Kong X B, Scheck A C, Price R W, Vidal P M, Fanucchi M P, Watanabe K A, Fox J J, Chou T C
Laboratory of Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Antiviral Res. 1988 Dec 1;10(4-5):153-66. doi: 10.1016/0166-3542(88)90028-9.
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 microM exposure for 4 h were 10-fold higher in HSV-1-infected vs mock-infected cells for 2'-fluoro-5-difluoromethyl-Ara-U (F2FMAU); 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 F2FMAU, 99.6% for EdU, 83.5% for FEAU and 98.3% for FMAU; from mock-infected cells, they were 63.6% for F2FMAU, 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 microM FEAU, 92% by 10 microM F2FMAU, 90% by 2 microM FMAU and 80% by 50 microM 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, F2FMAU was extensively metabolized; (c) FEAU and F2FMAU were among the most selective inhibitors of HSV-1 DNA synthesis while allowing cellular DNA synthesis to continue.
通过高效液相色谱法（HPLC）以及对分离出的DNA进行氯化铯等密度梯度分析，研究了2'-氟-5-取代阿拉伯糖基嘧啶类似物的掺入和代谢情况，以及它们对单纯疱疹病毒1型（HSV-1）感染和未感染的非洲绿猴肾（Vero）细胞中病毒DNA合成的选择性抑制作用。在10微摩尔浓度下暴露4小时后，作为母体化合物掺入的放射性标记类似物的量，在HSV-1感染细胞中比未感染细胞高10倍（对于2'-氟-5-二氟甲基阿糖脲苷（F2FMAU））；高4.3倍（对于5-乙基脱氧尿苷（EdU））；高2.6倍（对于2'-氟-5-甲基阿糖脲苷（FMAU））；高1.7倍（对于胸腺嘧啶脱氧核苷（dThd））。对于2'-氟-5-乙基阿糖脲苷（FEAU），每10^6个HSV-1感染细胞中掺入3.0皮摩尔未改变的部分，但在未感染细胞中未检测到掺入。HPLC分析表明，从HSV-1感染细胞中提取的DNA中作为母体化合物掺入的放射性标记类似物的百分比，F2FMAU为31.0%，EdU为99.6%，FEAU为83.5%，FMAU为98.3%；从未感染细胞中提取的DNA中，F2FMAU为63.6%，EdU为96.7%，FMAU为97.3%，未检测到FEAU掺入DNA。对分离出的DNA进行氯化铯密度梯度分析表明，10微摩尔的FEAU可抑制病毒DNA合成98%，10微摩尔的F2FMAU可抑制92%，2微摩尔的FMAU可抑制90%以及50微摩尔的EdU可抑制80%，而细胞DNA合成分别被抑制53%、44%、61%、66%和54%。我们得出以下结论：（a）未检测到FEAU掺入宿主细胞DNA，但FEAU被选择性地掺入HSV感染细胞；（b）FMAU和FEAU在代谢上稳定；然而，F2FMAU被广泛代谢；（c）FEAU和F2FMAU是HSV-1 DNA合成的最具选择性的抑制剂之一，同时允许细胞DNA合成继续进行。
