Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego St., 02-106 Warszawa, Poland.
Antiviral Res. 2010 Nov;88(2):176-81. doi: 10.1016/j.antiviral.2010.08.011. Epub 2010 Aug 23.
3'-Deoxy-3'-fluorothymidine (FLT, alovudine(®)) belongs to the most potent agents inhibiting HIV-1 replication. Its 5'-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5'-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5'-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and β (pol β) was investigated. It was shown that 3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate (4-SFLTTP) and 3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with K(i)(app) values of 0.091 and 0.022 μM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with K(i)(app) of 0.174 μM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with K(i)(app) of 0.055 μM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol β, with K(i)(app) values of 16.84 and 4.04 μM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3'-fluorothymidine 5'-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989; Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT. Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.
3'-脱氧-3'-氟胸苷(FLT,阿昔洛韦(®))属于抑制 HIV-1 复制最有效的药物之一。其 5'-三磷酸酯(FLTTP)是 HIV-1 逆转录酶(HIV RT)的有效抑制剂。不幸的是,FLT 在体外和体内均表现出明显的血液学毒性。据推测,这种毒性可能与核苷类似物 5'-三磷酸酯抑制人类 DNA 聚合酶有关,特别是线粒体 DNA 聚合酶 γ,导致 DNA 合成终止和线粒体功能障碍。为了降低 FLT 的毒性,先前合成了其硫代类似物 4-SFLT 和 2-SFLT,并证明它们是具有低体外细胞毒性的强效 HIV-1 抑制剂。为了解释这一现象,本研究进行了硫代 FLT 类似物 5'-三磷酸酯的合成,并研究了它们与重组 HIV-1 RT 以及人类 DNA 聚合酶 γ(pol γ)和 β(pol β)的相互作用。结果表明,3'-脱氧-3'-氟-4-硫代胸苷 5'-三磷酸酯(4-SFLTTP)和 3'-脱氧-3'-氟-2-硫代胸苷 5'-三磷酸酯(2-SFLTTP)与 FLTTP 相似,均为 HIV-1 RT 的强竞争性抑制剂,K(i)(app) 值分别为 0.091 和 0.022 μM。有趣的是,2-SFLTTP,一种在糖苷键周围呈异常顺式构象的化合物,是人类线粒体 DNA pol γ 的非竞争性抑制剂,K(i)(app) 为 0.174 μM,而反式构象的 4-SFLTTP 与 FLTTP 相似,即非竞争性,K(i)(app) 为 0.055 μM。4-SFLTTP 和 2-SFLTTP 均为人类 DNA pol β 的竞争性抑制剂,K(i)(app) 值分别为 16.84 和 4.04 μM。结果表明,硫代 3'-氟胸苷 5'-三磷酸酯类似物对 HIV RT 具有部分选择性抑制作用。特别值得注意的是,与 4-SFLTTP 和 FLTTP 相比,呈顺式构象的 2-SFLTTP 对人类线粒体 pol γ 的抑制作用较弱,均为反式构象,对 HIV-1 RT 的抑制活性高于 4-SFLTTP。此外,具有顺式构象的母体核苷 2-SFLT 显示出比其反式构象的 4-硫代异构体更强的抗 HIV-1 活性和更好的选择性指数(Matthes 等人,1989 年;Poopeiko 等人,1995 年),2-SFLT 是一种强效且选择性的抗 HIV-1 药物,其选择性指数比 FLT 高 4 倍。讨论了 FLT 及其硫代类似物的抗病毒和细胞毒性活性的机制。