Activities of Certain 5-Substituted 4′-Thiopyrimidine Nucleosides against Orthopoxvirus Infections

Synthetic chemistry.1-(2-Deoxy-4-thio-β-d-ribofuranosyl)-5-fluoro-uracil (Fig. 1, compound 1), 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-thymine (compound 2), 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-bromouracil (compound 3), 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-trifluoromethyluracil (compound 4), and 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-iodouracil (compound 5) were synthesized according to the procedures previously published by Secrist et al. (32) and Rahim et al. (31). 1-(2-Deoxy-4-thio-β-d-ribofuranosyl)-5-phenyluracil (compound 6) was prepared by Suzuki coupling of compound 9 with phenylboronic acid (unpublished results). 1-(4-Thio-β-d-arabinofuranosyl)-5-fluorouracil (compound 7), 1-(4-thio-β-d-arabinofuranosyl)-cytosine (compound 10), 1-(4-thio-β-d-arabinofuranosyl)-5-fluorocytosine (compound 11), 1-(4-thio-β-d-arabinofuranosyl)-5-chlorocytosine (compound 12), and 1-(4-thio-β-d-arabinofuranosyl)-5-methylcytosine (compound 13) were prepared as described by Tiwari et al. (38, 39); and 1-(4-thio-β-d-arabinofuranosyl)-5-iodouracil (compound 8) was synthesized by the same procedure. A prodrug of compound 5 was synthesized by routine acetylation to obtain 1-(2-deoxy-3, 5-di-O-acetyl-4-thio)-5-iodouridine (compound 9). 1-(2-Deoxy-4-thio-β-d-ribofuranosyl)-cytosine (compound 14), 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-bromocytosine (compound 15), 1-(2-deoxy-4-thio-β-d-ribofuranosyl)-5-chlorocytosine (compound 16), and 1-(2-deoxy-4-thio-β-D-ribofuranosyl)-5-methylcytosine (compound 17) were prepared by the same methodology described by Secrist et al. (32).

Viruses and cells.Stock pools of vaccinia virus strain Copenhagen and cowpox virus strain Brighton were obtained from John W. Huggins (Department of Viral Therapeutics, Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fredrick, MD). Vaccinia virus strain Western Reserve (WR) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The CDV-resistant vaccinia virus strain (CDV^r strain 15A) and the two cowpox virus strains used in the TK assay, ΔcrmA lacZ (TK positive [TK⁺]) and TK:GFP lacZ (TK negative [TK⁻]), as well as vaccinia virus VVTK::luc, were obtained from Peter C. Turner and Marie N. Becker (University of Florida, Gainesville, FL), and all strains were described previously (1, 3). The mutant ST-246-resistant vaccinia virus (strain VV911) was provided by Robert Jordan (SIGA Technologies, Inc., Corvallis, OR). Working stocks of the vaccinia virus and cowpox virus strains were propagated in Vero cells obtained from ATCC. Human foreskin fibroblast (HFF) cells were prepared as primary cultures from freshly obtained newborn human foreskins. Culture medium for both cell lines was minimal essential medium (MEM) with Earle's salts containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 20 U/ml of penicillin, and 25 μg/ml of gentamicin.

Drug susceptibility assays for vaccinia virus strains and cowpox virus.Plaque reduction and cytopathic effect (CPE) assays were used to determine drug susceptibility, as described previously (14). For the plaque reduction assays, HFF cells were seeded into six-well plates, and the plates were incubated at 37°C with 5% CO₂ and 90% humidity. Two days later, drug at twice the final desired concentration was serially diluted 1:5 in 2× MEM with 10% FBS to provide six concentrations. Aspiration of the culture medium from triplicate wells for each drug concentration was followed by addition of 0.2 ml per well of diluted virus, which gave 20 to 30 plaques per well in MEM containing 10% FBS, or 0.2 ml medium for drug toxicity wells. The plates were incubated for 1 h to allow the virus to adsorb and were shaken every 15 min. An equal amount of 1% agarose was added to each drug dilution, and the mixture was added to each well in 2-ml volumes. Infected monolayers were incubated for 3 days, stained with a solution of neutral red in phosphate-buffered saline (PBS), and incubated for an additional 5 to 6 h. The stain was aspirated, the plaques were counted by using a stereomicroscope at ×10 magnification, and the 50% effective concentrations (EC₅₀s) were calculated by standard methods.

Cowpox virus β-galactosidase assay.Dependence on the viral TK was assessed by previously published methods (23, 24). Briefly, monolayers of HFF cells in 96-well plates were incubated at 37°C for 24 h in a humidified incubator. The experimental compounds were then diluted in the plates, and either TK⁺ or TK⁻ strains of cowpox virus were added at a multiplicity of infection of 0.05 PFU/cell. At 48 h postinfection, the medium was removed and the β-galactosidase substrate chlorophenol red-β-galactopyranoside was added at a final concentration of 50 μg/ml in PBS. The conversion of the colorimetric substrate was determined by measuring the absorbance at 570 nm, and EC₅₀s were calculated by standard methods (21). The ratio of the EC₅₀ for TK⁻ viruses and the EC₅₀ for TK⁺ viruses was calculated and used as a measure of TK dependence.

DNA synthesis inhibition assays.Monolayers of HFF cells were infected with vaccinia virus at an multiplicity of infection of 3 PFU/cell. After 24 h, the cells were harvested and total DNA was purified with a Wizard DNA purification kit (Promega, Madison, WI). The genome copy number was determined by real-time PCR with primers 5′-CTG CTG TGT GTA TGA AAT GCT TTA AG-3′ and 5′-TCT CGG TTT CCT CAC CCA AT-3′ and the strain VVTK-specific probe 6-carboxyfluorescein-5′-AGG CTT CCT TTT CTA AAC-3′-6-carboxytetramethylrhodamine (Applied Biosystems, Foster City, CA).

Cytotoxicity assays. (i) Neutral red uptake assay in stationary cells.HFF cells were seeded into 96-well plates at a concentration of 2.5 × 10⁴ cells per well, and the plates were incubated for 24 h. The medium was aspirated; and 125 μl of each drug concentration was added to designated wells in triplicate, while 100 μl of MEM with 2% FBS was added to all other wells. Serial 1:5 dilutions were made with a Beckman BioMek liquid handling system, and the plates were incubated for 7 days in a CO₂ incubator at 37°C. Aspirations of the well contents were followed by the addition of 100 μl/well of a solution of neutral red in PBS and incubation for 1 h. The cells were washed with PBS by using a Nunc plate washer, and 200 μl/well of 50% ethanol containing 1% glacial acetic acid was added. The plates were placed on a rotary shaker for 15 min, and the optical densities at 540 nm were read on a Bio-Tek plate reader. The concentration of drug that reduced cell viability by 50% (CC₅₀) was then calculated.

(ii) Cell proliferation assay.Twenty-four hours prior to assay, HFF cells were seeded into six-well plates at a concentration of 2.5 × 10⁴ cells per well in MEM containing FBS. On the day of the assay, the compounds were diluted serially in medium at increments of 1:5, resulting in concentrations covering a range from 100 to 0.03 μM. The medium from the wells was aspirated, and 2 ml of each drug concentration was added to duplicate wells. The cells were incubated in a CO₂ incubator at 37°C for 3 days. After incubation, the medium-drug solutions were removed, 1 ml of 0.25% trypsin-EDTA was added to each well, and the plates were incubated until the cells started to detach from the plate. Vigorous pipetting was used to obtain a homogeneous cell suspension, 0.2 ml of the mixture was added to 9.8 ml of Isoton III solution, and counts were obtained with a Coulter counter. The counts for each sample were obtained three times, and two replicate wells were used for each sample. The 50% inhibitory concentration (IC₅₀) was calculated by standard methods.

Antiviral activity in mice.Female BALB/c mice (age, 3 weeks) were obtained from Charles River Laboratories, Raleigh, NC. The mice were housed in groups in microisolator cages and were utilized at a quantity of 15 mice per treatment group. They were obtained, housed, utilized, and euthanized according to the regulatory policies of USDA and AAALAC. All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee prior to the initiation of the studies.

Antiviral compounds.CDV (Vistide; Gilead Sciences, Foster City, CA) was diluted in sterile saline to yield the desired doses in a 0.1-ml volume. It was administered as a fine suspension intraperitoneally (i.p.) once daily for 5 days. 4′-ThioIDU was provided as a dry powder, dissolved in a constant amount of dimethyl sulfoxide (DMSO), and then suspended in 0.4% carboxymethyl cellulose (CMC) to yield the desired doses in a 0.1-ml volume for i.p. injection or a 0.2-ml volume for oral gavage. Each dose was administered twice daily for 5 days beginning at various times after viral inoculation. Uninfected mice served as toxicity controls and were treated similarly.

Experimental infections.Vaccinia virus strain WR and cowpox virus infections were initiated by intranasal inoculation of anesthetized (ketamine-xylazine) BALB/c mice with approximately 90% lethal doses, which corresponded to 4 × 10⁴ PFU and 1.6 × 10⁴ PFU for cowpox virus and vaccinia virus, respectively (25, 27). The virus inoculum was instilled into both nostrils with a micropipette at a total volume of 40 μl per animal. For the mortality experiments, the animals were checked at least once daily for 21 days. Pathogenesis studies were performed with both vaccinia virus and cowpox virus in order to determine the effect of 4′-thioIDU on viral replication in the target organs of the mice. Three mice each from the control and the treated groups were euthanized on day 1, 3, 5, 7, or 10 postinoculation for collection of the lung, liver, spleen, and kidney. Samples were pooled by tissue type, homogenized in a 10% (wt/vol) suspension, and frozen until they were assayed for virus. The virus titers were determined by cocultivation of homogenates on Vero cells, and plaques were enumerated after 5 days of incubation.

Statistical evaluations.Mortality rates were analyzed by Fisher's exact test, and the mean day of death was determined by the Mann-Whitney U rank-sum test. A P value of ≤0.05 was considered significant.

Compounds active against vaccinia and cowpox viruses.Seventeen 4′-thiopyrimidine nucleosides were synthesized and were initially screened by CPE assays to determine the efficacies of the compounds against both vaccinia and cowpox viruses. Seven of the compounds found to be efficacious by the CPE assays were subject to confirmatory plaque reduction assays, and these results, along with cytotoxicity data, are presented in Table 1. All seven of these compounds had activities against both vaccinia and cowpox viruses that ranged from being 20- to 1,400-fold greater than the activity of CDV. The activity of one compound selected from the series, 4′-thioIDU, was also tested against vaccinia virus WR, and its activities against the Copenhagen strain of vaccinia virus and cowpox virus were compared. Selectivity indices (CC₅₀/EC₅₀) ranged from >200 to 2,000 for 4′-thioIDU; in contrast, the values for CDV were >9 to >32. All of the compounds exhibited minimal toxicity in stationary cells, as measured by the neutral red uptake assay, but cytotoxicity against proliferating cells was considerable (Table 1). The remaining 10 compounds were inactive when they were tested at 100 μM.

Activity against drug-resistant viruses.The antiviral activities of these compounds observed in vitro prompted additional studies of their activities against strains of vaccinia virus that were resistant to CDV or ST-246, and 4′-thioIDU was used as the representative compound from this series. This molecule retained its antiviral activity against a drug-resistant strain of CDV that contained mutations in the E9L DNA polymerase gene (3), as well as a strain resistant to ST-246 that contained mutations in the F13L gene (38) (Table 2). These data indicate that the mechanism of action of this molecule is distinct from the mechanisms of action of CDV and ST-246 and suggest that the molecule might be useful for the treatment of drug-resistant virus infections. The susceptibility of a recombinant virus lacking the J2R gene encoding the viral TK was also evaluated, since 4′-thioIDU is a thymidine analog. This recombinant virus appeared to be comparatively resistant to both 4′-thioIDU and IDU, while it remained fully sensitive to CDV, which does not require phosphorylation by this viral enzyme. These results initially suggested that the mechanism of action of 4′-thioIDU may be similar to that of IDU and that the viral TK may contribute to the phosphorylation of the drug.

Mechanism-of-action studies.Additional studies were conducted to investigate the effects of the drug on viral infection. Previous results from our laboratory showed that the activity of IDU against cowpox virus was dependent on the viral TK, suggesting that 4′-thioIDU might act by a similar mechanism (25). These studies were repeated and IDU exhibited TK dependence, while CDV and ST-246 were equally effective against both strains of virus (Table 3). The antiviral activity of 4′-thioIDU also proved to be largely dependent on the viral TK, which is consistent with our hypothesized mechanism. Subsequent studies examined its effects on viral DNA synthesis and confirmed that it is a potent inhibitor of viral DNA synthesis (Table 3). When they are taken together, the results of these studies suggest that this compound is likely phosphorylated by the viral TK and that at least one of the phosphorylated metabolites affects the synthesis of viral DNA. Nevertheless, the TK-resistant mutants were still partially sensitive to the drug, raising the possibility that other viral or cellular enzymes may also contribute to the phosphorylation of the compound.

In vivo activity of 4′-thioIDU.To determine if 4′-thioIDU was active in an animal model infection, mice were inoculated intranasally with either vaccinia or cowpox virus. 4′-ThioIDU given i.p. twice daily beginning at 24 h after viral inoculation was highly effective (P < 0.001) at reducing or eliminating mortality when it was used at a dose of 50, 15, or 5 mg/kg of body weight against vaccinia virus or 15, 5, or 1.5 mg/kg against cowpox virus. As a comparison, CDV was used as a positive control and was found to provide complete protection at a dose of 15 mg/kg (Table 4). Since it would be highly desirable for a drug for use for the treatment of poxvirus infections to be active when it was given by the oral route, we next determined the activity of 4′-thioIDU when it was given orally. The drug was administered twice daily beginning at 1, 2, 3, or 4 days after viral inoculation at a concentration of 15, 5, or 1.5 mg/kg and was highly effective (P ≤ 0.0001) in preventing mortality against cowpox virus infection when treatment was initiated at as late as 4 days after infection but was ineffective when treatment with 5 mg/kg was begun at 5 days after infection (Table 5). The positive control, CDV, at 15 mg/kg significantly reduced the rate of mortality against cowpox virus infection, even if the initiation of therapy was delayed until 8 days postinfection. In other experiments whose results are not presented here, 4′-thioIDU significantly reduced the rate of mortality when it was given orally at 0.3 or 1.0 mg/kg and when treatment was initiated at 24 h after cowpox virus infection.

To determine the effect of oral treatment with 4′-thioIDU compared with that of CDV on the replication of vaccinia or cowpox virus in the target organs of mice, animals were inoculated with virus and were treated orally with 15 mg of 4′-thioIDU per kg twice daily beginning at 24 h after infection; CDV was given at 15 mg/kg i.p. once daily. Both of the treatment regimens resulted in significant decreases in the rates of mortality of the control mice (data not shown). In mice infected with vaccinia virus, the virus titers in the liver, spleen, and kidney tissues of both 4′-thioIDU- and CDV-treated mice were reduced to undetectable levels, about a 5-log₁₀-unit reduction, but there was only about a 1-log₁₀-unit decrease in the virus titers in lung tissue (Fig. 2). When mice were infected with cowpox virus, treatment with 4′-thioIDU resulted in a 2-log₁₀-unit reduction in virus titers in the liver, spleen, and kidney tissues (data not shown).

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FIG. 2.

Effects of oral treatment with 4′-thioIDU on pathogenesis of vaccinia virus infection in mice.