The novel nucleoside analog R1479 (4′-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture
The novel nucleoside analog R1479 (4′-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture
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DOI：
10.1074/jbc.m510195200
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发表时间：
2006-02-17
影响因子：
4.8
通讯作者：
Nájera, I
中科院分区：
文献类型：
--
作者：
Klumpp, K;Lévêque, V;Nájera, I
丙型肝炎病毒 (HCV) 聚合酶活性对于 HCV 复制至关重要。核苷类似物的靶向筛选鉴定出 R1479（4'-叠氮胞苷）是 HCV 亚基因组复制子系统中 HCV 复制的特异性抑制剂（IC50 = 1.28 μM），与 2'-C-甲基胞苷（IC50 = 1.13 μM）相比具有相似的效力。 R1479 在浓度高达 2 mM 时对 HCV 复制子或 Huh-7 细胞的细胞活力或增殖没有影响。与 R1479 长时间孵育后，HCV 复制子 RNA 可以从复制子细胞中完全清除。相应的 5'-三磷酸衍生物 (R1479-TP) 是从复制子细胞中分离的天然 HCV 复制酶和重组 HCV 聚合酶 (NS5B) 介导的 RNA 合成活性的有效抑制剂。 R1479-TP 作为 CTP 竞争性抑制剂抑制 RNA 合成，K-i 为 40 nM。在 HCV RNA 衍生的模板底物（互补的内部核糖体进入位点）上，R1479-TP 显示出与 3'-dCTP 相似的 NS5B 抑制效力。在反应条件下，R1479-TP 通过 HCV 聚合酶掺入新生 RNA 中，并以与 3'-dCTP 相似的效率进一步减少延伸。 NS5B 编码序列中的 S282T 点突变赋予对 2'-C-MeATP 和其他 2'-甲基核苷酸抑制的抗性。相比之下，S282T 突变并未赋予对 R1479 的交叉抗性。
Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC50 = 1.28 mu M) with similar potency compared with 2'-C-methylcytidine (IC50 = 1.13 mu M). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K-i of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.