Cruchaga Carlos, Anso Elena, Font María, Martino Virginia S, Rouzaut Ana, Martinez-Irujo Juan J
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Navarra, 31008 Pamplona, Spain.
Biochem J. 2007 Jul 1;405(1):165-71. doi: 10.1042/BJ20061831.
Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.
由HIV-1逆转录酶（RT）催化的切除反应抑制剂是对抗HIV的一种有前景的方法，因为这些分子会干扰该酶对链终止核苷酸的主要耐药机制。迄今为止，仅有限数量的化合物被证明能抑制此反应，包括非核苷类逆转录酶抑制剂（NNRTIs）和某些焦磷酸类似物。我们之前发现，从三叶榄仁树叶中提取的抗病毒化合物2-O-没食子酰石榴皮苷（2GP）能够抑制HIV-1 RT的RT和核糖核酸酶H活性，而不影响细胞增殖或活力。在本研究中，我们表明2GP在亚微摩尔浓度下还能抑制野生型和对叠氮胸苷（AZT）耐药的酶所催化的依赖ATP和焦磷酸（PP(i)）的磷酸解反应。动力学和直接结合分析表明，2GP是核苷酸底物的非竞争性抑制剂，而它与RT和模板引物的结合存在竞争（解离常数K(d)=85 nM）。从其作用机制预期，2GP对赋予对NNRTIs和AZT耐药性的突变具有活性。当在焦磷酸存在下进行测试时，AZT与2GP的组合具有高度协同性，表明RT催化的磷酸解反应的抑制是所发现协同作用的原因。尽管已经描述了其他与模板引物竞争的RT抑制剂，但这是首次证明这些化合物可用于阻断链终止核苷酸的切除，为它们与核苷类似物联合使用提供了理论依据。
