Inhibition of the human immunodeficiency virus type I integrase by guanosine quartet structures
Inhibition of the human immunodeficiency virus type I integrase by guanosine quartet structures
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DOI：
10.1021/bi960541u
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发表时间：
1996-10-29
期刊：
影响因子：
2.9
通讯作者：
Pommier, Y
中科院分区：
文献类型：
--
作者：
Mazumder, A;Neamati, N;Pommier, Y
一个完全由脱氧鸟苷和胸腺嘧啶组成的寡核苷酸（T30177）在钾的存在下折叠成一个高度稳定的四链DNA结构，其中包含两个堆叠的脱氧鸟苷区（G4s）。T30177还能保护宿主细胞免受人类免疫缺陷病毒1型（HIV-1）的细胞病变影响。我们报道这种G4寡核苷酸是迄今为止鉴定的最有效的HIV-1整合酶抑制剂，其IC50值在纳摩尔范围内。形成的四分之一的数量和四分之一之间的环的顺序对最佳活性都很重要。T30177不经加工就与HIV-1整合酶结合，并阻断正常病毒DNA底物与该酶的结合。正常的DNA底物不能竞争T30177与HIV-1整合酶的结合，表明G4s与该酶紧密结合。用截断的HIV-1整合酶进行的实验表明，T30177的抑制作用需要含有锌指的n端区域，并且T30177与含有锌指区域的全长或缺失突变体整合酶的结合优于与仅包含中心催化结构域的缺失突变体的结合。在锌存在的情况下，仅整合酶的n端区域能够有效地与T30177结合，而不能与线性病毒DNA底物结合。因此，G4s代表了通过与酶n端结构域相互作用来抑制HIV-1整合酶的第一类化合物。T30177在含镁缓冲液中比含锰缓冲液具有更大的抑制效能，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HIV-1相似，但抑制猫和猴免疫缺陷病毒整合酶的浓度更高，表明可以实现选择性。我们提出新的艾滋病治疗方法可以基于鸟苷四重奏作为HIV-I整合酶抑制剂。
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 quarters (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 quarters 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 quartets as inhibitors of HIV-I integrase.