Daly Michele B, Roth Megan E, Bonnac Laurent, Maldonado José O, Xie Jiashu, Clouser Christine L, Patterson Steven E, Kim Baek, Mansky Louis M
Center for Drug Discovery, Department of Pediatrics, Emory Center for AIDS Research, Emory University, Children's Healthcare of Atlanta, 1760 Haygood Dr., Atlanta, GA, 30322, USA.
Institute for Molecular Virology, University of Minnesota, 18-242 Moos Tower, 515 Delaware St SE, Minneapolis, MN, 55455, USA.
Retrovirology. 2016 Mar 24;13:20. doi: 10.1186/s12977-016-0254-0.
HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages.
Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages.
Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.
HIV-1复制动力学本质上取决于细胞脱氧核苷酸三磷酸（dNTP）用于病毒DNA合成的可利用性。在活化的CD4(+) T细胞和其他快速分裂的细胞中，dNTP浓度很高，HIV-1逆转录以高效方式发生。相比之下，诸如巨噬细胞等非分裂细胞具有较低的dNTP库，这限制了高效逆转录。氯法拉滨是一种经美国食品药品监督管理局（FDA）批准的核糖核苷酸还原酶抑制剂，在转化细胞系中已显示出强大的抗逆转录病毒活性。在此，我们探讨氯法拉滨在人类原发性HIV-1靶细胞（活化的CD4(+) T细胞和巨噬细胞）中的效力、毒性及作用机制。
氯法拉滨在活化的CD4(+) T细胞和巨噬细胞中均为强效HIV-1抑制剂。由于其在巨噬细胞中的毒性极小，氯法拉滨在这种非分裂细胞类型中显示出超过300的选择性指数。氯法拉滨的抗HIV-1活性与细胞dNTP水平和病毒DNA合成的显著降低相关。此外，我们观察到三磷酸氯法拉滨被HIV-1逆转录酶直接掺入DNA并阻断连续DNA合成，特别是在巨噬细胞中发现的低dNTP水平时。
综上所述，这些数据提供了有力的机制证据，表明氯法拉滨是HIV-1复制的双重作用抑制剂，既限制病毒DNA合成所需的dNTP底物，又直接抑制HIV-1逆转录酶的DNA聚合酶活性。
