Molecular designing of nucleoside analogues for inhibitor of telomerase

核苷和相应的5‘-三磷酸作为潜在的人类端粒酶选择性抑制剂的分子设计已经被考虑。首先，我们详细地研究了伸展PCR检测HeLa细胞端粒酶的方法。所得结果可用于后续实验，包括利用Lineweaver-Burk图进行酶动力学分析。与HIV-1逆转录酶相比，天然底物dGTP和dTTP的对映体L-dGTP和L-dTIP对端粒酶的抑制作用较小。这一结果清楚地表明，端粒酶能够严格识别底物的对映体，另一方面，5-苯乙酰取代的dUTP类似物对端粒酶表现出很强的亲和力，因此该酶更倾向于疏水和空间取代基无碱基团。在此基础上，合成了5-叠氮甲基、氨甲基、羟甲基和甲氧基甲基等dTTP类似物，并合成了具有不同苷元含量的糖取代呋喃核苷。作为端粒酶的另一个来源，樱桃鲑鱼试验发现端粒酶活性很强。这些发现将为从蛋白质结构和功能水平上进一步研究该酶提供依据。

Molecular designing of nucleosides and corresponding 5'-triphosphates as potential selective inhibitor for human telomerase have been considered.First, we have examined the stretch PCR method for determination of HeLa cell telomerase in detail. The results obtained could be used in following experiments including enzyme kinetic analysis by Lineweaver-Burk plots. L-dGTP and L-dTIP, enantiomers of natural substrates dGTP and dTTP, showed inhibitory effect on telomerase in lesser extent when compared with HIV-1 reverse transcriptase. This result clearly indicated that telomerase can strictly recognize enantiomer for its substrate, On the other hand, a 5-styryl-substituted dUTP analogue has shown strong affinity to the telomerase, thus this enzyme was prefer hydrophobic and steric substituent no base moieties. Based on these findings, several dTTP analogues such as 5-azidomethyl-, aminomethyl-, hydroxymethyl- and methoxymethly-derivatives were synthesized, additionally, sugar replacement lyxofuranosyl nucleotides having various aglycon moieties were also prepared. As the other source for telomerase, cherry salmon tests were investigated and found strong telomerase activity. These findings should be applicable to further studies of the enzyme on the level of protein structure and function.