Vol. 53, Issue 1, 157-165, January 1998

Intracellular Metabolism of the N7-Substituted Acyclic Nucleoside Analog 2-Amino-7-(1,3-dihydroxy-2-propoxymethyl)purine, a Potent Inhibitor of Herpesvirus Replication

Johan Neyts, Jan Balzarini, Graciela Andrei, Zhu Chaoyong, Robert Snoeck, Albert Zimmermann, Thomas Mertens, Anna Karlsson and Erik De Clercq

Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium (J.N., J.B., G.A., R.S., E.D.C.), Universität Ulm, Klinikum, Abteilung Virologie, 89081 Ulm, Germany (A.Z., T.M.), and Karolinska Institute, S-171 77 Stockholm, Sweden (Z.C., A.K.)

We investigated the intracellular metabolism of S2242 (2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine), the only known antivirally active acyclic nucleoside analogue with the side chain substituted at the N7 position of the purine ring. Uptake of S2242 by CEM cells increased linearly with increasing extracellular concentrations of the compound and was blocked by inhibitors of nucleoside transport. S2242 was phosphorylated in a time- and concentration-dependent manner to its monophosphates, diphosphates, and triphosphates. Intracellular half-life of the diphosphates and triphosphates in CEM cells was ~3-6 hr. A strong correlation was found between the cytostatic action of the compound and its phosphorylation in different cell lines. In accord with the findings that (1) the cytostatic potential of S2242 is reversed by deoxycytidine (dCyd) and (2) the growth of deoxycytidine kinase-deficient (dCK) cells is refractory to the inhibitory effect of S2242, the amount of metabolites formed from S2242 in the dCK cell line was approximately one hundredth of that in the wild-type cells. The observation that purified dCK phosphorylates S2242 to its monophosphate further corroborates these results. The activity of S2242 against herpes simplex virus, varicella-zoster virus, and human herpesvirus type 6 was reversed by 50-100-fold on the addition of exogenous dCyd. Compound S2242 was not preferentially phosphorylated in herpes simplex virus 1-, varicella-zoster virus-, or human herpesvirus type 6-infected cells (Vero, human embryonic lung, and HSB-2 cells, respectively), and exogenously added dCyd reduced substantially the formation of S2242 metabolites in these cells. In human cytomegalovirus (HCMV)-infected human embryonic lung cells, a 5-25-fold increase in S2242 metabolite formation was observed compared with the noninfected cells, suggesting that an HCMV-encoded or -induced enzyme causes the specific phosphorylation of S2242. Exogenously added dCyd had little effect on the activity of S2242 against HCMV and on the phosphorylation of the compound in HCMV-infected cells. S2242 was not specifically phosphorylated by the HCMV-encoded UL-97 kinase in cells infected with a vaccinia/UL-97 recombinant. S2242 was found to be a substrate (K_m = 90 µM) for purified human deoxyguanosine kinase; the latter enzyme was stimulated 3-4-fold in HCMV-infected cells.