KINETIC-STUDIES WITH THE NONNUCLEOSIDE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE INHIBITOR U-90152E
KINETIC-STUDIES WITH THE NONNUCLEOSIDE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE INHIBITOR U-90152E
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DOI：
10.1016/0006-2952(94)90077-9
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发表时间：
1994-06-01
影响因子：
5.8
通讯作者：
REUSSER, F
中科院分区：
文献类型：
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作者：
ALTHAUS, IW;CHOU, JJ;REUSSER, F
双杂芳基哌嗪 U-90152E 是人类免疫缺陷病毒 1 型 (HIV-1) 逆转录酶 (RT) 的有效抑制剂，在组织培养物中生长的 HIV-1 感染淋巴细胞中具有出色的抗 HIV 活性。该化合物抑制 HIV-1 RT 的 RNA 和 DNA 导向的 DNA 聚合酶功能。进行动力学研究以阐明 U-90152E 的 RT 抑制机制。米氏动力学基于酶及其底物之间快速平衡的建立，但事实证明不足以分析实验数据。因此，使用 Briggs-Haldane 动力学分析数据，假设反应顺序为模板：引物首先与酶结合，然后添加 dNTP，并且聚合酶是持续酶。基于这些假设，导出了速度方程，该方程允许计算酶、其底物和抑制剂之间发生的反应的所有基本的前向和后向速率常数。获得的结果表明，U-90152E 专门充当模板的混合抑制剂：酶的 RNA 和 DNA 导向的 DNA 聚合酶结构域的引物和 dNTP 结合位点。该抑制剂对酶-底物复合物的结合亲和力明显高于对游离酶的结合亲和力，因此不会直接损害底物结合位点的功能。因此，U-90152E 似乎会损害酶-底物复合物形成后发生的事件，该事件涉及磷酸酯键形成的抑制或酯键形成后酶相对于其模板：引物的易位。
The bisheteroarylpiperazine U-90152E is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-directed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived, which allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template:primer and dNTP binding sites for both the RNA- and DNA-directed DNA polymerase domains of the enzyme. The inhibitor shows a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently does not directly impair the functions of the substrate binding sites. Therefore, U-90152E appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond.