Introduction

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Anti-herpes simplex virus agents, mainly nucleoside analogs such as ACV, have been developed (Elion et al., 1977) and used for the treatment of HSV infection in humans (Dunkle et al., 1991; Fiddian et al., 1984; Meyers et al., 1982; Whitley et al., 1991). However, the appearance of ACV-resistant HSV strains has become evident in immunosuppressed patients, such as organ transplant recipients and patients with acquired immunodeficiency syndrome (Birch et al., 1990; Coen, 1994; Erlich et al., 1989; Norris et al., 1988; Nugier et al., 1992; Oliver et al., 1989; Pelosi et al., 1992; Reusser et al., 1996; Sibrack et al., 1982). Those ACV-resistant viruses were also resistant to the other nucleoside analogs (Nugier et al., 1992). Thus the development of new therapeutic agents with different mode of anti-HSV action is required.

We previously selected 12 herbal extracts with oral therapeutic antiviral activity against cutaneous HSV-1 infection in mice from 142 herbal medicines (Kurokawa et al., 1993b). Among the 12 herbal extracts, oral administration of four extracts augmented the therapeutic efficacy of ACV in mice and showed potent anti-HSV-1 activity against ACV-PAA-resistant (AP^r) HSV-1 and wild HSV-2 strains in vitro and in vivo (Kurokawa et al., 1995a, b). These four herbal extracts also exhibited prophylactic efficacy against recurrent HSV-1 disease in mice (Kurokawa et al., 1997). Their anti-HSV action inhibited HSV DNA synthesis and the mode of anti-HSV action was different from those of ACV and PAA (Kurokawa et al., 1995b). Among these four herbal extracts, the EtOAc-extractable fraction of the herbal extract of Geum japonicum Thunb. exhibited anti-HSV-1 activity in a yield-reduction assay in vitro and a cutaneous HSV-1 infection model in mice (Kurokawa et al., 1993a). Thus, the EtOAc-extractable fraction may contain anti-HSV compounds that are absorbed from alimentary tracts after its oral administration.

In this study, we isolated and identified an anti-HSV compound, eugeniin, from the herbal extracts of not only G. japonicum but also Syzygium aromaticum (L.) MERR. et PERRY, which are two of the four herbal extracts with anti-HSV activity in vitro and in vivo reported previously (Kurokawa et al., 1993a, 1993b, 1995a, 1995b, 1997). Thus the action of the anti-HSV compound isolated coincidentally from the two different medicinal herbs was characterized. One of the major target sites of inhibitory action of eugeniin was found to be viral DNA synthesis. It showed higher specificity for the inhibition of HSV-1 DNA polymerase activity than that of cellular DNA polymerase activities. Its specificity for HSV-1 DNA polymerase as well as its therapeutic index was higher than those of Vidarabine (Ara-A) that has been used clinically for the treatment of herpetic disease (Coen et al., 1982; Seidlin and Straus, 1984; Whitley et al., 1986). The inhibitory action of eugeniin for HSV-1 DNA polymerase was noncompetitive, such as a non-nucleoside inhibitor of reverse transcriptase of HIV-1, Nevirapine (Carr and Cooper, 1996; Kilby and Saag, 1996; Kopp et al., 1991; Romero et al., 1991). Thus eugeniin may be a promising novel anti-HSV agent which is different from anti-HSV nucleoside analogs.

	Materials and Methods

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Viruses and cells. The HSV strains used were the wild-type 7401H HSV-1 (Kurokawa et al., 1993b), TK B2006 HSV-1 (Dubbs and Kit, 1964), AP^r HSV-1 (Kurokawa et al., 1995a) and the wild-type HSV-2 (Ito-1262) (Kurokawa et al., 1995a, 1995b). These virus stocks were prepared from infected Vero cells as reported previously (Kurokawa et al., 1995a, 1995b). Vero cells were grown and maintained in Eagle's MEM supplemented with 5% and 2% calf serum, respectively. For the preparation of HSV-1 DNA polymerase and cellular DNA polymerases  and , HEL cells were grown and maintained in MEM supplemented with 10% and 2% fetal bovine serum, respectively.

Fractionation of herbal extracts and identification of purified compounds. Anti-HSV compounds were purified from herbal extracts with use of different chromatographic fractionations guided by anti-HSV activity. Hot-water extracts were prepared from dried G. japonicum and then extracted with EtOAc as described previously (Kurokawa et al., 1993a, 1993b). The EtOAc-extractable fraction was further extracted with methanol. The methanol-soluble fraction was separated by Sephadex LH-20 column chromatography eluting 50% methanol in water and increasing the percentage of methanol. Each fraction separated was dried in vacuo. The residue was dissolved in DMSO at 20 mg/ml and examined for anti-HSV-1 activity by the plaque-reduction assay with Vero cells as described below. The fractions showing the strongest anti-HSV-1 activity among all fractions separated were further resolved by a reverse-phase HPLC (Shim-pack Prep-ODC(H) column, Shimadzu, Kyoto, Japan) with 27.5% of acetonitrile in 1/15 M phosphate buffer, pH 3.7. The fractions separated were extracted three times with EtOAc, and the collected EtOAc-soluble phases were dried in vacuo. The residue was dissolved in DMSO and examined for anti-HSV-1 activity. The chemical structure for a compound obtained from a fraction with the strongest anti-HSV-1 activity among the HPLC fractions was determined by comparing its NMR spectral, optical rotation data and mass spectrometric profiles with those in the literature (Lee et al., 1990; Nonaka et al., 1980). Eugeniin (fig. 1) was identified as an anti-HSV compound in the herbal extract of G. japonicum. This compound was also isolated as an anti-HSV compound from the herbal extract of S. aromaticum by EtOAc and methanol extractions and successive chromatographic separations as reported by Takechi and Tanaka (1981).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Structure of eugeniin.

Plaque-reduction assay and cytotoxicity assay. Fractions separated in each step for fractionations of herbal extracts were examined for anti-HSV activity in the plaque-reduction assay. Duplicate cultures of Vero cells in 60-mm plastic dishes were infected with 100 PFU of wild HSV-1 for 1 h. The cells were overlaid with 5 ml of nutrient methylcellulose (0.8%) medium containing various concentrations of samples and then cultured at 37°C for 2 days. The cells were fixed and stained, and the numbers of plaques were counted as described previously (Kurokawa et al., 1995a). The effective concentrations for 50% plaque reduction (EC₅₀) were determined from a curve relating the plaque number to the concentration of samples.

Yield-reduction assay. Eugeniin was compared for its antiviral activity against wild HSV-1, AP^r HSV-1, TK HSV-1 and wild HSV-2 strains in the yield-reduction assay. Monolayers of Vero cells in 25-cm² plastic flasks were infected with each HSV strain at 5 to 10 PFU/cell for 1 h. The cells were washed three times with MEM and incubated in maintenance medium containing various concentrations of eugeniin at 37°C for 24 h. The cultures were frozen and thawed and then centrifuged at 3,000 rpm for 15 min. Virus titers in culture medium were determined by the plaque assay on Vero cells (Kurokawa et al., 1995a).

Analysis of viral DNA synthesis. The effects of eugeniin and PAA on viral DNA synthesis were compared in Vero cells infected with wild HSV-1 or AP^r HSV-1 strains. Monolayers of Vero cells in 60-mm plastic dishes were infected with the HSV strains (5 PFU/cell) for 1 h and then incubated in maintenance medium containing various concentrations of eugeniin or 150 µg/ml of PAA at 37°C for 8 h. The cells were lysed in the presence of 0.5% SDS and 100 µg/ml of proteinase K, and then DNA was prepared from the lysates as described previously (Kurokawa et al., 1990, 1995b). DNA was blotted to nylon filters (Hybond-N, Amersham) by a slot-blot apparatus (Bio-Rad, Hercules, CA) and fixed by UV irradiation. The filters were prehybridized at 60°C for 4 h and then incubated with denatured radioactive probes at 60°C overnight. The radioactive probes were synthesized from the DNA fragments of the HSV-1 TK gene by a random primer DNA labeling kit (Takara Biochemicals, Kusatu, Japan) (Kurokawa et al., 1995b). The hybridized filters were washed, dried and exposed to X-ray films at 80°C (Kurokawa et al., 1990).

Analysis of viral protein synthesis. Eugeniin was examined for its effect on viral protein synthesis in Vero cells infected with wild HSV-1, AP^r HSV-1 or wild HSV-2 strains. The cells were mock-infected or infected with the HSV strains and incubated in the presence of various concentrations of eugeniin or 150 µg/ml of PAA at 37°C. HSV-infected and mock-infected cells were labeled with [³⁵S]methionine and [³⁵S]cysteine (43.5 TBq/mmol, NEN, Boston, MA) for 5.5 to 7.5 h postinfection in the presence of eugeniin or PAA as described above. The labeled cells were lysed and viral proteins were immunoprecipitated with immunoglobulin for human use (Miles Inc., Elkart, IN) (Kurokawa et al., 1995b). The immunoprecipitates and total cell lysates were subjected to SDS-polyacrylamide gel electrophoresis followed by fluorography (Kurokawa et al., 1990, 1995b). The dried gels were exposed to X-ray films at 80°C. Protein bands on the exposed films were scanned with a Bio-Rad's Image Analysis Systems with Fluor-S^TM MultiImager and quantitatively analyzed by Macintosh software, Multi-Analyst 1.0.

Preparation of DNA polymerases. HSV-1 DNA polymerase and cellular DNA polymerases  and  were prepared to examine the enzymological properties against eugeniin. HSV-1 DNA polymerase was partially purified from wild HSV-1-infected HEL cells by high-salt extraction, DEAE-cellulose and phosphocellulose column chromatography as described elsewhere (Nishiyama et al., 1984; Powell and Purifoy, 1977; Yoshida et al., 1974). Cellular DNA polymerases  and  were partially purified from HEL cells by DEAE-cellulose and phosphocellulose column chromatography as reported elsewhere (Ono, 1987; Ostrander and Cheng, 1980; Powell and Purifoy, 1977). The specific activities of HSV-1 DNA polymerase and cellular DNA polymerases  and  were 3,587, 29 and 76 U/mg of protein, respectively. One unit of activity was defined as an amount that catalyzes the incorporation of 1 nmol of [³H]dTMP into an acid-insoluble fraction in an hour at 37°C under the conditions specific for each enzyme with 6.84 µM [³H]dTTP (2.04-2.85 TBq/mmol, Moravek Biochemicals, Inc., Brea, CA) and 80 µM dATP, 80 µM dCTP and 80 µM dGTP.

Assay of DNA polymerase activity. The reaction mixture (25 µl) for an assay of partially purified HSV-1 DNA polymerase activity contained 50 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, 0.5 mM DTT, 100 mM ammonium sulfate, 80 µM dATP, 80 µM dGTP and 80 µM dCTP, 0.214 to 3.42 µM [³H]dTTP, 50 µg/ml activated calf thymus DNA (Sigma), various concentrations of eugeniin and enzyme. The reaction mixtures were incubated at 37°C for 10 min, and the acid-insoluble radioactivity was measured as described elsewhere (Nishiyama et al., 1984; Yoshida et al., 1974). The reaction mixture for an assay of partially purified DNA polymerase  activity was the same as for the HSV-1 DNA polymerase, except that ammonium sulfate was omitted and [³H]dTTP was used at concentrations of 0.428 to 6.84 µM. The reaction mixture (25 µl) for assay of partially purified DNA polymerase  activity contained 85 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 5 mM DTT, 0.5 mg/ml heat-inactivated BSA, 30 mM KCl, 50 µg/ml activated calf thymus DNA, 80 µM dATP, 80 µM dGTP and 80 µM dCTP, 0.855 to 6.89 µM [³H]dTTP, various concentrations of eugeniin and enzyme.

	Results

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Purification of eugeniin from herbal extracts. Eugeniin was isolated from the herbal extract of G. japonicum, and the chemical structure was identified by physicochemical analyses. The herbal extract was first extracted with EtOAc and the EtOAc-extractable fraction was further extracted with methanol to remove methanol-insoluble materials, mainly ellagic acid with cytotoxicity. The methanol-soluble fraction was separated into 63 fractions by Sephadex LH-20 column chromatography. The fractions 50 and 51 showed the strongest anti-HSV-1 activity among the 63 fractions, and their EC₅₀ values were 16.5 and 17.5 µg/ml, respectively (table 1). These fractions were separated further into six fractions (P1 to P6, table 1) by HPLC. Among the six fractions, fraction P4 exhibited the strongest anti-HSV-1 activity (EC₅₀ value, 5.0 ± 0.61 µg/ml). Its CC₅₀ values for Vero and HEL cells were 69.3 ± 9.4, 66 and 77 µg/ml. These CC₅₀ values were 13.9-, 13.2- and 15.4-fold, respectively, higher than the EC₅₀ value. Chemical structure for a compound obtained from fraction P4 was identified as eugeniin (fig. 1, 1,2,3-trigalloyl 4,6-hexahydroxydiphenoyl -D-glucopyranose, molecular weight, 938 daltons), which is a light yellow amorphous solid showing a quasi molecular ion peak at m/z: 939 [M+H]⁺; []_D+64.5° (c = 0.067, acetone) and soluble in water. This compound was also isolated as an anti-HSV compound from the herbal extract of S. aromaticum with anti-HSV activity in vitro and in vivo (Kurokawa et al., 1993a, 1995a, 1995b, 1997) (data not shown). We coincidentally purified eugeniin as a major anti-HSV compound from both extracts of G. japonicum and S. aromaticum, which belong to different families of plants.

Anti-HSV activity of eugeniin. Anti-HSV activity of eugeniin was compared with wild HSV-1, AP^r HSV-1, TK HSV-1 and wild HSV-2 strains in the yield-reduction assay with Vero cells (fig. 2). All four strains used were similarly susceptible to eugeniin and their virus yields were reduced more than 1,100-fold in the presence of 20 µg/ml eugeniin compared with its absence. Thus this compound exhibited potent antiviral activity against AP^r HSV-1, TK HSV-1 and wild HSV-2 strains as well as the wild HSV-1 strain.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Inhibition of the growth of various HSV strains by eugeniin. Eugeniin was compared for its antiviral activity with wild HSV-1, APr HSV-1, TK HSV-1 and wild HSV-2 strains in the yield-reduction assay. Vero cells were infected with the HSV strains at 5 to 10 PFU/cell and incubated in the presence of 0, 10, 20 and 30 µg/ml of eugeniin at 37°C for 24 h. The cultures were frozen, thawed and then centrifuged at 3,000 rpm for 15 min. Virus titers in the supernatants were determined by the plaque assay. The titers represent the average values with standard error for triplicate samples.

Effect of eugeniin on viral DNA synthesis. Eugeniin was examined for the inhibition of viral DNA synthesis of wild HSV-1 and AP^r HSV-1 strains in Vero cells by slot-blot hybridization to assess the mode of its anti-HSV action. As shown in figure 3, eugeniin dose-dependently inhibited DNA synthesis of wild HSV-1 and AP^r HSV-1 strains, although AP^r HSV-1 DNA was synthesized in the presence of PAA. These viral DNA syntheses were strongly suppressed in the presence of 20 µg/ml eugeniin, and the levels of DNA amounts detected were similar to those in the HSV-1-infected cells immediately after virus adsorption and in the presence of 150 µg/ml PAA, which indicated the complete inhibition of viral DNA synthesis. DNA syntheses of TK HSV-1 and wild HSV-2 strains were not detectable in the presence of 20 µg/ml eugeniin either (data not shown). Eugeniin inhibited DNA synthesis of all HSV strains examined.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Inhibition of DNA synthesis of various HSV strains by eugeniin. Effects of eugeniin and PAA on viral DNA synthesis were examined in Vero cells infected with wild HSV-1 or APr HSV-1 strains. The cells were infected with the HSV-1 strains (5-10 PFU/cell) and then incubated in the presence of 0 (lane 2), 5 (lane 3), 10 (lane 4) and 20 (lane 5) µg/ml of eugeniin and 150 µg/ml of PAA (lane PAA) at 37°C for 8 h. The cells were lysed and then DNA was prepared from the lysates. DNA was blotted to nylon filters at 6.25, 25, 100 and 400 ng/slot and fixed by UV irradiation. In lane 1, DNA was prepared from infected cells immediately after virus adsorption for 1 h. In lane 6, DNA was prepared from uninfected cells as a negative control. In lane pTK, plasmid pTK involving the DNA fragment of the HSV-1 TK gene was blotted at 104, 105, 106 and 107 copies/slot as a positive control for HSV-1 DNA. In lane PAA, DNA prepared from infected cells in the presence of 150 µg/ml of PAA was blotted at 400 ng/slot. The filters were hybridized with denatured radioactive probes as described in the text. The hybridized filters were washed and the dried filters were exposed to X-ray films at 80°C.

Effect of eugeniin on viral protein synthesis. Because PAA inhibits the synthesis of HSV DNA and late HSV proteins but permits the synthesis of early HSV proteins (Honess and Roizman, 1973; Honess and Watson, 1977), the synthesis of HSV proteins was examined in the presence of eugeniin and PAA to compare the effects of PAA and eugeniin on protein syntheses. As shown in figure 4 and table 2, the synthesis of late viral proteins was noticeably reduced in cells infected with wild HSV-1 and HSV-2 strains in the presence of 150 µg/ml PAA compared with in its absence. Eugeniin dose-dependently inhibited the synthesis of viral proteins such as 53-, 60-, 67-, 89- and 155-kdalton proteins for wild HSV-1 and 48-, 57-, 64-, 71-, 80-, 115- and 145-kdalton proteins for wild HSV-2. The quantitative analysis of autoradiograms showed that the intensities of those bands of wild HSV-1 and HSV-2 proteins synthesized in the presence of 20 µg/ml eugeniin were reduced similarly to those in the presence of 150 µg/ml PAA (table 2). In AP^r HSV-1-infected cells, the synthesis of AP^r HSV-1 proteins was not markedly modified in the presence or absence of 150 µg/ml PAA, but eugeniin dose-dependently reduced the synthesis of late viral proteins of AP^r HSV-1 strain. The concentrations (5-20 µg/ml) of eugeniin used did not inhibit the synthesis of host cellular proteins (fig. 4 and table 2). Thus eugeniin as well as PAA selectively inhibited the synthesis of late HSV proteins without apparent cytotoxicity.

View larger version (59K): [in this window] [in a new window]   Fig. 4.   Inhibition of protein synthesis of various HSV strains by eugeniin. Eugeniin was examined for its effect on viral protein synthesis in Vero cells infected with HSV strains. The cells were mock-infected (M)- or infected with wild HSV-1, APr HSV-1 or wild HSV-2 strains and incubated in the presence of 0 (lane 1), 5 (lane 3), 10 (lane 4) and 20 (lane 5) µg/ml of eugeniin or 150 µg/ml of PAA (lane 2) at 37°C. The infected and mock-infected cells were labeled with [35S]methionine and [35S]cysteine for 5.5 to 7.5 h postinfection in the presence of eugeniin or PAA as described in the text. Immunoprecipitates from wild HSV-1, APr HSV-1 and wild HSV-2 infected cells (Wild HSV-1, APr HSV-1 and Wild HSV-2) and total cell lysates of mock-infected cells (Cell lysate) were subjected to SDS-polyacrylamide gel electrophoresis followed by fluorography. Marker proteins (30, 46, 69, 97.4 and 200 kdaltons) were co-electrophoresed in each gel. Arrow heads indicate late viral proteins whose production was reduced in the presence of eugeniin as well as PAA.

Effect of eugeniin on DNA polymerase activity. To examine the effect of eugeniin on the activity of HSV DNA polymerase, HSV-1 DNA polymerase was partially purified from wild HSV-1-infected HEL cells. As shown in figure 5A, eugeniin inhibited the activity of HSV-1 DNA polymerase (apparent K_m value, 1.67 µM) noncompetitively with respect to dTTP, which indicates that the viral DNA polymerase is a target of eugeniin. Further cellular DNA polymerases  and  were partially purified from HEL cells to clarify the difference in the enzymological properties against eugeniin between HSV-1 DNA polymerase and cellular DNA polymerases. This compound inhibited the activity of cellular DNA polymerases  (apparent K_m value, 7.02 µM) and  (apparent K_m value, 13.1 µM) with respect to dTTP (fig. 5, B and C). However apparent K_i values (4.82 and 3.44 µM) of the  and  DNA polymerases for eugeniin were 8.2- and 5.8-fold higher, respectively, than that (0.59 µM) of the HSV-1 DNA polymerase. Thus eugeniin noncompetitively inhibited the activity of HSV-1 DNA polymerase more strongly than the activities of host cellular DNA polymerases.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Enzymological properties of partially purified HSV-1 (A), a (B) and b (C) DNA polymerases against eugeniin. a) Reaction velocities were measured at various concentrations of [3H]dTTP and apparent Km values were graphically evaluated from Lineweaver-Burk plots [A-a: , 25 µM eugeniin (correlation coefficient = 0.998); µ, no eugeniin (correlation coefficient = 0.985); B-a: , 25 µM eugeniin (correlation coefficient = 0.993); µ, no eugeniin (correlation coefficient = 0.992); C-a: , 37.5 µM eugeniin (correlation coefficient = 1.00); µ, no eugeniin (correlation coefficient = 0.995)]. b) Apparent Ki values were graphically determined by measuring reaction veracities in the presence and absence of fixed concentrations of eugeniin [A-b: , 0.428 µM [3H]dTTP (correlation coefficient = 0.943); µ, 3.42 µM [3H]dTTP (correlation coefficient = 0.993); B-b: , 1.71 µM [3H]dTTP (correlation coefficient = 0.883); µ, 6.84 µM [3H]dTTP (correlation coefficient = 0.948); C-b: , 0.855 µM [3H]dTTP (correlation coefficient = 0.982); µ, 3.42 µM [3H]dTTP (correlation coefficient = 0.948)]. Km and Ki values were calculated with two different eugeniin and [3H]dTTP concentrations, respectively. These kinetic values were confirmed to be similar in repeated experiments.

	Discussion

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We have isolated an anti-HSV compound, eugeniin, from the extracts of G. japonicum and S. aromaticum with differential and chromatographic fractionations guided by antiviral assay in this study. In each step of the fractionations, we selected fractions showing the lowest EC₅₀ value for the step of next fractionation. Thus eugeniin (EC₅₀, 5 µg/ml in table 1), a phenolic compound with diphenyl group, was isolated as a major anti-HSV-1 compound which was quantitatively separable from the herbal extracts and of which the chemical structure was identified, although it is possible that the two herbs contain other minor anti-HSV compounds with much lower EC₅₀ values than the EC₅₀ of eugeniin. The CC₅₀ values of eugeniin determined by [³H]thymidine uptake and/or growth inhibition assays with Vero and HEL cells were 66 to 77 µg/ml (table 1), which indicates the coincidence of cytotoxic concentrations by the different assays. The analysis of protein synthesis of host cells (fig.4 and table 2) showed that eugeniin inhibited late HSV protein synthesis but not cellular protein synthesis at 20 µg/ml and supported our EC₅₀ and CC₅₀ values. However Kashiwada et al. (1992) have reported a much lower cytotoxic concentration (2.74 µg/ml) for tumor cells by use of an assay condition with a longer exposure time (3 days) of eugeniin. Similarly, Fukuchi et al. (1989) reported that the cytotoxic concentration of geraniin, an anti-HSV compound with the EC₅₀ value of 0.093 µg/ml, is more than 30 µg/ml, whereas Kishiwada et al. (1992) reported that it is 0.35 µg/ml. It is necessary to clarify that an antiviral agent should show direct antiviral activity by inhibiting virus replication cycle but not by cytotoxicity. Therefore the anti-HSV activity assay and cytotoxic assay should be done in the same assay conditions. Thus the culture cells and exposure time used by Kishiwada et al. (1992) may not be appropriate for anti-HSV activity assay, and the discrepancy in cytotoxicity may be caused by the difference in these assay conditions. In our experiments, eugeniin was not cytotoxic in the condition that anti-HSV activity was examined, which indicates that the anti-HSV activity of eugeniin was not caused by its cytotoxicity.

Eugeniin inhibited the growth of AP^r HSV-1, TK HSV-1 and wild HSV-2 strains as well as wild HSV-1 (fig. 2). This suggests that eugeniin showed a different mode of anti-HSV action from ACV and PAA. In this respect, the relationship between eugeniin and ACV may correspond to that between nucleoside analogs (zidovudine and dideoxyinosine) and a non-nucleoside inhibitor (Nevirapine) in the treatment of HIV-1 infection (Hammer, 1997); ACV and zidovudine as well as dideoxyinosine are strong chain terminators, whereas eugeniin and Nevirapine are noncompetitive inhibitors of DNA polymerase and reverse transcriptase, respectively. Eugeniin may be a possible candidate or mother compound of a promising anti-HSV agent with a different anti-HSV action from nucleoside analogs.

Eugeniin exhibited anti-HSV activity by the inhibition of HSV DNA synthesis (figs. 2 and 3). The synthesis of late HSV proteins were inhibited in the presence of 20 µg/ml of eugeniin as well as PAA at 150 µg/ml, in which the growth of HSV and synthesis of HSV DNA were inhibited almost completely (figs. 2, 3 and 4). Because PAA specifically inhibits the synthesis of HSV DNA but permits the synthesis of early HSV proteins (Honess and Roizman, 1973; Honess and Watson, 1977), eugeniin was suggested to have anti-HSV action similar to PAA that inhibits the synthesis of HSV DNA selectively. Eugeniin inhibited the activity of partially purified HSV DNA polymerase in vitro (fig. 5), and therefore, one of the major sites of its inhibitory action may be viral DNA synthesis.

Eugeniin inhibited the activity of HSV-1 DNA polymerase more strongly than cellular DNA polymerases  and  (fig. 5). The apparent K_m values for HSV-1 DNA polymerase and cellular DNA polymerases  and  used in this study were within the range of those reported elsewhere (Allaudeen, 1985; Coen et al., 1982; Frank et al., 1985; Larder et al., 1983; Merta et al., 1990; Nishiyama et al., 1984), which indicates that they were successfully purified. The ratios of K_i values of cellular DNA polymerases  and  for eugeniin to that of HSV-1 DNA polymerase were 5.8 and 8.2, respectively. In contrast, the ratio for Ara-A ranges 3 to 4 (Coen et al., 1982). The higher specificity of eugeniin for HSV-1 DNA polymerase was consistent with a higher therapeutic index (CC₅₀/EC₅₀, 13.2 to 15.4, table 1) of eugeniin than that of Ara-A (1, De Clercq et al., 1980). Thus the inhibitory activity of eugeniin on HSV-1 DNA polymerase was confirmed with higher specificity than that of Ara-A. Eugeniin behaved as a noncompetitive inhibitor of both HSV-1 and cellular polymerases. Noncompetitive-type inhibition has been reported for the inhibition of DNA polymerase  by Evans blue (Nakane et al., 1988). Recently, Nevirapine, a unique noncompetitive-type and non-nucleoside inhibitor of reverse transcriptase of HIV-1, has been used for the treatment of HIV-1 infection (Carr and Cooper, 1996; Grob et al., 1997; Kilby and Saag, 1996; Kopp et al., 1991; Romero et al., 1991; Smerdon et al., 1994). The activity of these polymerases was mainly inhibited allosterically in the presence of those inhibitors. Because eugeniin exerted differential inhibitory activity between HSV-1 and cellular DNA polymerases in the reaction mixtures with the same DNA template, nonspecific interaction between DNA and eugeniin such as intercalation into DNA may not occur strongly. Because K_i value of DNA polymerase  for eugeniin in the presence of BSA at 500 µg/ml was similar to that of DNA polymerase  in its absence (fig. 5), eugeniin may not interact with proteins nonspecifically. Thus eugeniin may interact selectively with DNA polymerases and/or the template-primer itself. Biochemical analysis of the inhibitory mechanism is underway.