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Dynamics of the antiviral activity of N-methanocarbathymidine against herpes simplex virus type 1 in cell culture
International Journal of Antimicrobial Agents 25 (2005) 427–432
Dynamics of the antiviral activity of N-methanocarbathymidine against herpes simplex virus type 1 in cell culture Mahmoud Huleihel a, ∗ , Marina Talishanisky a , Harry Ford Jr. b , Victor E. Marquez b , James A. Kelley b , David G. Johns b , Riad Agbaria c a
c
The Institute for Applied Biosciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA Department of Clinical Pharmacology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Received 8 November 2004; accepted 12 January 2005
Abstract N-Methanocarbathymidine [(N)-MCT], a thymidine analogue, exhibits potent activity in cell culture against herpes simplex virus1 (HSV-1). (N)-MCT showed higher antiviral activity than ganciclovir (GCV). Continuous treatment of Vero cells with (N)-MCT immediately or 10 h post-infection (p.i.) fully prevented the development of viral infection. However, when infected cells were treated with (N)-MCT at 12 h p.i., there was only a partial inhibition (ca. 50%). Additionally, continuous treatment of infected cells with (N)-MCT for about 48 h was sufficient to achieve full prevention of viral infection without further treatment. These findings suggest the complete loss of herpes simplex thymidine kinase (HSV-tk) activity occurs after 48 h of treatment with (N)-MCT. This study helps to understand the mechanism and dynamics of antiHSV activity of (N)-MCT, which is necessary for its future development as an antiviral drug. © 2005 Published by Elsevier B.V. and the International Society of Chemotherapy. Keywords: Herpes simplex virus; Herpes simplex thymidine kinase; N-Methanocarbathymidine; Ganciclovir; Antiviral activity; Phosphorylation
1. Introduction Viruses are considered to be one of the main causes of serious human and animal infectious diseases. Despite numerous studies seeking to find relevant antiviral therapy, there is still a need for new antiviral drugs. Nucleoside analogues, such as ganciclovir (GCV) and acyclovir (ACV) and other agents, such as inhibitors of the herpes simplex virus (HSV) helicase–primase have been found to possess potent antiviral activity against various members of herpes family of viruses (HSV-1 and HSV-2) [1–7]. The antiviral mechanism of action of the nucleoside analogues is through their selective phosphorylation in HSV-infected cells, by herpes simplex thymidine kinase ∗
Corresponding author. Tel.: +972 8 646 1999; fax: +972 8 647 2970. E-mail address: [email protected] (M. Huleihel).
(HSV-tk) [1,8–12] and subsequent inhibition of herpes DNA polymerase by the drug’s 5 -triphosphate form [13–15]. N-Methanocarbathymidine [(N)-MCT], a nucleoside analogue with a pseudosugar moiety rigidly fixed in the north conformation (Fig. 1), was previously synthesized [16] and shown by our group to exhibit potent antiviral activity against HSV-1 and HSV-2 with higher potency than ACV and GCV [16,17]. In addition, we have recently demonstrated high levels of phosphorylated (N)-MCT in HSV-1- and HSV-2infected cells, suggesting that such phosphorylation is involved in its antiviral activity [17]. (N)-MCT was also shown to have antitumour activity against murine and human tumour cells transduced with the HSV-tk gene in vitro and in vivo without any cytotoxic effect on the non-transduced tumour cells [18]. To further understand the dynamics of the antiviral activity and mechanism of action of (N)-MCT against herpes viruses,
0924-8579/$ – see front matter © 2005 Published by Elsevier B.V. and the International Society of Chemotherapy. doi:10.1016/j.ijantimicag.2005.01.013
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2.3. Cell infection and determination of cytopathic effect (CPE)
Fig. 1. Chemical structure of N-methanocarbathymidine [(N)-MCT].
we examined its antiviral activity as a function of the time of addition or removal of (N)-MCT from the cell culture media. Our results show that addition of (N)-MCT to infected cells in culture with 1 multiplicity of infection (m.o.i.) of HSV-1 at no later than 10 h p.i., completely prevented the development of viral infection. These findings indicate that (N)-MCT is most effective against the development of infection by HSV-1, when it is added prior to the end of viral DNA synthesis. Furthermore, the results are in full agreement with the role of the phosphorylated form of (N)-MCT in inhibiting HSV DNA synthesis as we reported previously [17].
2. Materials and methods 2.1. Materials (N)-MCT was synthesized as previously described [16]. GCV (cytovene IV) was obtained from Hoffman La Roche Laboratories (Nutley, NJ) and [3 H] GCV (22 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). The radiochemical purity of this substance, as determined by HPLC, was >99%. Other nucleoside and nucleotide standards were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents and chemicals were of the highest quality obtainable. 2.2. Cells and virus African green monkey kidney (Vero) cells were purchased from the American Type Culture Collection (ATCC), Rockville, MD. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal calf serum (FCS), 1% glutamine, 50 U/ml penicillin and 50 g/ml streptomycin and incubated at 37 ◦ C in humidified air containing 5% CO2 . HSV-1 was obtained from ATCC (VR735). These viruses were propagated to >107 to 108 plaqueforming units (PFU)/ml in Vero cells, and the titre was estimated by a standard plaque assay as described previously [19].
Monolayers of Vero cells (seeded at 1.5 × 105 cells/well on 24-well culture plates) were incubated with HSV-1 at the appropriate multiplicity of infection in RPMI medium containing 2% FCS at 37 ◦ C for 2 h. The unabsorbed virus particles were removed, fresh medium containing 2% FCS was added, and the cell monolayers were incubated at 37 ◦ C. At various times p.i., the cytopathic effect in the cell culture was examined under an inverted microscope. The cytopathic effect was expressed as percentage of damaged cells in the inspection field. The drug concentrations conferring 50% (IC50 ) or 95% (IC95 ) inhibition of CPE, were calculated on quadruplicate HSV-1-infected cell monolayers using CalcuSyn software [20]. 2.4. (N)-MCT and GCV cytotoxicity The cytotoxic effects of (N)-MCT and the reference standard GCV were determined as described previously [17]. Briefly, exponentially growing Vero cells (105 cells) were cultured in 24-well plates overnight. Cells were then washed with increasing concentrations (0–150 M) of (N)-MCT or the reference compound GCV. After an additional 48 h, cells were harvested by trypsinization, collected and counted in a coulter counter. The cell growth rate was expressed as a percentage of the increase in cell number of the untreated control cultures and the CC50 (drug concentration causing 50% inhibition of non-infected cell growth) was calculated using Calc CalcuSyn software as mentioned above. 2.5. Determination of GCV phosphorylation and metabolites analysis Vero cell cultures (3 × 106 to 5 × 106 cells/(25-cm2 flasks)) were infected with 1 m.o.i. of HSV-1 for 2 h. Thereafter, medium was removed and replaced by fresh medium with or without (N)-MCT. At the end of this incubation, the medium was replaced with fresh medium containing [3 H] GCV. After 2 h incubation, cells were washed three times with PBS to remove any residual drug, then trypsinized and pelleted. The cell pellets were suspended in 250 l of 60% methanol (HPLC grade) and heated for 3 min at 95 ◦ C. After centrifugation at 12,000 × g for 10 min, the clear supernatant fractions were collected, evaporated under nitrogen and redissolved in 250 l of distillated water. Aliquots of the latter reconstituted samples were subjected to anion-exchange chromatography as described below. 2.6. HPLC separation of GCV metabolites 2.6.1. Gradient anion-exchange HPLC The separation of GCV and its phosphorylated metabolites was carried out using a Hewlett Packard 1100 HPLC with a diode-array ultraviolet absorption detector as de-
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scribed by Zaleh et al. [17]. A Partisil-10 SAX column (250 mm × 4.6 mm) (Whatman Inc., NJ) was used with the following elution programme: 0–5 min, 100% buffer A (0.01 M ammonium phosphate, native pH); 5–20 min, linear gradient to 25% buffer B (0.7 M ammonium phosphate with 10% methanol); 20–30 min, linear gradient to 100% buffer B; 30–40 min, 100% buffer B; 40–55 min, linear gradient to 100% buffer A and equilibration. The flow rate was 2 ml/min; 1-min fractions were collected and radioactivity was determined by scintillation spectrometry. The retention time of GCV and its phosphates were as follows: GCV, 3 min; GCV-MP, 9 min; GCV-DP, 24 min; GCV-TP, 33 min. Fractions containing radiolabelled GCV nucleotides were quantitated based on the known specific activity of the parent tritiated nucleoside.
3. Results 3.1. Antiviral activity of (N)-MCT against HSV-1 The antiviral activity of (N)-MCT against HSV-1 was tested by examining its effect on the CPE formation by this virus. As a positive control, the antiviral activity of GCV against the same viruses was also tested. Vero cell monolayers were treated with increasing concentrations of the drugs for 1 h before infection with 1 m.o.i. of HSV-1 virus. Treatment with the drug continued after infection up to the end of the experiment. Under these experimental conditions (N)MCT and GCV showed a significant and reproducible antiviral activity against HSV-1 with the respective IC50s , 0.036 and 0.267 M (Fig. 2A). These results showed also that the IC95 is 0.147 for MCT and 0.475 for GCV. Both drugs had no significant cytotoxicity against uninfected Vero cells, CC50 >150 M (Fig. 2B). 3.2. Effect of time of (N)-MCT addition on the development of HSV-I infection Vero cells were infected with 1 m.o.i of HSV-1 and treated with 1 M of (N)-MCT or GCV at various times p.i. Treatment with the drug continued up to the end of the experiment. The results showed that when either (N)-MCT or GCV was added to the infected cell culture immediately or 8 h p.i., there was full prevention of CPE development (Fig. 3A and B). In contrast, when (N)-MCT was added at 12 h p.i., it partially inhibited the development of viral infection (ca. 60% protection) as seen in Fig. 3A. It can also be seen that both drugs were able to stop progression of CPE at day 3 p.i., an event that was followed by a significant decrease in CPE as a function of time (Fig. 3A and B). When cells were infected with 0.01 m.o.i. of HSV-1 and treated at various times p.i. with 1 M of (N)-MCT or GCV, they provided full protection against the development of the CPE even when they were added at 24 h p.i. However, when the drug was added at 48 h p.i., only a small fraction of the
Fig. 2. Antiviral activity and cytotoxicity of (N)-MCT and GCV: (A) Vero cell monolayers were treated with increasing concentrations (0–1 M) of (N)-MCT (䊉) or GCV () at 1 h before infection with 1 m.o.i. of HSV-1. Treatment with the drug was continued up to the end of the experiment. CPE was determined at day 3 p.i., and drug IC50s were calculated as described in Section 2. (B) Drug cytotoxicity in uninfected Vero cells was determined by culturing Vero cells for 48 h with increasing concentrations (0–150 M) of (N)-MCT (䊉) or GCV (). At the end of incubation, cell growth rate was determined, and drug CC50s were calculated as described in Section 2. Data are mean ± S.D. (n = 4).
cells (15–20%) developed CPE, although the development of CPE stopped at day 4 p.i. and started to decrease, reaching basal levels at day 8 (Fig. 4A and B). 3.3. Effect of time of (N)-MCT removal on HSV-1 infection development Vero cells were infected with either 0.01 or 1 m.o.i. of HSV-1 and treated with 1 M of either (N)-MCT or GCV at the time of infection. The drug was removed from the culture medium at various times p.i. The results showed that in cell cultures infected with 1 m.o.i. of HSV-1, there was no inhibitory effect on viral CPE development when the drug (MCT or GCV) was removed at 2 h p.i. (Fig. 5A and B). When the drug was removed at 10 or 24 h p.i., there was a significant delay (3–4 days) in CPE development followed by a rise in CPE, although at a slower rate compared with untreated cultures. Alternatively, when (N)-MCT was removed at 48 h p.i. or later, there was full prevention of CPE development (Fig. 5A), whilst in the case of GCV, there was a full prevention of CPE development only, when it was removed at 60 h or later (Fig. 5B).
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Fig. 3. Effect of time of (N)-MCT addition on the kinetics of CPE development after infection with HSV-1 (1 m.o.i.). Vero cells were infected with 1 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at 8 h (), 12 h (), 18 h () or 24 h () p.i. Treatment continued up to the end of the experiment. As a control, HSV-1-infected cells without treatment with the drug were used (䊉). Data are mean ± S.D. (n = 4).
Fig. 5. Effect of time of (N)-MCT removal on the kinetics of CPE development after infection with HSV-1 (1 m.o.i.). Vero cells were infected with 1 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at the time of infection. The medium containing (N)-MCT was removed and replaced with drug-free medium at 2 h (), 10 h (), 24 h (), 48 h () or 60 h () p.i. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 3).
In the case of cells infected with 0.01 m.o.i. of HSV-1, there was full prevention of CPE development even when the drug was removed 24 h p.i. in the case of (N)-MCT (Fig. 6A) or 36 h p.i. in the case of GCV (Fig. 6B). Furthermore, when the drug was removed at 10 h p.i., there was a considerable delay in the appearance of CPE and a significant reduction in the rate of CPE development (Fig. 6A and B). 3.4. Levels of GCV phosphates in HSV-1-infected cells after various periods of time of treatment with (N)-MCT.
Fig. 4. Effect of time of (N)-MCT addition on the kinetics of CPE development after infection with HSV-1 (0.01 m.o.i.). Vero cells were infected with a 0.01 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at 24 h () or 48 h () p.i. Treatment was continued until the end of the experiment. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 4).
The phosphorylation rate of GCV was examined in HSV1-infected cells after different intervals of treatment with (N)MCT as an indication for the amount of active virus particles after such a treatment with (N)-MCT. First, Vero cells were infected with 1 m.o.i. of HSV-1 and treated immediately with 1 M of drug. At 24 or 48 h p.i., cells were washed three times and incubated with fresh medium for an additional 48 h. Thereafter, the cells were incubated for 2 h with fresh medium containing [3 H] GCV, 10 M, 5 Ci/ml and at the end of the incubation, the levels of GCV-phosphorylated metabolites in cell extracts were measured as detailed above. The results showed that infected cells treated continuously with (N)-MCT for 48 h produced only basal levels of di- or triphosphate metabolites similar to those measured in non-infected cells (Table 1). However, cells treated for 24 h with (N)-MCT showed considerable levels of di- or triphosphate metabo-
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Fig. 6. Effect of time of (N)-MCT removal on the kinetics of CPE development after infection with HSV-1 (0.01 m.o.i.). Vero cells were infected with 0.01 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at the time of infection. The medium containing (N)-MCT was removed and replaced with drug-free medium at 2 h (), 10 h (), 24 h () or 36 h () p.i. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 3).
lites of (N)-MCT, although these levels were significantly lower compared with their levels in infected untreated cells (Table 1). These results are consistent with the data presented in Fig. 5 showing that treatment of HSV-1-infected cells with (N)-MCT for 48 h completely inactivated the infecting virus, whereas treatment for 24 h offered only partial inactivation.
4. Discussion In the present study, we have demonstrated that (N)-MCT significantly inhibits HSV-1 infection in Vero cells. (N)MCT showed higher potency (IC50 = 0.036 M) compared with GCV (IC50 = 0.267 M), which was used as a positive control, with no cytotoxic effects against the non-infected
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cells even at concentrations 1000 times greater than those necessary for inhibiting the virus development (Fig. 2). Our previous studies have demonstrated the production of high levels of the 5 -triphosphate metabolite of (N)-MCT in HSV-1-infected cells [17] and in HSV-tk-transduced cancer cells [21]. These data strongly support the possibility that the formation of the 5 -triphosphate metabolite of (N)-MCT plays a role in its antiviral activity against herpes viruses. The favourable substrate kinetics and binding properties of (N)-MCT with the viral kinase (HSV-tk) and the limited activity with cytoplasmic thymidine kinase [17,22] supports this hypothesis. Furthermore, we have shown that when Vero cells were infected with 1 m.o.i. of HSV-1, full prevention of virus infection was achieved when (N)-MCT was added 8 h p.i. On the other hand, when (N)-MCT was added at 12 h p.i., there was only partial inhibition of viral infection (Fig. 3). Based on previous reports that showed that the majority of HSV–DNA synthesis occurs between 8 and 12 h p.i. [23], our observations strongly support the notion that formation of the 5 -triphosphate metabolite of (N)-MCT is crucial for its antiviral activity, in keeping with the known activity of other 5 -triphosphate metabolites of nucleoside drugs capable of inhibiting the viral DNA synthesis [13–15]. These results also fit very well with the well-known mechanism of action of GCV and ACV where the herpes DNA polymerase is inhibited by the drugs’ 5 -triphosphates [13–15]. The results displayed in Figs. 5 and 6 also demonstrate that the inhibitory effect of (N)-MCT could be reversed when treatment with (N)-MCT was terminated 10 or 24 h p.i., depending on the titre of the infecting virus. Conversely, the (N)-MCT inhibitory effect was completely irreversible, when the treatment was terminated at 24 or 48 h p.i., respectively. Our experiments which examined the ability of HSV-1infected cells to produce phosphorylated metabolites of GCV after 24 or 48 h of treatment with (N)-MCT showed that when the treatment was terminated at 48 h p.i., there was no significant formation of GCV-phosphorylated metabolites. When the treatment was terminated at 24 h p.i., only low levels of phosphorylated metabolites were formed (Table 1). The lack of GCV-phosphorylated metabolites may be a result of losing HSV-tk activity. These results may suggest that HSV-tk (and probably other viral components) could persist in the infected cells without losing activity, only for limited time. However,
Table 1 Levels of GCV phosphates in uninfected and HSV-1-infected Vero cells after various treatment intervals with (N)-MCT GCV metabolites
GCV-MP GCV-DP GCV-TP
Without (N)-MCT
With (N)-MCT
Uninfected cells
Infected cells
Infected cells (24 h)
Infected cells (48 h)
0.53 ± 0.04 0.60 ± 0.06 0.75 ± 0.06
1.68 ± 0.18 6.50 ± 0.52 11.00 ± 0.33
0.61 ± 0.02 0.92 ± 0.07 1.88 ± 0.12
0.54 ± 0.06 0.52 ± 0.08 0.51 ± 0.03
All values are pmole/106 cells. Uninfected and HSV-1-infected Vero cells were cultured in the presence or absence of (N)-MCT (1 M) for 24 or 48 h. At the end of incubation, cells were washed three times with drug-free medium and grown in fresh medium for an additional 48 h. Thereafter, cells were washed with fresh medium and reincubated with [3 H] GCV (10 M, 5 Ci/ml) for 2 h. Cells were collected and extracted for GCV phosphate analysis as detailed in Section 2. Data are mean ± S.D. (n = 3).
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these data are not in agreement with the results reported by Cheng et al. [24], who showed that GCV could be removed from the infected cells at 8 h p.i. without a subsequent increase in viral replication. In our study, the inhibitory effect of GCV was irreversible only when it was removed from the infected culture at 36 or 60 h p.i., depending on the amount of infecting virus (Figs. 5B and 6B). The discrepancy could be attributed to experimental differences, such as the titre of the infecting virus and the nature of the host cells. Additional differences between (N)-MCT and GCV could be due to the slower decay rate of (N)-MCT-TP compared with GCV-TP as previously shown [17]. In conclusion, our results in this study showed that treatment of HSV-1-infected cell culture with (N)-MCT for only limited time (24–48 h) may offer full irreversible prevention of viral infection development in cell culture.
References [1] Elion GB, Furman PA, Fyfe JA, et al. The selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. Proc Natl Acad Sci USA 1977;74:5716–20. [2] Reefschlager J, Barwolff D, Herrmann G, et al. Antiherpes activity of some novel analogoues of (E)-5-(2-bromovinyl)-2 -deoxyuridine (E-BrVUdR) in two different cell lines. Acta Virol 1984;28: 1–10. [3] Larsson A, Sundqvist A, Parnerud AM. Inhibition of herpes virusinduced DNA polymerases and cellular DNA polymerase alpha by triphosphates of acyclic guanosine analogs. Mol Pharmacol 1986;29:614–21. [4] Macchia, Antonelli G, Calvani F, et al. Synthesis and antiviral properties of 9-(2-methyleneaminoxyethoxy) guanine derivatives as novel cyclovir analogues. Il Farmacol 2000;55:104–8. [5] Betz UA, Fischer R, Kletmann G, et al. Potent in vitro antiviral activity of the herpes simplex virus helicase: primase inhibitor BAY 57-1293. Antimicrob Agents Chemother 2002;46:1766–72. [6] Cruse JJ, Grygon CA, Hargrave KD, et al. Herpes simplex virus helicase–primase inhibitors are active in animal models of human disease. Nat Med 2002;8:386–91. [7] Kleymann G, Fischer R, Betz UA, et al. New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat Med 2002;8:392–8. [8] Fyfe JA, Keller PM, Furman PA, et al. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound 9-(2hydroxyethoxymethyl)guanine. J Biol Chem 1978;253:8721–7. [9] De Clercq E, Descamps J, Verhelst G, et al. Comparative efficacy of antiherpes drugs against different strains of herpes simplex virus. J Infect Dis 1980;141:563–74.
[10] Field AK, Davies ME, DeWitt C, et al. 9-([2-Hydroxy-1hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci USA 1983;80:4139–43. [11] Smee DF, Martin JC, Verheyden JP, et al. Anti-herpesvirus activity of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine. Antimicrob Agents Chemother 1983;23:676–82. [12] Snoeck R. Antiviral therapy of herpes simplex. Int J Antimicrob Agents 2000;16:157–9. [13] Derse D, Cheng YC, Furman PA, et al. Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate: effects on primertemplate function. J Biol Chem 1981;256:11447–51. [14] Germershausen J, Bostedor R, Field AK. A comparison of the antiviral agents 2 -nor-2 -deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates. Biochem Biophys Res Commun 1983;6:360–7. [15] Frank KB, Chiou JF, Cheng YC. Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2propoxymethyl)guanine triphosphate. J Biol Chem 1984;259:1566–9. [16] Marquez VE, Siddiqui MA, Ezzitouni A. Nucleosides with a twist: can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides? J Med Chem 1996;39:3739–47. [17] Zalah L, Huleihel M, Konson A, et al. Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type-1-infected vero cells. Antiviral Res 2002;55:63–75. [18] Noy R, Ben-Zvi Z, Manor E, et al. Antitumor activity and metabolic activation of (N)-methanocarbathymidine, a novel thymidine analog with a pseudosugar rigidly fixed in the northern conformation, in murine colon cancer cells expressing herpes simplex thymidine kinase. Mol Cancer Ther 2002;1:585–93. [19] Huleihel M, Ishanu V, Tal J, et al. Antiviral effect of red microalgal polysaccharides on herpes simplex and varicella zoster viruses. J Appl Phycol 2001;13:127–34. [20] Chou TC, Haball M. CalcuSyn for Windows: multiple-drug doseeffect analyzer and manual. Cambridge, UK: Biosoft; 1996. [21] Agbaria R, Mullen CA, Hartman NR. Effects of IMP dehydrogenase inhibitors on the phosphorylation of ganciclovir in MOLT-4 cells before and after herpes simplex virus thymidine kinase gene transduction. Mol Pharmacol 1994;45:777–82. [22] Prota A, Vogt J, Pilger B, et al. Kinetic and crystal structure of the wild-type and engineered Y101F mutant of the herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarbathymidine. Biochemistry 2000;39:9597–603. [23] Dulbecco R, Ginsberg H. Herpes simplex viruses replication. In: Virology. 2nd ed. Philadelphia: J.B. Lippincott Company; 1988. p. 164–65. [24] Cheng YC, Grill SP, Dutschman GE, et al. Metabolism of 9(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells. J Biol Chem 1983;258:12460–4.
Dynamics of the antiviral activity of N-methanocarbathymidine against herpes simplex virus type 1 in cell culture Mahmoud Huleihel a, ∗ , Marina Talishanisky a , Harry Ford Jr. b , Victor E. Marquez b , James A. Kelley b , David G. Johns b , Riad Agbaria c a
c
The Institute for Applied Biosciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel b Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA Department of Clinical Pharmacology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Received 8 November 2004; accepted 12 January 2005
Abstract N-Methanocarbathymidine [(N)-MCT], a thymidine analogue, exhibits potent activity in cell culture against herpes simplex virus1 (HSV-1). (N)-MCT showed higher antiviral activity than ganciclovir (GCV). Continuous treatment of Vero cells with (N)-MCT immediately or 10 h post-infection (p.i.) fully prevented the development of viral infection. However, when infected cells were treated with (N)-MCT at 12 h p.i., there was only a partial inhibition (ca. 50%). Additionally, continuous treatment of infected cells with (N)-MCT for about 48 h was sufficient to achieve full prevention of viral infection without further treatment. These findings suggest the complete loss of herpes simplex thymidine kinase (HSV-tk) activity occurs after 48 h of treatment with (N)-MCT. This study helps to understand the mechanism and dynamics of antiHSV activity of (N)-MCT, which is necessary for its future development as an antiviral drug. © 2005 Published by Elsevier B.V. and the International Society of Chemotherapy. Keywords: Herpes simplex virus; Herpes simplex thymidine kinase; N-Methanocarbathymidine; Ganciclovir; Antiviral activity; Phosphorylation
1. Introduction Viruses are considered to be one of the main causes of serious human and animal infectious diseases. Despite numerous studies seeking to find relevant antiviral therapy, there is still a need for new antiviral drugs. Nucleoside analogues, such as ganciclovir (GCV) and acyclovir (ACV) and other agents, such as inhibitors of the herpes simplex virus (HSV) helicase–primase have been found to possess potent antiviral activity against various members of herpes family of viruses (HSV-1 and HSV-2) [1–7]. The antiviral mechanism of action of the nucleoside analogues is through their selective phosphorylation in HSV-infected cells, by herpes simplex thymidine kinase ∗
Corresponding author. Tel.: +972 8 646 1999; fax: +972 8 647 2970. E-mail address: [email protected] (M. Huleihel).
(HSV-tk) [1,8–12] and subsequent inhibition of herpes DNA polymerase by the drug’s 5 -triphosphate form [13–15]. N-Methanocarbathymidine [(N)-MCT], a nucleoside analogue with a pseudosugar moiety rigidly fixed in the north conformation (Fig. 1), was previously synthesized [16] and shown by our group to exhibit potent antiviral activity against HSV-1 and HSV-2 with higher potency than ACV and GCV [16,17]. In addition, we have recently demonstrated high levels of phosphorylated (N)-MCT in HSV-1- and HSV-2infected cells, suggesting that such phosphorylation is involved in its antiviral activity [17]. (N)-MCT was also shown to have antitumour activity against murine and human tumour cells transduced with the HSV-tk gene in vitro and in vivo without any cytotoxic effect on the non-transduced tumour cells [18]. To further understand the dynamics of the antiviral activity and mechanism of action of (N)-MCT against herpes viruses,
0924-8579/$ – see front matter © 2005 Published by Elsevier B.V. and the International Society of Chemotherapy. doi:10.1016/j.ijantimicag.2005.01.013
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2.3. Cell infection and determination of cytopathic effect (CPE)
Fig. 1. Chemical structure of N-methanocarbathymidine [(N)-MCT].
we examined its antiviral activity as a function of the time of addition or removal of (N)-MCT from the cell culture media. Our results show that addition of (N)-MCT to infected cells in culture with 1 multiplicity of infection (m.o.i.) of HSV-1 at no later than 10 h p.i., completely prevented the development of viral infection. These findings indicate that (N)-MCT is most effective against the development of infection by HSV-1, when it is added prior to the end of viral DNA synthesis. Furthermore, the results are in full agreement with the role of the phosphorylated form of (N)-MCT in inhibiting HSV DNA synthesis as we reported previously [17].
2. Materials and methods 2.1. Materials (N)-MCT was synthesized as previously described [16]. GCV (cytovene IV) was obtained from Hoffman La Roche Laboratories (Nutley, NJ) and [3 H] GCV (22 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). The radiochemical purity of this substance, as determined by HPLC, was >99%. Other nucleoside and nucleotide standards were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents and chemicals were of the highest quality obtainable. 2.2. Cells and virus African green monkey kidney (Vero) cells were purchased from the American Type Culture Collection (ATCC), Rockville, MD. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal calf serum (FCS), 1% glutamine, 50 U/ml penicillin and 50 g/ml streptomycin and incubated at 37 ◦ C in humidified air containing 5% CO2 . HSV-1 was obtained from ATCC (VR735). These viruses were propagated to >107 to 108 plaqueforming units (PFU)/ml in Vero cells, and the titre was estimated by a standard plaque assay as described previously [19].
Monolayers of Vero cells (seeded at 1.5 × 105 cells/well on 24-well culture plates) were incubated with HSV-1 at the appropriate multiplicity of infection in RPMI medium containing 2% FCS at 37 ◦ C for 2 h. The unabsorbed virus particles were removed, fresh medium containing 2% FCS was added, and the cell monolayers were incubated at 37 ◦ C. At various times p.i., the cytopathic effect in the cell culture was examined under an inverted microscope. The cytopathic effect was expressed as percentage of damaged cells in the inspection field. The drug concentrations conferring 50% (IC50 ) or 95% (IC95 ) inhibition of CPE, were calculated on quadruplicate HSV-1-infected cell monolayers using CalcuSyn software [20]. 2.4. (N)-MCT and GCV cytotoxicity The cytotoxic effects of (N)-MCT and the reference standard GCV were determined as described previously [17]. Briefly, exponentially growing Vero cells (105 cells) were cultured in 24-well plates overnight. Cells were then washed with increasing concentrations (0–150 M) of (N)-MCT or the reference compound GCV. After an additional 48 h, cells were harvested by trypsinization, collected and counted in a coulter counter. The cell growth rate was expressed as a percentage of the increase in cell number of the untreated control cultures and the CC50 (drug concentration causing 50% inhibition of non-infected cell growth) was calculated using Calc CalcuSyn software as mentioned above. 2.5. Determination of GCV phosphorylation and metabolites analysis Vero cell cultures (3 × 106 to 5 × 106 cells/(25-cm2 flasks)) were infected with 1 m.o.i. of HSV-1 for 2 h. Thereafter, medium was removed and replaced by fresh medium with or without (N)-MCT. At the end of this incubation, the medium was replaced with fresh medium containing [3 H] GCV. After 2 h incubation, cells were washed three times with PBS to remove any residual drug, then trypsinized and pelleted. The cell pellets were suspended in 250 l of 60% methanol (HPLC grade) and heated for 3 min at 95 ◦ C. After centrifugation at 12,000 × g for 10 min, the clear supernatant fractions were collected, evaporated under nitrogen and redissolved in 250 l of distillated water. Aliquots of the latter reconstituted samples were subjected to anion-exchange chromatography as described below. 2.6. HPLC separation of GCV metabolites 2.6.1. Gradient anion-exchange HPLC The separation of GCV and its phosphorylated metabolites was carried out using a Hewlett Packard 1100 HPLC with a diode-array ultraviolet absorption detector as de-
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scribed by Zaleh et al. [17]. A Partisil-10 SAX column (250 mm × 4.6 mm) (Whatman Inc., NJ) was used with the following elution programme: 0–5 min, 100% buffer A (0.01 M ammonium phosphate, native pH); 5–20 min, linear gradient to 25% buffer B (0.7 M ammonium phosphate with 10% methanol); 20–30 min, linear gradient to 100% buffer B; 30–40 min, 100% buffer B; 40–55 min, linear gradient to 100% buffer A and equilibration. The flow rate was 2 ml/min; 1-min fractions were collected and radioactivity was determined by scintillation spectrometry. The retention time of GCV and its phosphates were as follows: GCV, 3 min; GCV-MP, 9 min; GCV-DP, 24 min; GCV-TP, 33 min. Fractions containing radiolabelled GCV nucleotides were quantitated based on the known specific activity of the parent tritiated nucleoside.
3. Results 3.1. Antiviral activity of (N)-MCT against HSV-1 The antiviral activity of (N)-MCT against HSV-1 was tested by examining its effect on the CPE formation by this virus. As a positive control, the antiviral activity of GCV against the same viruses was also tested. Vero cell monolayers were treated with increasing concentrations of the drugs for 1 h before infection with 1 m.o.i. of HSV-1 virus. Treatment with the drug continued after infection up to the end of the experiment. Under these experimental conditions (N)MCT and GCV showed a significant and reproducible antiviral activity against HSV-1 with the respective IC50s , 0.036 and 0.267 M (Fig. 2A). These results showed also that the IC95 is 0.147 for MCT and 0.475 for GCV. Both drugs had no significant cytotoxicity against uninfected Vero cells, CC50 >150 M (Fig. 2B). 3.2. Effect of time of (N)-MCT addition on the development of HSV-I infection Vero cells were infected with 1 m.o.i of HSV-1 and treated with 1 M of (N)-MCT or GCV at various times p.i. Treatment with the drug continued up to the end of the experiment. The results showed that when either (N)-MCT or GCV was added to the infected cell culture immediately or 8 h p.i., there was full prevention of CPE development (Fig. 3A and B). In contrast, when (N)-MCT was added at 12 h p.i., it partially inhibited the development of viral infection (ca. 60% protection) as seen in Fig. 3A. It can also be seen that both drugs were able to stop progression of CPE at day 3 p.i., an event that was followed by a significant decrease in CPE as a function of time (Fig. 3A and B). When cells were infected with 0.01 m.o.i. of HSV-1 and treated at various times p.i. with 1 M of (N)-MCT or GCV, they provided full protection against the development of the CPE even when they were added at 24 h p.i. However, when the drug was added at 48 h p.i., only a small fraction of the
Fig. 2. Antiviral activity and cytotoxicity of (N)-MCT and GCV: (A) Vero cell monolayers were treated with increasing concentrations (0–1 M) of (N)-MCT (䊉) or GCV () at 1 h before infection with 1 m.o.i. of HSV-1. Treatment with the drug was continued up to the end of the experiment. CPE was determined at day 3 p.i., and drug IC50s were calculated as described in Section 2. (B) Drug cytotoxicity in uninfected Vero cells was determined by culturing Vero cells for 48 h with increasing concentrations (0–150 M) of (N)-MCT (䊉) or GCV (). At the end of incubation, cell growth rate was determined, and drug CC50s were calculated as described in Section 2. Data are mean ± S.D. (n = 4).
cells (15–20%) developed CPE, although the development of CPE stopped at day 4 p.i. and started to decrease, reaching basal levels at day 8 (Fig. 4A and B). 3.3. Effect of time of (N)-MCT removal on HSV-1 infection development Vero cells were infected with either 0.01 or 1 m.o.i. of HSV-1 and treated with 1 M of either (N)-MCT or GCV at the time of infection. The drug was removed from the culture medium at various times p.i. The results showed that in cell cultures infected with 1 m.o.i. of HSV-1, there was no inhibitory effect on viral CPE development when the drug (MCT or GCV) was removed at 2 h p.i. (Fig. 5A and B). When the drug was removed at 10 or 24 h p.i., there was a significant delay (3–4 days) in CPE development followed by a rise in CPE, although at a slower rate compared with untreated cultures. Alternatively, when (N)-MCT was removed at 48 h p.i. or later, there was full prevention of CPE development (Fig. 5A), whilst in the case of GCV, there was a full prevention of CPE development only, when it was removed at 60 h or later (Fig. 5B).
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Fig. 3. Effect of time of (N)-MCT addition on the kinetics of CPE development after infection with HSV-1 (1 m.o.i.). Vero cells were infected with 1 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at 8 h (), 12 h (), 18 h () or 24 h () p.i. Treatment continued up to the end of the experiment. As a control, HSV-1-infected cells without treatment with the drug were used (䊉). Data are mean ± S.D. (n = 4).
Fig. 5. Effect of time of (N)-MCT removal on the kinetics of CPE development after infection with HSV-1 (1 m.o.i.). Vero cells were infected with 1 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at the time of infection. The medium containing (N)-MCT was removed and replaced with drug-free medium at 2 h (), 10 h (), 24 h (), 48 h () or 60 h () p.i. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 3).
In the case of cells infected with 0.01 m.o.i. of HSV-1, there was full prevention of CPE development even when the drug was removed 24 h p.i. in the case of (N)-MCT (Fig. 6A) or 36 h p.i. in the case of GCV (Fig. 6B). Furthermore, when the drug was removed at 10 h p.i., there was a considerable delay in the appearance of CPE and a significant reduction in the rate of CPE development (Fig. 6A and B). 3.4. Levels of GCV phosphates in HSV-1-infected cells after various periods of time of treatment with (N)-MCT.
Fig. 4. Effect of time of (N)-MCT addition on the kinetics of CPE development after infection with HSV-1 (0.01 m.o.i.). Vero cells were infected with a 0.01 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at 24 h () or 48 h () p.i. Treatment was continued until the end of the experiment. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 4).
The phosphorylation rate of GCV was examined in HSV1-infected cells after different intervals of treatment with (N)MCT as an indication for the amount of active virus particles after such a treatment with (N)-MCT. First, Vero cells were infected with 1 m.o.i. of HSV-1 and treated immediately with 1 M of drug. At 24 or 48 h p.i., cells were washed three times and incubated with fresh medium for an additional 48 h. Thereafter, the cells were incubated for 2 h with fresh medium containing [3 H] GCV, 10 M, 5 Ci/ml and at the end of the incubation, the levels of GCV-phosphorylated metabolites in cell extracts were measured as detailed above. The results showed that infected cells treated continuously with (N)-MCT for 48 h produced only basal levels of di- or triphosphate metabolites similar to those measured in non-infected cells (Table 1). However, cells treated for 24 h with (N)-MCT showed considerable levels of di- or triphosphate metabo-
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Fig. 6. Effect of time of (N)-MCT removal on the kinetics of CPE development after infection with HSV-1 (0.01 m.o.i.). Vero cells were infected with 0.01 m.o.i. of HSV-1 and treated with 1 M of (N)-MCT (A) or GCV (B) at the time of infection. The medium containing (N)-MCT was removed and replaced with drug-free medium at 2 h (), 10 h (), 24 h () or 36 h () p.i. As a control, HSV-1-infected cells without treatment with (N)-MCT were used (䊉). Data are mean ± S.D. (n = 3).
lites of (N)-MCT, although these levels were significantly lower compared with their levels in infected untreated cells (Table 1). These results are consistent with the data presented in Fig. 5 showing that treatment of HSV-1-infected cells with (N)-MCT for 48 h completely inactivated the infecting virus, whereas treatment for 24 h offered only partial inactivation.
4. Discussion In the present study, we have demonstrated that (N)-MCT significantly inhibits HSV-1 infection in Vero cells. (N)MCT showed higher potency (IC50 = 0.036 M) compared with GCV (IC50 = 0.267 M), which was used as a positive control, with no cytotoxic effects against the non-infected
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cells even at concentrations 1000 times greater than those necessary for inhibiting the virus development (Fig. 2). Our previous studies have demonstrated the production of high levels of the 5 -triphosphate metabolite of (N)-MCT in HSV-1-infected cells [17] and in HSV-tk-transduced cancer cells [21]. These data strongly support the possibility that the formation of the 5 -triphosphate metabolite of (N)-MCT plays a role in its antiviral activity against herpes viruses. The favourable substrate kinetics and binding properties of (N)-MCT with the viral kinase (HSV-tk) and the limited activity with cytoplasmic thymidine kinase [17,22] supports this hypothesis. Furthermore, we have shown that when Vero cells were infected with 1 m.o.i. of HSV-1, full prevention of virus infection was achieved when (N)-MCT was added 8 h p.i. On the other hand, when (N)-MCT was added at 12 h p.i., there was only partial inhibition of viral infection (Fig. 3). Based on previous reports that showed that the majority of HSV–DNA synthesis occurs between 8 and 12 h p.i. [23], our observations strongly support the notion that formation of the 5 -triphosphate metabolite of (N)-MCT is crucial for its antiviral activity, in keeping with the known activity of other 5 -triphosphate metabolites of nucleoside drugs capable of inhibiting the viral DNA synthesis [13–15]. These results also fit very well with the well-known mechanism of action of GCV and ACV where the herpes DNA polymerase is inhibited by the drugs’ 5 -triphosphates [13–15]. The results displayed in Figs. 5 and 6 also demonstrate that the inhibitory effect of (N)-MCT could be reversed when treatment with (N)-MCT was terminated 10 or 24 h p.i., depending on the titre of the infecting virus. Conversely, the (N)-MCT inhibitory effect was completely irreversible, when the treatment was terminated at 24 or 48 h p.i., respectively. Our experiments which examined the ability of HSV-1infected cells to produce phosphorylated metabolites of GCV after 24 or 48 h of treatment with (N)-MCT showed that when the treatment was terminated at 48 h p.i., there was no significant formation of GCV-phosphorylated metabolites. When the treatment was terminated at 24 h p.i., only low levels of phosphorylated metabolites were formed (Table 1). The lack of GCV-phosphorylated metabolites may be a result of losing HSV-tk activity. These results may suggest that HSV-tk (and probably other viral components) could persist in the infected cells without losing activity, only for limited time. However,
Table 1 Levels of GCV phosphates in uninfected and HSV-1-infected Vero cells after various treatment intervals with (N)-MCT GCV metabolites
GCV-MP GCV-DP GCV-TP
Without (N)-MCT
With (N)-MCT
Uninfected cells
Infected cells
Infected cells (24 h)
Infected cells (48 h)
0.53 ± 0.04 0.60 ± 0.06 0.75 ± 0.06
1.68 ± 0.18 6.50 ± 0.52 11.00 ± 0.33
0.61 ± 0.02 0.92 ± 0.07 1.88 ± 0.12
0.54 ± 0.06 0.52 ± 0.08 0.51 ± 0.03
All values are pmole/106 cells. Uninfected and HSV-1-infected Vero cells were cultured in the presence or absence of (N)-MCT (1 M) for 24 or 48 h. At the end of incubation, cells were washed three times with drug-free medium and grown in fresh medium for an additional 48 h. Thereafter, cells were washed with fresh medium and reincubated with [3 H] GCV (10 M, 5 Ci/ml) for 2 h. Cells were collected and extracted for GCV phosphate analysis as detailed in Section 2. Data are mean ± S.D. (n = 3).
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these data are not in agreement with the results reported by Cheng et al. [24], who showed that GCV could be removed from the infected cells at 8 h p.i. without a subsequent increase in viral replication. In our study, the inhibitory effect of GCV was irreversible only when it was removed from the infected culture at 36 or 60 h p.i., depending on the amount of infecting virus (Figs. 5B and 6B). The discrepancy could be attributed to experimental differences, such as the titre of the infecting virus and the nature of the host cells. Additional differences between (N)-MCT and GCV could be due to the slower decay rate of (N)-MCT-TP compared with GCV-TP as previously shown [17]. In conclusion, our results in this study showed that treatment of HSV-1-infected cell culture with (N)-MCT for only limited time (24–48 h) may offer full irreversible prevention of viral infection development in cell culture.
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