Incorporation and metabolism of 2′-fluoro-5-substituted arabinosyl pyrimidines and their selective inhibition of viral DNA synthesis in herpes simplex virus type 1 (HSV-1)-infected and mock-infected Vero cells

Abstract

The incorporation and metabolism of 2′-fluoro-5-substituted arabinosyl pyrimidine analogs, and their selective inhibition of viral DNA synthesis in herpes simplex virus type 1 (HSV-1)-infected and mock-infected Vero cells were studied by HPLC and CsCl isopycnic density gradient analysis of isolated DNAs. The amounts of radiolabeled analogs incorporated as parent compound following 10 μM exposure for 4 h were 10-fold higher in HSV-1-infected vs mock-infected cells for 2′-fluoro-5-difluoromethyl-Ara-U (F 2FMAU); 4.3-fold higher for 5-ethyl deoxyuridine (EdU); 2.6-fold higher for 2′-fluoro-5-methyl-Ara-U (FMAU) and 1.7-fold higher for dThd. For 2′-fluoro-5-ethyl-Ara-U (FEAU), 3.0 pmole of unchanged moiety was incorporated per 10 6 HSV-1-infected cells but no incorporation was detected in mock-infected cells. HPLC profiles showed that the percentages of radiolabeled analogs incorporated as parent compound in the DNA extracted from HSV-1-infected cells were 31.0% for F 2FMAU, 99.6% for EdU, 83.5% for FEAU and 98.3% for FMAU; from mock-infected cells, they were 63.6% for F 2FMAU, 96.7% for EdU, 97.3% for FMAU and no incorporation into DNA for FEAU was detected. CsCl density gradient analyses of isolated DNA showed that viral DNA synthesis was inhibited 98% by 10 μM FEAU, 92% by 10 μM F 2FMAU, 90% by 2 μM FMAU and 80% by 50 μM EdU, whereas cellular DNA synthesis was inhibited by 53, 44, 61, 66 and 54%, respectively. We conclude that: (a) FEAU incorporation into host-cell DNA was not detectable but FEAU was selectively incorporated into HSV-infected cells; (b) FMAU and FEAU were metabolically stable; however, F 2FMAU was extensively metabolized; (c) FEAU and F 2FMAU were among the most selective inhibitors of HSV-1 DNA synthesis while allowing cellular DNA synthesis to continue.