Miller M D, Lamy P D, Fuller M D, Mulato A S, Margot N A, Cihlar T, Cherrington J M
Gilead Sciences, Foster City, California 94404, USA.
Mol Pharmacol. 1998 Aug;54(2):291-7. doi: 10.1124/mol.54.2.291.
Adefovir dipivoxil [9-(2-(bispivaloyloxymethyl)phosphonylmethoxyethyl)adenine (bis-POM PMEA)], an oral prodrug of adefovir (PMEA), is currently in phase III clinical testing for the treatment of human immunodeficiency virus-1 (HIV-1) infection. Previous in vitro experiments have shown that HIV-1 recombinant viruses expressing either a K65R or a K70E mutation in reverse transcriptase (RT) have reduced sensitivity to PMEA and that the K70E mutant also has impaired replication capacity in vitro. Genotypic analyses of samples from patients enrolled in a phase I/II clinical trial of adefovir dipivoxil demonstrated that the K70E RT mutation developed in two of 29 patients during extended therapy. To further investigate the molecular mechanisms involved in the resistance to PMEA, we cloned, expressed, and purified HIV-1 RT enzymes carrying either the K65R or K70E and, for comparison, the M184V mutation. The Km values of dNTPs for these mutant enzymes were not significantly altered from wild-type RT. The Ki values for the K65R mutant were increased from wild-type by 2-5-fold against a variety of inhibitors, whereas the Ki values for the M184V mutant were increased 12-fold specifically for 2', 3'-dideoxy-3'-thiacytidine (3TC) triphosphate. The Ki values for the K70E mutant were increased for PMEA diphosphate and 3TC triphosphate by 2-3-fold. These results are in agreement with antiviral drug susceptibility assay results. The three recombinant enzymes were also evaluated for their specific activities and processivities. All mutants were reduced in specific activity with respect to wild-type RT. In single-cycle processivity studies, the M184V mutant was, as expected, notably impaired. The K70E mutant was also slightly impaired, whereas the K65R mutant was slightly more processive than wild-type. These results with recombinant K70E RT are consistent with the reduced in vitro replication capacity of the K70E RT mutant of HIV-1 and further demonstrate that the K70E mutation confers minor PMEA and 3TC resistance to HIV-1.
阿德福韦酯[9-(2-(双特戊酰氧甲基)膦酰甲氧基乙基)腺嘌呤(bis-POM PMEA)]，是阿德福韦(PMEA)的口服前体药物，目前正处于治疗人类免疫缺陷病毒1型(HIV-1)感染的III期临床试验阶段。先前的体外实验表明，在逆转录酶(RT)中表达K65R或K70E突变的HIV-1重组病毒对PMEA的敏感性降低，并且K70E突变体在体外的复制能力也受损。对参加阿德福韦酯I/II期临床试验患者的样本进行基因分型分析表明，在29名患者中有2名在延长治疗期间出现了K70E RT突变。为了进一步研究对PMEA耐药的分子机制，我们克隆、表达并纯化了携带K65R或K70E以及作为对照的M184V突变的HIV-1 RT酶。这些突变酶的dNTPs的Km值与野生型RT相比没有显著改变。K65R突变体对多种抑制剂的Ki值比野生型增加了2至5倍，而M184V突变体对2'，3'-双脱氧-3'-硫代胞苷(3TC)三磷酸的Ki值增加了12倍。K70E突变体对二磷酸阿德福韦酯和3TC三磷酸的Ki值增加了2至3倍。这些结果与抗病毒药物敏感性试验结果一致。还评估了这三种重组酶的比活性和持续合成能力。所有突变体相对于野生型RT的比活性均降低。在单循环持续合成能力研究中，正如预期的那样，M184V突变体明显受损。K70E突变体也略有受损，而K65R突变体的持续合成能力比野生型略强。这些重组K70E RT的结果与HIV-1的K70E RT突变体在体外复制能力降低一致，并进一步证明K70E突变赋予HIV-1对PMEA和3TC的轻微耐药性。
