Inhibition of Hepatitis B Virus (HBV) Replication by Pyrimidines Bearing an Acyclic Moiety: Effect on Wild-Type and Mutant HBV

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The main clinical limitation of a current antiviral drug for HBV, lamivudine, is the emergence of drug-resistant viral strains upon prolonged therapy. A group of 5-, 6-, or 5,6-substituted acyclic pyrimidine nucleosides with a 1-[(2-hydroxyethoxy)methyl] moiety were synthesized and evaluated for antiviral activities. The target compounds were prepared by the reaction of silylated uracils possessing a variety of substituents at the C-5 or C-6 positions or both with 1,3-dioxolane in the presence of potassium iodide and chlorotrimethylsilane by a convenient and single-step synthesis. Among the compounds tested, 5-chloro and 5-bromo analogues possessing an acyclic glycosyl moiety were the most effective and selective antiviral agents in the in vitro assays against wild-type duck HBV (EC50 = 0.4−2.2 and 3.7−18.5 μM, respectively) and human HBV-containing 2.2.15 cells (EC50 = 4.5−45.4 and 18.5−37.7 μM, respectively). These compounds were also found to retain sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and double mutations (L180M/M204V). The compounds investigated did not show cytotoxicity to host HepG2 and Vero cells, up to the highest concentration tested. The results presented here confirm and accentuate the potential of acyclic pyrimidine nucleosides as anti-HBV agents and extend our previous observations. We herein report the capability of acyclic pyrimidine nucleosides to inhibit the replication of both wild-type and drug-resistant mutant HBV

Synthesis and Antiviral Activity of Novel Acyclic Nucleoside Analogues of 5-(1-Azido-2-haloethyl)uracils

We present the discovery of a novel category of 5-substituted acyclic pyrimidine nucleosides as potent antiviral agents. A series of 1-[(2-hydroxyethoxy)methyl] (5−7), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] (8−10), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] (11−13) derivatives of 5-(1-azido-2-haloethyl)uracil were synthesized and evaluated for their biological activity in cell culture. 1-[4-Hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil (12) was the most effective antiviral agent in the in vitro assays against DHBV (EC50 = 0.31−1.55 μM) and HCMV (EC50 = 3.1 μM). None of the compounds investigated showed any detectable toxicity to several stationary and proliferating host cells

Effect of Various Pyrimidines Possessing the 1-[(2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl] Moiety, Able To Mimic Natural 2‘-Deoxyribose, on Wild-type and Mutant Hepatitis B Virus Replication

Hepatitis B virus (HBV) is the most common cause of chronic liver disease worldwide. Development of drug resistance against clinical anti-HBV drug lamivudine due to long-term use and rebound of viral DNA after cessation of treatment has been a major setback of the current therapy. We have synthesized a series of pyrimidine nucleosides possessing a variety of substituents at the C-5 position, and a 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] flexible acyclic glycosyl moiety at the N-1 position, that have the ability to mimic the natural 2‘-deoxyribosyl moiety. Some of these potential antiviral compounds included variations at both C-5 and C-6 positions of the uracil base. Other variations of the uracil derivatives were the 6-aza congeners. 4-Amino and 4-methoxy pyrimidine derivatives were also made. Compounds in which the base moiety was substituted by 5-chloro- (25), 5-(2-bromovinyl)- (32), or 5-bromo-6-methyl- (37) groups possess significant activity against duck-HBV, wild-type human HBV (2.2.15 cells), and lamivudine-resistant HBV containing single and double mutations. No cytotoxicity was seen in host HepG2 and Vero cells, up to the highest concentration tested. The anti-HBV activity exhibited by compounds 25, 32, and 37 was superior for human HBV and comparable for DHBV to that of the corresponding purine nucleoside, ganciclovir. Further, they were only 10−15-fold less inhibitory against human HBV in 2.2.15 cells than the reference drug, lamivudine. Other compounds in the series were moderately inhibitory against DHBV and wild-type human HBV. The size of the halogen and the electronegativity of the substituents at the 5- and 6-positions are important for antiviral activity toward HBV. These compounds were also evaluated for their antiviral activity for West Nile virus, respiratory syncytial virus, SARS-coronavirus, and hepatitis C virus. They were generally inactive in these antiviral assay systems (at concentrations up to 100 μg/mL). 1-[(2-Hydroxy-1-(hydroxymethyl) ethoxy)methyl]-5-fluorocytosine (34) showed some inhibitory activity against hepatitis C virus. Taken together, these data support our previous observations that the 5-substituted pyrimidine nucleosides containing acyclic glycosyl moieties have potential to serve as a new generation of potent, selective, and nontoxic anti-HBV agents for wild-type and lamivudine-resistant mutant HBV

Memory, but not naïve B cells from cryoglobulin positive HCV patients express higher levels of activation markers compared to B cells from cryoglobulin negative HCV patients.

<p>PBMCs were freshly isolated and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068308#pone-0068308-g003" target="_blank">Figure 3</a> except that this analysis compares cryoglobulin+ (n = 20, cryo+) and cryoglobulin- (n = 34, cryo-) HCV patients. Antibodies to CD86, CD71, HLA-DR and CD183 were added in combinations with CD19 and CD27 to gate on memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. The percent positive was calculated based on mouse isotype controls and the geometric mean fluorescent intensities (MFI) for each marker were determined. Horizontal lines on graphs represent median values. For CD71 analysis, 18 cryo+ and 30 cryo- HCV samples were stained. ***, P<0.001, **, P<0.01, *, P<0.05, n.s., not significant.</p

Gating strategy for flow cytometry analysis of memory and naïve B cell activation.

<p>Lymphocytes were gated by FSC/SSC properties and memory B cells (CD19+CD27+) and naïve B cells (CD19+CD27−) were analyzed within the lymphocyte gate for the expression of 6 different markers described in the Materials and Methods. Histograms show a representative example of the expression of CD183 (numbers represent geometric mean fluorescent intensity) on memory and naïve B cells from a healthy control (HCV-) and chronic HCV patient (HCV+).</p

Geometric mean fluorescent intensities of each marker on memory and naïve B cells.

<p>Abbreviation: MFI, mean fluorescent intensity.</p

B cell subset frequencies are unchanged in HCV patients compared to controls.

<p>(<b>A–C</b>) 7.5×10⁵ freshly isolated PBMCs from chronically infected HCV patients (n = 54) or healthy controls (n = 50) were incubated with fluorescently labeled antibodies to CD19, CD27 and CD5 for multi-color flow cytometry analysis. (<b>A</b>) The total percent of CD19+ B cells within the lymphocyte gate and number of B cells per ml of blood based on our isolations. (<b>B</b>) The percent of memory (CD19+CD27+) and naïve (CD19+CD27−) and number of each subset per ml of blood. <b>(C)</b> The percent and number per ml of CD5+CD19+ B cells. Horizontal lines on graphs represent means +/− SEM (percentages) or median (number of cells/ml) values. In (C) only 48 HCV and 47 healthy control samples were tested for CD5. *, P<0.05; n.s., not significant.</p

Memory, but not naïve B cells are significantly activated in chronically infected HCV patients.

<p>PBMCs were freshly isolated and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068308#pone-0068308-g002" target="_blank">Figure 2</a>. Antibodies to CD183, CD71, CD86, CD69, HLA-DR and CD40 were added in combinations with CD19 and CD27 to gate on memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. The percent positive was calculated based on mouse isotype controls and the geometric mean fluorescent intensities (MFI) for each marker were determined. HLA-DR and CD40 are expressed by all B cells therefore only the MFI is shown. Horizontal lines on graphs represent median values. For CD71 analysis, only 48 HCV and 47 healthy control samples were stained. ***, P<0.001, **, P<0.01, *, P<0.05, n.s., not significant.</p

Clinical characteristics of HCV+ patients and healthy controls in study.

<p>Abbreviations: N/A, not applicable, ALT, alanine aminotransferase (normal values = <50), GGT, gamma-glutamyl transpeptidase (normal levels = <70 (males), <55 (females)), IU, International units, IFN, interferon.</p>*<p>determined from biopsy or Fibroscan.</p

PBMC and B cell quantification and clinical characteristics of cryoglobulin positive versus cryoglobulin negative HCV patients.

<p>(<b>A–B</b>) PBMCs were freshly isolated and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068308#pone-0068308-g002" target="_blank">Figure 2</a> except that this analysis compares cryoglobulin+ (n = 20, cryo+) and cryoglobulin- (n = 34, cryo-) HCV patients. (<b>A</b>) The total percent of CD19+ B cells within the lymphocyte gate and number of B cells per ml of blood based on our isolations. (<b>B</b>) The percent of memory (CD19+CD27+) and naïve (CD19+CD27−) and number of each subset per ml of blood. (<b>C</b>) The number of total PBMCs isolated per ml of blood. (<b>D</b>) The alanine aminotransferase (ALT) and gamma-glutamyl transpeptidase (GGT) levels were measured in the serum in units/L (U/L) for 54 (ALT) or 48 (GGT) HCV patients. (<b>E</b>) Serum HCV RNA titers were quantified by qRT-PCR as described in the Materials and Methods for all 54 HCV patients. Horizontal lines on graphs represent mean +/− SEM (A-B, percentages, C) or median (A–B numbers, D, E) values. *, P<0.05, n.s., not significant. IU/ml, international units/ml.</p