Olsen D B, Carroll S S, Culberson J C, Shafer J A, Kuo L C
Department of Biological Chemistry, Merck Research Laboratories, Merck & Co., West Point, PA 19486.
Nucleic Acids Res. 1994 Apr 25;22(8):1437-43. doi: 10.1093/nar/22.8.1437.
The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.
通过使用异源引物-模板系统，研究了RNA二级结构对HIV-1逆转录酶催化聚合反应以及吡啶-2-酮抑制剂3-(4,7-二氯苯并恶唑-2-基甲基氨基)-5-乙基-6-甲基吡啶-2(1H)-酮（L-697,661）效力的影响。我们的数据表明，RNA模板中的茎环发夹二级结构可能会强烈阻碍HIV-1逆转录酶催化反应中的逆转录过程，导致中等长度（暂停）聚合产物的积累。L-697,661的存在极大地增强了暂停产物的积累，这表明酶从暂停产物上移位的速率可能比聚合反应直至暂停位点的速率受到更有效的抑制。使用含有茎环发夹的合成RNA模板进行的模型实验表明，聚合至二级结构的四个碱基范围内时，L-697,661的抑制效力增加了2倍。当引物延伸穿过茎环的双链区域时，抑制剂效力增强了6倍以上。
