Vero cells (Table 1). 18,19 In an initial set of compounds, unusual 6-modifications such as introduction of a phosphonate ester or an ethoxy vinyl group were counterproductive and lead to inactive nucleosides and monophosphate prodrugs 14, 15, 34, and 35.…”

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

“…Reassessment of the D-isomer has recently found it to have some promising antiviral characteristics . With previously discovered NRTIs being re-examined in a new mechanistic light and new compounds including 2′-substituted (Carroll et al, 2003) and base-modified (Stuyver et al, 2003) cytidine analogues effective against the hepatitis C virus the potential for new antiviral drug discovery in this area of nucleoside research is promising.…”