Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase
Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase
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DOI：
10.1074/jbc.m106743200
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发表时间：
2001-11-02
影响因子：
4.8
通讯作者：
Johnson, KA
中科院分区：
文献类型：
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作者：
Johnson, AA;Ray, AS;Johnson, KA
为了研究线粒体聚合酶(POL伽马)在临床观察到的用于治疗艾滋病的核苷类似物毒性中的作用，我们研究了美国食品和药物管理局(FDA)批准的每个类似物加上1-(2-脱氧2-氟-β-D-阿拉伯呋喃葡萄糖基)-5-碘尿嘧啶(FIAU)、β-L-(-)-2‘，3’-二脱氧-3‘-硫代胞苷(-)3TC和(R)-9-(2-膦甲氧丙基)腺嘌呤(PMPA)在波尔伽马催化下的掺入动力学。我们使用重组核酸外切酶缺陷型(E200A)，重组人多聚酶在单周转动力学研究中测定K-d(K-m)和k(Poll)(k(CAT))，以估算每个核苷类似物三磷酸的特异性常数(k(Cat)/K-m)。系列ddC>；dda(Ddi)远大于2‘，3’-二氢-2‘，3’-二脱氧胸苷(D4T)远大于(+)3TC(-)3TC>；PMPA>；叠氮胸苷(AZT)Carbovir(CBV)，其特异性常数变化超过500,000倍。阿巴卡韦(CBV的前体药物)和PMPA是预计毒性最低的两种新药。值得注意的是，d4T、ddC和dda的高毒性源于它们与正常dNTP的结合强度是正常dNTP的13-36倍，尽管它们的参入率与PMPA和AZT相当。我们还检测了掺入后每个类似物的核酸外切酶去除率。FIAU>；(+)3TC(-)3TC>；CBV>；AZT>；PMPA类似于d4T的比率从0.06%到0.0004 S(-1)不等，远大于dda(Ddi)。DdC的去除速度太慢，无法测量(
To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy2-fluoro-beta -D-arabinofuranosyl)-5-iodouracil (FIAU), beta -L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Poly holoenzyme in single turnover kinetic studies to measure K-d (K-m) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K-m) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) much greater than 2',3'-didehydro-2',3'-dideoxythymidine (d4T) much greater than (+)3TC (-)3TC > PMPA > azidothymidine (AZT) Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC (-)3TC > CBV > AZT > PMPA similar to d4T much greater than ddA (ddI) much greater than ddC. Removal of ddC was too slow to measure (