Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.
Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.
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线粒体转录和RNA聚合酶II依赖性核转录对抗病毒核糖核苷的敏感性。
DOI：
10.1371/journal.ppat.1003030
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Cameron CE
中科院分区：
文献类型：
--
作者：
Arnold JJ;Sharma SD;Feng JY;Ray AS;Smidansky ED;Kireeva ML;Cho A;Perry J;Vela JE;Park Y;Xu Y;Tian Y;Babusis D;Barauskus O;Peterson BR;Gnatt A;Kashlev M;Zhong W;Cameron CE
核糖核苷类似物具有作为抗病毒剂、抗寄生虫剂、抗细菌剂和抗癌剂的潜在用途。然而，它们的临床应用受到脱靶效应的限制。用于治疗丙型肝炎病毒（HCV）感染的抗病毒核糖核苷的开发因临床试验期间出现的毒性而受到阻碍，这些毒性在临床前研究期间逃避了检测。众所周知，人类线粒体 DNA 聚合酶是脱氧核糖核苷逆转录酶抑制剂的脱靶点。在这里，我们检验了这样的假设：治疗性核糖核苷类似物的三磷酸化代谢物是细胞 RNA 聚合酶的底物。我们使用具有抗 HCV 活性的核糖核苷类似物作为治疗性核糖核苷的模型化合物。我们已经纳入了含有 2'-C-甲基、4'-甲基和 4'-叠氮基取代基的核糖核苷类似物，这些取代基是 HCV RNA 聚合酶的非专性链终止子。我们在体外证明所有抗 HCV 核糖核苷类似物都是人线粒体 RNA 聚合酶 (POLRMT) 和真核核心 RNA 聚合酶 II (Pol II) 的底物。出乎意料的是，含有2'-C-甲基、4'-甲基和4'-叠氮基取代基的类似物是POLRMT和Pol II的抑制剂。重要的是，TFIIS 的校对活动能够从 Pol II 转录本中切除这些类似物。对细胞中转录的评估证实了 POLRMT 对抗病毒核糖核苷的敏感性，而 Pol II 仍然主要是难治性的。我们引入了一个称为米托韦（抗病毒核糖核苷引起的线粒体功能障碍）评分的参数，该评分可以在量化化合物的线粒体毒性潜力的临床前研究中轻松获得。我们认为，由于线粒体或核转录缺陷，在临床试验中表现出不良反应的患者可能更容易受到核苷类似物的损害。这里报道的范例应该促进具有较低毒性潜力的核糖核苷的开发。核糖核苷类似物具有作为抗病毒剂、抗寄生虫剂、抗细菌剂和抗癌剂的潜在用途。然而，它们的临床应用受到来源不明的副作用的限制。在这里，我们在生化和细胞研究中表明，抗病毒核糖核苷酸类似物是体外人线粒体 RNA 聚合酶 (POLRMT) 和真核核心 RNA 聚合酶 II (Pol II) 的底物。终止病毒 RNA 聚合酶合成 RNA 的类似物也会抑制这些细胞 RNA 聚合酶。重要的是，Pol II 的 TFIIS 校对活动能够从 Pol II 转录本中切除这些类似物。我们引入了一个称为米托韦（抗病毒核糖核苷引起的线粒体功能障碍）评分的参数，该评分可以在量化化合物的线粒体毒性潜力的临床前研究中轻松获得。我们认为，由于线粒体或核转录缺陷，在临床试验中表现出不良反应的患者可能更容易受到核苷类似物的损害。这里报道的范例应该促进具有较低毒性潜力的核糖核苷的开发。
Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity. Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by side effects of unknown origin. Here we show in biochemical and cell-based studies that antiviral ribonucleotide analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Analogues that terminate RNA synthesis by viral RNA polymerases also inhibit these cellular RNA polymerase. Importantly, the TFIIS proofreading activity of Pol II is capable of excising these analogues from Pol II transcripts. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.
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影响因子：
4.8
作者：
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通讯作者：
Edwards, AM
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FLETCHER, J
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Unadkat, JD