Dresler S L, Frattini M G
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Biochem Pharmacol. 1988 Mar 15;37(6):1033-7. doi: 10.1016/0006-2952(88)90506-0.
The purine base and nucleoside analogues N2-(p-n-butylphenyl)-guanine (BuPh-Gua) and N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPh-dGuo) are strong inhibitors of isolated mammalian DNA polymerase alpha, but are less potent that expected as inhibitors of DNA replication in intact cultured cells [G. E. Wright, L. W. Dudycz, Z. Kazimierczuk, N. C. Brown and N. N. Khan, J. med. Chem. 30, 109 (1987)]. The mechanistic basis for these observations was explored using permeable human fibroblasts. DNA replication in the permeable cells was inhibited only slightly by BuPh-Gua and BuPh-dGuo at 100 microM, the highest concentration which could be attained. Similar results were obtained for ultraviolet-induced DNA repair synthesis, a process which is though to involve the same DNA polymerase as replication. More detailed studies were performed using the corresponding nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh-dGTP), which is much more water-soluble than the base and nucleoside. The apparent Ki values for BuPh-dGTP inhibition of both replication and ultraviolet-induced repair synthesis in permeable cells were approximately 3 microM. These values are several hundred-fold greater than the apparent Ki for BuPh-dGTP inhibition of isolated human DNA polymerase alpha, which is approximately 10 nM. We conclude that BuPh-Gua and BuPh-dGuo are poor inhibitors of DNA replication in intact cells not because of permeability barriers, but because, unlike polymerase alpha, cellular DNA synthesis is relatively insensitive to this group of inhibitors. These results suggest that polymerase alpha may not be a good general model for predicting the potency of base, deoxyribonucleoside and deoxyribonucleotide analogues as inhibitors of mammalian cellular DNA replication. The fact that the permeable cell systems accurately reflect the relative insensitivity to butylphenyl-guanine derivatives of mammalian DNA replication suggests that permeable cells may be useful tools in future studies of base and nucleoside analogues.
嘌呤碱基和核苷类似物N2 -（对正丁基苯基）-鸟嘌呤（BuPh - Gua）和N2 -（对正丁基苯基）-2'-脱氧鸟苷（BuPh - dGuo）是分离的哺乳动物DNA聚合酶α的强抑制剂，但作为完整培养细胞中DNA复制的抑制剂，其效力低于预期[G. E. Wright，L. W. Dudycz，Z. Kazimierczuk，N. C. Brown和N. N. Khan，J. med. Chem. 30, 109 (1987)]。使用可渗透的人成纤维细胞探索了这些观察结果的机制基础。在可渗透细胞中，BuPh - Gua和BuPh - dGuo在100 microM（可达到的最高浓度）时仅轻微抑制DNA复制。紫外线诱导的DNA修复合成也得到了类似结果，该过程被认为涉及与复制相同的DNA聚合酶。使用相应的核苷酸类似物N2 -（对正丁基苯基）-2'-脱氧鸟苷-5'-三磷酸（BuPh - dGTP）进行了更详细的研究，它比碱基和核苷的水溶性大得多。在可渗透细胞中，BuPh - dGTP抑制复制和紫外线诱导的修复合成的表观Ki值约为3 microM。这些值比BuPh - dGTP抑制分离的人DNA聚合酶α的表观Ki值（约10 nM）大几百倍。我们得出结论，BuPh - Gua和BuPh - dGuo在完整细胞中是较差的DNA复制抑制剂，不是因为渗透屏障，而是因为与聚合酶α不同，细胞DNA合成对这组抑制剂相对不敏感。这些结果表明，聚合酶α可能不是预测碱基、脱氧核糖核苷和脱氧核糖核苷酸类似物作为哺乳动物细胞DNA复制抑制剂效力的良好通用模型。可渗透细胞系统准确反映了哺乳动物DNA复制对丁基苯基鸟嘌呤衍生物的相对不敏感性，这一事实表明可渗透细胞可能是未来碱基和核苷类似物研究中的有用工具。
