Metabolism of 9-( 1,3-Dihydroxy-2-propoxymethyl)guanine, a New Anti-herpes Virus Compound, in Herpes Simplex Virus-infected Cells*

The metabolism of 9-( 1,3-dihydroxy-Z-propoxy- methy1)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells in- fected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus- infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 "). the

DHPG,' a newly synthesized guanosine analog, was found to have potent activity against HSV (1)(2)(3), varicella-zoster virus (I), cytomegalovirus (2,3), and Epstein-Barr virus (3). DHPG is more potent and the spectrum of susceptible viruses to the compound is broader than that to acyclovir. Furthermore, HSV mutants, which are resistant to acyclovir because of altered thymidine kinase or DNA polymerase, were as sensitive to DHPG as the parental HSV (3). This unique spectrum of activity suggests the potential use of DHPG in the clinic for the treatment of herpesvirus infection. In order to have a better understanding of the mechanism of action of DHPG, it is important to explore its metabolism in virusinfected cells. This paper presents evidence of phosphorylation of DHPG, leading to the formation of DHPGTP, and of incorporation of the nucleoside analog into DNA.

MATERIALS AND METHODS
Chemicals and Enzymes-DHPG, DHPGMP, and [3H]DHPG (16 Ci/mmol) were donated by Syntex (U.S.A.), Inc., Palo Alto, CA. Other nucleosides, nucleotides, and dithiothreitol were purchased from Sigma. Proteinase K and micrococcal nuclease were obtained * This work was supported by Grant CH-29 from the American Cancer Society and a gift from Syntex (U. S. A.) Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
from Boehringer Mannheim Gmbh. Spleen phosphodiesterase was from Worthington and Escherichia coli alkaline phosphatase was from Sigma.
Cells and Virus-Cells were grown at 37 "C in RPMI 1640 medium (GIBCO) containing 100 pg/ml of Kanamycin supplemented with 5% horse serum and 5% fetal calf serum for HeLa SB and Vero cells, respectively. HSV-1 (strain KOS) was maintained as described previously (4).
Preparation of HSV-I Thymidine Kinase and Human Erythrocyte Lysate-A highly purified enzyme was prepared from HSV-1-infected HeLa BU (TK-) cells using affinity column as described previously (5). Twenty-five ml of fresh heparinized blood was diluted with phosphate-buffered saline (0.14 M NaC1, 4 mM KCI, 0.94 mM Na2HP0,, 0.15 M KH2P0,). Erythrocytes were pelleted by centrifugation and 4 volumes of cold water were added to lyse the cells. After centrifugation at 12,000 X g for 20 min, supernatant was collected and solid ammonium sulfate was added to 70% saturation. Precipitate was collected by centrifugation, dissolved in 10 ml of 5 mM Tris-HC1 (pH 7.5), 2 mM dithiothreitol, 10% glycerol, and used for phosphorylation of DHPGMP.
Preparation ofdcid-soluble and -insoluble Fractions-HeLa S, cells in 25-cm2 flasks were infected with 3 plaque-forming units/cell of HSV-1. Various concentrations of [3H]DHPG were added at 0 h postinfection and the cells were harvested at 6 h and washed three times with phosphate-buffered saline. The cell pellets (5 X lo6 cells) were extracted with 50 r l of 1.5 M perchloric acid at 0 "C for 30 min. The extract was neutralized with 1 N potassium hydroxide (77 pl) and 23 p1 of 0.5 M potassium phosphate buffer (pH 7.4) was added. Precipitate was removed by centrifugation and the supernatant was used as the acid-soluble fraction. The precipitate from the perchloric acid extraction were washed twice with 1.5 N perchloric acid and designated as the acid-insoluble fraction.
Alkaline Hydrolysis ofdcid-insoluble Fractions-The acid-insoluble fraction was dissolved in 100 pl of 1 N NaOH and incubated for 2 h at 37 "C. Four hundred pl of 5% trichloroacetic acid was added to the solution. The supernatant and an additional 200-pl wash of trichloroacetic acid were neutralized with KOH solution and counted in a liquid scintillation counter as the alkali-labile fraction. The pellet was redissolved in dimethyl sulfoxide and counted as the alkali-stable material.
HPLC Analysis-An anion exchange column, Partisil 10 SAX/25 (Whatman), was used in a solvent system with a gradient of potassium phosphate buffer (pH 6.6) from 0.03 to 0.15 M. The solvent system for a reverse phase column, CS (ALLTECH), was acetonitrile, 0.03 M acetic acid (1-5000, v/v).
For cesium sulfate density gradient centrifugation, the mixture was extracted with '/z volume of phenol and the aqueous phase was further extracted with ether. One-tenth volume of 3 M sodium acetate buffer (pH 5.5) and 2.5 volume of ethanol were added to the aqueous phase This is an Open Access article under the CC BY license. and the mixture was kept at -80 "C for 1 h. The precipitate was dissolved in 10 mM Tris-HC1 (pH 7.4), 1 mM EDTA and cesium sulfate was added to the solution to a density of 1.53 g/cm3. Centrifugation was at 25,000 rpm for 60 h using the same rotor as above.
Gradient solution was fractionated from top to bottom with a Buchler Auto-Densi Flow pump-dripper. Density was calculated from refractive index measured with an American Optical ABBE refractometer.

Uptake of DHPG in Virus-infected and Mock-infected HeLa
Cells-HeLa cells were infected with HSV-1 and various amounts of ['HIDHPG were added at 0 h post-virus adsorption. The cells were harvested at 6 h post-virus adsorption. The uptake of DHPG into an acid-soluble and -insoluble fraction of these cells was analyzed. The results are presented in Table I. The more DHPG added, the more uptake into both fractions was observed, with the majority of [3H]DHPG uptake taking place in the acid-soluble fraction. When 5 p~ DHPG was added to mock-infected HeLa cells, the uptake of DHPG into the acid-soluble fraction was similar to that in infected cells exposed to 0.5 KM DHPG. The uptake of DHPG into the acid-insoluble fraction was about the same as that of infected cells exposed to between 1.0 and 2.0 p~ DHPG. More than three-quarters of ['HJDHPG in the acid-insoluble fraction could be rendered acid-soluble following treatment with 1 N KOH.
HPLC Analysis of DHPG Metabolites in Acid-soluble Fraction-The acid-soluble fraction of virus-infected and mockinfected HeLa cells after the treatment with ['HIDHPG was analyzed by HPLC using a Partisil 10 SAX column system. The results are shown in Fig. 1. In addition to the radioactivity associated with the DHPG marker fraction (Fraction 5 ) , three other metabolites of DHPG were found in either virus-infected or mock-infected cells. One of the metabolites (Fraction 20) was co-eluted with a DHPGMP marker. The amount of each metabolite formed in infected cells was dependent on the extracellular concentration of DHPG. In comparison with the virus-infected cells, very small amounts of metabolites could be found in the mock-infected cells and most of the radioactivity was in the DHPG fraction. When the extract from HSV-infected cells was treated with alkaline phosphatase for 4 h and then analyzed with the same HPLC system, the radioactivity associated with each of these metabolites was decreased with a corresponding increase of radioactivity associated with the DHPG fraction (Fig. 1D). When alkaline phosphatase-treated sample was subjected to the reverse phase Ce HPLC column system, the majority of the radioactivity was found to be associated with DHPG.' In order to identify these three metabolites further, each compound was isolated by HPLC and subjected to DEAE-Sephadex column chromatography in urea-containing buffer (Fig. 2). This allows the separation of nucleotides based on the number of ionic charges on phosphate groups. The metabolites associated with fractions 19 and 20 (Compound I), 25 and 26 (Compound Z ) , and 52 to 55 (Compound 3) in Fig. 1 eluted slightly faster than GMP, GDP, and GTP, respectively. These results indicate that Compounds 1, 2, and 3 were mono-, di-, and triphosphate derivatives of DHPG.
I n Vitro Formation of Metabolites-Highly purified HSV-1 thymidine kinase was incubated with ['HIDHPG in the presence of ATP. The major metabolite formed co-eluted with DHPGMP on HPLC/Partisil 10 SAX system as shown in Fig. 3. ["HIDHPGMP was isolated and then incubated with human erythrocyte lysate in the presence of ATP. As shown Y.-C. Cheng, S. P. Grill, G. E. Dutschman, K. Nakayama, and K. F. Bastow, unpublished observations.   D,o). The radiospecificity of [3H]DHPG used was 1.6 X lo3 cpm/pmol. The sample injected was equivalent to the amount extracted from 5 X IO5 cells.
in Fig. 4, it was converted to metabolites which gave the same retention times as those of in vivo metabolites, Compounds 2 and 3, respectively. This conversion was inhibited by GMP, but not by TMP, UMP, or AMP,2 suggesting that the enzyme responsible for the conversion of DHPGMP to DHPGDP is GMP kinase. The decrease of Compound 2 and the increase of Compound 3 with longer incubation suggested that Compound 2 is the precursor of Compound 3 (Fig. 3, B and C).
Incorporation of DHPG into DNA-HSV-1-infected cells were treated with 2 p~ ['HIDHPG for 8 h and the cell lysate was subjected to sodium iodide density gradient centrifugation. Although this method allows separation of virus DNA (density = 1.52) from host cell DNA (density 1.50), they were not distinguishable for ['HIDHPG-labeled DNA, which distributed into the fractions between the two densities (Fig.  5A). It is quite possible that ['HIDHPG-labeled DNA has different density from normal DNA, which makes the virus and host DNA indistinguishable by density.
When these fractions were treated with alkali, a portion of radioactivity became acid-soluble and the radioactivity in the [3H]DHPG (53 pCi, 9.3 nmol) for 8 h. Extraction of nucleic acid and centrifugation were as described under "Materials and Methods." Seventy-five pl of each fraction were collected. Twenty-five p1 of each fraction were absorbed into fiber glass discs (Whatman GF/A) and the discs were washed three times with ice-cold 5% trichloroacetic acid and once with ethanol. When dry, the discs were counted in ACS scintillant (A). Five p1 of 5 N NaOH was added to another 25 pl of each fraction. The mixtures were incubated at 37 "C for 2 h, absorbed into discs, and washed as above ( B ) .
Nucleic acid extracted from ["IDHPG-treated infected cells was analyzed by cesium sulfate density gradient centrifugation. Most of the radioactivity was associated with DNA (Fig. 6A). The radioactive DNA was digested with micrococcal nuclease and spleen phosphodiesterase and the products were analyzed by HPLC/Partisil 10 SAX. This method allows us to distinguish terminally incorporated nucleoside, which gives nucleoside, from internally incorporated nucleoside, which gives nucleoside 3'-monophosphate. AS shown in Fig. 6B, there was little DHPG produced, while a significant amount of radioactivity was associated with DHPGMP and other material, which was possibly dinucleotide resulting from incomplete digestion. These results indicate that DHPG was internally incorporated into DNA and therefore does not act as pure chain terminator for DNA synthesis.

DISCUSSION
Like several other selective anti-HSV nucleoside analogs (6-15), DHPG could be preferentially phosphorylated in HSV-infected cells. The primary metabolic scheme is shown in Fig. 7. In spite of two symmetrical -CH20H groups at carbon 2 of the propoxyl group of DHPG, the phosphorylation of DHPG could take place at one of those two -OH groups by virus-induced thymidine kinase. Two stereoisomers at carbon 2 could be anticipated. It is probable that only one stereoisomer of DHPGMP which could be utilized for the further metabolism was formed since all the DHPGMP was converted to DHPGDP and DHPGTP by the erythrocyte lysate. However, the possibility that the enzyme(s) responsible for further phosphorylation could not distinguish the two stereoisomers requires further experimentation.
It seems likely that the isomer formed should have a steric conformation similar to dGMP, but regardless of the outcome of this issue, the host cells apparently have an efficient system to carry out further phosphorylation as indicated in Fig. 1E. There was no accumulation of DHPGMP in mock-infected cells, although DHPG could be found in the acid-insoluble fraction. This implies that a cell enzyme could perform the initial phosphorylation of DHPG but in a relatively inefficient manner since other human herpesviruses are less susceptible to DHPG than HSV and these viruses do not induce virusspecific thymidine kinase (1-3). The host enzymes responsible for the phosphorylation of DHPGMP to DHPGDP could be GMP kinases since GMP could inhibit this process. A similar suggestion was also made recently (16). Although there are several GMP kinase isozymes (17), it is not clear whether there is a preference for a particular GMP kinase isozyme in this process.
The amount of accumulated DHPGTP in infected cells treated with DHPG at concentrations of 0.5, 1, 2, and 5 FM was 0.5, 1.2, 2.8, and 8.4 pmol/106 cells, respectively. The amount of DHPG incorporated into the acid-insoluble fraction was found to be less than 5-propyl-dUrd (13). This suggested the posssibility that DHPG is acting as a chain terminator such as acyclovir or possibly a pseudochain terminator such as arabinosyl adenine (19). A detailed study of the mode of DHPG (18) on virus DNA replication is in progress. Since a portion of DHPG incorporated into the acidinsoluble fraction is alkali-labile, the possibility is raised that either DHPG could be incorporated into RNA which is alkaline-labile or into DNA with an alkali-labile linkage. Using the CszSOs isopycnic centrifugation technique, all the radioactivity of [3H]DHPG in the nucleic acid from the infected cells was found to be with DNA. DHPG was also demonstrated to be incorporated into the internucleotide chain of DNA. The unpublished results of this laboratory using an in uitro DNA synthesis system catalyzed by HSV DNA polymerase further confirms the incorporation of DHPG into the internucleotide chain of DNA. The alkaline lability of DHPG in the internucleotide DNA chain or at the terminal of DNA is currently under investigation.