Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977
Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977
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DOI：
10.1074/jbc.m110.161802
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发表时间：
2010-11-05
影响因子：
4.8
通讯作者：
Furman, Phillip A.
中科院分区：
文献类型：
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作者：
Murakami, Eisuke;Tolstykh, Tatiana;Furman, Phillip A.
2'-脱氧-2'-α-氟-β-C-甲基尿苷-5'-单磷酸的氨基磷酸酯前药 PSI-7851 在体外和体内均表现出有效的抗丙型肝炎病毒 (HCV) 活性。 PSI-7851 是两种非对映异构体 PSI-7976 和 PSI-7977 的混合物，其中 PSI-7977 是 HCV 复制子测定中更活跃的 HCV RNA 复制抑制剂。为了抑制 HCV NS5B RNA 依赖性 RNA 聚合酶，PSI-7851 必须代谢为活性三磷酸形式。第一步，通过人组织蛋白酶 A (CatA) 和/或羧酸酯酶 1 (CES1) 水解羧基酯，这是一个立体特异性反应。 Western blot分析显示CatA和CES1均在原代人肝细胞中表达。然而，在克隆 A 复制子细胞中检测不到 CES1 的表达。 CatA 和/或 CES1 抑制剂的研究表明，CatA 主要负责克隆 A 细胞中羧基酯的水解，尽管在原代人肝细胞中，CatA 和 CES1 均有助于水解。酯水解后，假定羧基对磷进行亲核攻击，导致苯酚自发消除并产生丙氨酰磷酸代谢物 PSI-352707，这是两种异构体共有的。 PSI-352707 的氨基酸部分由组氨酸三联体核苷酸结合蛋白 1 (Hint1) 催化去除，得到 5'-单磷酸形式 PSI-7411。 siRNA 介导的 Hint1 敲低研究进一步表明，Hint1 至少部分负责将 PSI-352707 转化为 PSI-7411。然后，PSI-7411 分别通过 UMP-CMP 激酶和核苷二磷酸激酶连续磷酸化为二磷酸 PSI-7410 和活性三磷酸代谢物 PSI-7409。
A phosphoramidate prodrug of 2'-deoxy-2'-alpha-fluoro-beta-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.