Probing the molecular mechanisms of AZT drug resistance mediated by HIV-1 reverse transcriptase using a transient kinetic analysis.
Probing the molecular mechanisms of AZT drug resistance mediated by HIV-1 reverse transcriptase using a transient kinetic analysis.
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使用瞬态动力学分析探讨 HIV-1 逆转录酶介导的 AZT 耐药的分子机制。
DOI：
10.1021/bi034435l
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Anderson,KarenS
中科院分区：
文献类型：
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作者：
Ray,AdrianS;Murakami,Eisuke;Basavapathruni,Aravind;Vaccaro,JosephA;Ulrich,Dagny;Chu,ChungK;Schinazi,RaymondF;Anderson,KarenS
已经提出了几种假说来解释抗艾滋病毒药物AZT的耐药性的发展。临床研究结果表明，艾滋病病毒逆转录酶（RT）中的AZT耐药突变不仅降低了对胸苷类似物的敏感性，而且还可能导致多双脱氧核苷耐药。在这份报告中，我们描述了瞬态动力学研究建立的生物化学效应的艾滋病病毒-1逆转录病毒的AZT耐药突变的掺入和去除天然和非天然脱氧核苷酸。虽然生理作用仍有待阐明，野生型和抗AZT HIV-1 RT之间的最大生化差异表现在ATP介导的脱氧核苷酸去除过程中。增强去除导致链终止子切除的最大速率的增加，这表明突变的残基在ATP介导的去除的底物的最佳对齐中发挥作用。焦磷酸解的效率并没有增加AZT耐药突变的存在。然而，在D4 TMP链终止的引物的焦磷酸解切割期间由下一个正确核苷酸引起的抑制程度的2倍降低可以说明该突变体如何利用焦磷酸来增强抗性。RT无法催化从RNA-RNA引物-模板中去除链终止子，这可能表明在DNA合成起始期间对AZTMP掺入的选择性的微小变化如何有助于高水平的抗性。总之，这些结果表明，这些突变可能赋予多种耐药模式。链终止剂去除的结构-活性研究表明，与RT活性位点中的残基形成紧密相互作用的类似物可能更容易发生去除介导的抗性机制。
Several hypotheses have been proposed to explain the development of resistance to the anti-HIV drug AZT. Clinical findings show that AZT resistance mutations in HIV-1 reverse transcriptase (RT) not only reduce susceptibility to thymidine analogues but may also confer multi-dideoxynucleoside resistance. In this report, we describe transient kinetic studies establishing the biochemical effects of AZT resistance mutations in HIV-1 RT on the incorporation and removal of natural and unnatural deoxynucleotides. While the physiological role remains to be elucidated, the largest biochemical difference between wild-type and AZT resistant HIV-1 RT manifested itself during ATP-mediated deoxynucleotide removal. Enhanced removal resulted from an increase in the maximum rate of chain terminator excision, suggesting that mutated residues play a role in the optimal alignment of substrates for ATP-mediated removal. The efficiency of pyrophosphorolysis was not increased by the presence of AZT resistance mutations. However, a 2-fold decrease in the extent of inhibition caused by the next correct nucleotide during pyrophosphorolytic cleavage of a D4TMP chain-terminated primer may illustrate how this mutant can utilize pyrophosphate to enhance resistance. The inability of RT to catalyze removal of a chain terminator from an RNA−RNA primer−template may show how slight changes in selectivity against AZTMP incorporation during the initiation of DNA synthesis can contribute to high-level resistance. Taken together, these results suggest that multiple modes of resistance may be conferred by these mutations. Structure−activity studies of chain terminator removal suggest that analogues that form tight interactions with residues in the RT active site may be more prone to resistance mechanisms mediated by removal.