Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors
Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors
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DOI：
10.1128/aac.01834-15
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发表时间：
2016-01-01
影响因子：
4.9
通讯作者：
Cihlar, Tomas
中科院分区：
文献类型：
--
作者：
Birkus, Gabriel;Bam, Rujuta A.;Cihlar, Tomas
富马酸替诺福韦艾拉酚胺（TAF）是HIV逆转录酶核苷酸抑制剂替诺福韦（TFV）的口服膦酰胺前药。先前的研究表明，溶酶体丝氨酸蛋白酶组织蛋白酶A（CatA）在TAF的细胞内活化中起主要作用。在这里，我们进一步研究了CatA和其他人水解酶在TAF代谢中的作用。在HEK293T细胞中过表达CatA或肝羧酸酯酶1（Ces1）分别使细胞内TAF水解增加2倍和5倍。在HeLa细胞中用RNA干扰（RNAi）敲低CatA表达使细胞内TAF代谢降低5倍。此外，抗HIV活性和CatA水解速率在大量TFV膦酰胺酯前药中显示出良好的相关性。共价丙型肝炎病毒（HCV）蛋白酶抑制剂（PI）telaprevir和boceprevir在体外可有效抑制CatA介导的TAF活化（50%抑制浓度[IC 50]分别为0.27和0.16 μ M），并在适当浓度下降低其在原代人CD 4（+）T淋巴细胞中的抗HIV活性（分别为21倍和3倍）。相比之下，使用cobicistat、非共价HIV和HCV PI或宿主丝氨酸蛋白酶的各种处方抑制剂时，未观察到CatA抑制或TAF对抗HIV活性的任何显著影响。总的来说，这些研究证实CatA在TAF的细胞内代谢中起关键作用，而肝脏酯酶Ces1可能有助于TAF的肝脏活化。此外，这项工作表明，广泛的病毒和宿主PI，除了特拉匹韦和boceprevir，不干扰TAF的抗逆转录病毒活性。
Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 mu M, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.