Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives
Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives
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DOI：
10.1074/jbc.m210891200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Sarisky, RT
中科院分区：
文献类型：
--
作者：
Gu, BH;Johnston, VK;Sarisky, RT
丙型肝炎病毒（HCV）NS 5 B蛋白编码RNA依赖性RNA聚合酶（RdRp），其是HCV复制酶复合物的主要催化酶。最近，两种苯并-1，2，4-噻二嗪化合物被证明是含有羧基末端21个残基截短的基因型1b HCV RdRp（Delta 21 HCV RdRp）的有效、高度特异性抑制剂（Dhanak，D.，Duffy，K.，约翰斯顿，V.K.，Lin-Goerke，J.，达西，M.，Shaw，A. N. G. B.，Silverman，C.，Gates，A. T.，Earnshaw，D. L.，Casper，D. J.，Kaura，A.，贝克，A.，格林伍德角，古特肖尔湖L.，Maley，D.，DelVecchio，A.，马卡龙河，霍夫曼，G.一、Alnoah，Z.，郑汉钧是的，陈，G.，Khandekar，S.，基南河M.，和Sarisky，R. T.（2002）J.Biol.Chem.277，38322-38327）。化合物4（C21 H21 N3 O 4S）通过其与病毒聚合酶的直接相互作用而不是通过核酸模板的非特异性滴定来减少病毒复制。在这项研究中，我们提出了几条证据，证明这种抑制剂干扰RNA合成的起始步骤，而不是作为一个延伸抑制剂。无论复制是否由引物依赖性或从头机制启动，初始磷酸二酯键形成的抑制都会发生。使用增加浓度的化合物4的滤器结合研究不干扰Delta 21 HCV RdRp与核酸相互作用的能力。此外，在引物延伸试验中改变试剂添加顺序显示抑制谱无明显差异。最后，表面等离子体共振分析提供的证据表明，一个三元复合物是能够形成之间的RNA模板，RdRp，和化合物4。总之，这些数据表明，这种杂环剂与脱辅基酶以及Delta 21 HCV RdRp的RNA结合形式相互作用，因此不会直接干扰RdRp-RNA相互作用以介导抑制。
The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (Delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C21H21N3O4S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of Delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of Delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.