Suguna Rachakonda

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

ABSTRACT A base-modified nucleoside analogue, β-d-N4-hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC90) of 5.4 μM, an observation that was confirmed by virus yield assays (EC90 = 2 μM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC90 of 5 μM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2′-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 μM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

Synthesis of beta-enantiomers of N4-hydroxy-3'-deoxypyrimidine nucleosides and their evaluation against bovine viral diarrhoea virus and hepatitis C virus in cell culture.

N4-Hydroxycytidine (NHC) was recently reported to have anti-pestivirus and anti-hepacivirus activity. It is thought that this nucleoside acts as a weak alternative substrate for the hepatitis C virus (HCV) polymerase. In addition to NHC, 3′-deoxyuridine (3′-dU) was found to inhibit bovine diarrhoea virus (BVDV) production by 1 log10 at 37.2 μM. These initial findings prompted the synthesis of β-D and β-L analogues of (i) base-modified 3′-deoxy-NHC; (ii) 3′-deoxyuridine; and 3′-deoxycytidine. The antiviral activity of these 42 nucleosides was evaluated against BVDV and HCV bicistronic replicon in cell culture. Among the NHC analogues, the antiviral activity observed for the β-L-3′-deoxy-5-fluoro-derivative 1-(3-deoxy-β-L-erythro-pentofuranosyl)-5-fluoro-4-hydrox-yaminopyrimidin-2(1H)-one and the β-D-3′-deoxy-5-iodo-derivative 1-(3-deoxy-β-D-erythro-pentofuranosyl)-5-iodocytosine in the replicon system (1 log10 reduction at 100 μM) was due to the concomitant toxicity towards intracellular ribosomal RNA levels (CC90 equal or lower than the EC90). In conclusion, none of the newly synthesized derivatives exhibited enhanced antiviral activity compared to the parent nucleoside NHC.

Phosphoramidate prodrugs of (-)-β-D-(2R,4R)-dioxolane-thymine (DOT) as potent anti-HIV agents.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) are an effective class of agents that has played a vital role in the treatment of HIV infections. (−)-β-D-(2R,4R)-dioxolane-thymine (DOT) is a thymidine analogue that is active against wild-type and NRTI-resistant HIV-1 mutants. It has been shown that the anti-HIV activity of DOT is limited due to poor monophosphorylation. Methods: To further enhance the anti-HIV activity of DOT, an extensive structure-activity relationship analysis of phosphoramidate prodrugs of DOT monophosphate was undertaken. These prodrugs were evaluated for anti-HIV activity using Hela CD4 β-gal reporter cells (P4-CCR5 luc cells). Results: Among the synthesized prodrugs, the 4-bromophenyl benzyloxy L-alanyl phosphate derivative of DOT was the most potent, with a 50% effective concentration of 0.089 μM corresponding to a 75-fold increase in activity relative to the parent nucleoside DOT with no increased cytotoxicity. The metabolic stability of a selected number of potent DOT phosphoramidates was also evaluated in simulated gastric fluid, simulated intestinal fluid, human plasma and liver S9 fractions. Conclusions: A series of new phosphoramidate prodrugs of DOT were prepared and evaluated as inhibitors of HIV replication in vitro. Metabolic stability studies indicated that these DOT phosphoramidate derivatives have the potential to show acceptable stability in the gastrointestinal tract, but they metabolize rapidly in the liver.

N4-Acyl-Modified D-2′,3′-Dideoxy-5-Fluorocytidine Nucleoside Analogues with Improved Antiviral Activity:

A series of 2,3-dideoxy (D2) and 2,3-didehydro-2,3-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5′-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5′-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.