Hamel E, Vaughns J, Getahun Z, Johnson R, Lin C M
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 1995 Oct 1;322(2):486-99. doi: 10.1006/abbi.1995.1492.
Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.
尽管相对于GTP而言，2',3'-二脱氧鸟苷5'-三磷酸（ddGTP）与微管蛋白的可交换核苷酸结合位点的亲和力有所降低，但它和鸟苷5'-[α,β-亚甲基]三磷酸[pp(CH2)pG]在促进微管蛋白组装方面具有很高的活性。抗有丝分裂药物紫杉醇与可交换位点GTP作用于β-微管蛋白分子的同一部位，这两种类似物都能增强成核反应并促进超稳定聚合物的形成。这些观察结果促使我们合成了双修饰类似物2',3'-二脱氧鸟苷5'-[α,β-亚甲基]三磷酸[pp(CH2)pddG]。我们比较了pp(CH2)pddG与ddGTP、pp(CH2)pG以及三种同源二磷酸在与微管蛋白相互作用时的效果。我们发现，pp(CH2)pddG在支持形成稳定性增加的聚合物方面与ddGTP和pp(CH2)pG一样具有活性，但其与可交换位点的亲和力低于两种单修饰类似物[pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP与可交换位点的相对亲和力为1:2.8:10:273]。这三种类似物与微管蛋白的相互作用存在显著差异，且pp(CH2)pddG的行为介于ddGTP和pp(CH2)pG之间。最重要的是，在所研究的反应条件下，用热处理的微管相关蛋白（MAPs）时，ddGTP诱导形成的聚合物由短微管组成，而pp(CH2)pddG和pp(CH2)pG形成的聚合物则由短片层组成。另一方面，在没有MAPs的情况下进行组装时，ddGTP和pp(CH2)pddG（0.5 mg/ml）使微管蛋白的临界浓度比pp(CH2)pG（2.5 mg/ml）低五倍。2',3'-二脱氧鸟苷5'-二磷酸很容易发生的从头组装，在α,β-亚甲基二磷酸GDP类似物中均未观察到。
