Bennett L L, Parker W B, Allan P W, Rose L M, Shealy Y F, Secrist J A, Montgomery J A, Arnett G, Kirkman R L, Shannon W M
Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255-5305.
Mol Pharmacol. 1993 Dec;44(6):1258-66.
CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.
卡波环胞苷（CdG）是2'-脱氧鸟苷的碳环类似物，对疱疹病毒、乙型肝炎病毒和人巨细胞病毒具有活性。我们研究了CdG的氚化对映体与单纯疱疹病毒1型特异性胸苷激酶（HSV-1 TK）的相互作用，并检测了它们在未感染和HSV-1感染细胞中的代谢情况。D-CdG和L-CdG作为部分纯化的HSV-1 TK催化胸苷（dThd）磷酸化的竞争性抑制剂效果相同（Ki值分别为2.1和3.4 microM），作为底物时也相同（Km值分别为17和26 microM，对映体的Vmax值相同，且比dThd的Vmax值大约高50%）。部分纯化的酶制剂含有细胞核苷酸激酶活性（测定培养基中也存在丙酮酸激酶），几乎将D-CdG全部转化为三磷酸形式，而将L-CdG几乎全部转化为单磷酸形式。同样，在病毒感染的细胞中，D-对映体主要转化为三磷酸形式，L-对映体主要转化为单磷酸形式。在未感染的细胞中，结果在定性上相似。竞争研究表明，在CEM细胞中，脱氧胞苷（dCyd）激酶（EC 2.7.1.74）似乎是主要负责两种对映体磷酸化的酶。因此，HSV-1 TK和细胞dCyd激酶（CEM细胞的）对CdG的对映体均无选择性。这种对映体特异性的缺乏对病毒增殖和细胞代谢抑制剂的设计具有明显的意义。
