Mazumder A, Neamati N, Ojwang J O, Sunder S, Rando R F, Pommier Y
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Biochemistry. 1996 Oct 29;35(43):13762-71. doi: 10.1021/bi960541u.
An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.
一种完全由脱氧鸟苷和胸苷组成的寡核苷酸（T30177）此前已被证明在钾存在的情况下会自身折叠成一种高度稳定的四链DNA结构，其中包含两个堆叠的脱氧鸟苷四联体（G4s）。T30177还能保护宿主细胞免受1型人类免疫缺陷病毒（HIV-1）的细胞病变效应。我们报告称，这种G4寡核苷酸是迄今为止鉴定出的最有效的HIV-1整合酶抑制剂，其IC50值在纳摩尔范围内。形成的四联体数量以及四联体之间环的序列对于最佳活性都很重要。T30177在未经加工的情况下与HIV-1整合酶结合，并阻断正常病毒DNA底物与该酶的结合。正常DNA底物无法竞争取代T30177与HIV-1整合酶的结合，这表明G4s与该酶紧密结合。对截短的HIV-1整合酶进行的实验表明，T30177抑制作用所需的含有假定锌指的N端区域，并且T30177与含有锌指区域的全长或缺失突变整合酶的结合要优于与仅由中央催化结构域组成的缺失突变体的结合。在锌存在的情况下，单独的整合酶N端区域能够有效地与T30177结合，但不能与线性病毒DNA底物结合。因此，G4s代表了第一类通过与酶的N端结构域相互作用来抑制HIV-1整合酶的化合物。与含锰缓冲液相比，T30177在含镁缓冲液中的抑制效力更强，这表明沿磷酸二酯主链的二价金属离子配位可能在抑制活性中起作用。T30177抑制HIV-2整合酶的效力与抑制HIV-1的效力相似，但在更高浓度下抑制猫和猴免疫缺陷病毒整合酶，这表明可以实现选择性。我们提出，新型艾滋病疗法可以基于鸟苷四联体作为HIV-1整合酶的抑制剂。
