Dueweke T J, Kézdy F J, Waszak G A, Deibel M R, Tarpley W G
Upjohn Laboratories, Kalamazoo, Michigan 49001-0199.
J Biol Chem. 1992 Jan 5;267(1):27-30.
The bisheteroarylpiperazines (BHAPs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and specifically block HIV-1 replication (Romero, D. L., Busso, M., Tan, C.-K., Reusser, F., Palmer, J. R., Poppe, S. M., Aristoff, P. A., Downey, K. M., So, A. G., Resnick, L., and Tarpley, W. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8806-8810). Here we show that the radiolabeled BHAP [3H]U-88204 binds specifically to HIV-1 RT with high affinity (KD of 50 nM) and a stoichiometry of 1 mol of U-88204 per 1 mol of p66/p51 RT heterodimer. Binding of [3H]U-88204 to RT is unaffected by the presence of saturating poly(rC).oligo (dG)12-18 template-primer. Direct measurement of competition between [3H]U-88204 and other RT inhibitors for binding to RT reveals mutually exclusive competition between [3H]U-88204 and the non-nucleoside RT inhibitor BI-RG-587 (Kopp, E. B., Miglietta, J. J., Shrutkowski, A. G., Shih, C.-K., Grob, P. M. and Skoog, M.T. (1991) Nucleic Acids Res. 19, 3035-3039), indicating that both share the same binding site. Phosphonoformate in concentrations up to 50 microM shows no competition with [3H]U-88204 for binding to RT either alone or in the presence of template-primer. Dideoxynucleotide RT inhibitors affect the binding of [3H]U-88204 to RT when complementary template-primer is present. [3H]U-88204 and the dideoxynucleotide ddGTP can bind RT simultaneously, but the presence of one ligand decreases the affinity of RT for the second. Inasmuch as ddGTP approximates the nucleotide substrate of RT, the direct demonstration of an RT-dideoxynucleotide-[3H]U-88204 complex validates the use of indirect kinetic methods to assess the strength of BHAP interaction with RT and suggests that RT inhibition by U-88204 is achieved via effects on nucleotide substrate binding.
双杂芳基哌嗪（BHAPs）是人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的有效抑制剂，可特异性阻断HIV-1复制（罗梅罗，D.L.，布索，M.，谭，C.-K.，罗伊瑟尔，F.，帕尔默，J.R.，波普，S.M.，阿里斯托夫，P.A.，唐尼，K.M.，索，A.G.，雷斯尼克，L.，以及塔尔普利，W.G.（1991年）《美国国家科学院院刊》88，8806 - 8810）。在此我们表明，放射性标记的BHAP [3H]U - 88204以高亲和力（解离常数KD为50 nM）特异性结合HIV-1 RT，化学计量比为每1摩尔p66/p51 RT异二聚体结合1摩尔U - 88204。[3H]U - 88204与RT的结合不受饱和聚（rC）·寡聚（dG）12 - 18模板 - 引物存在的影响。直接测量[3H]U - 88204与其他RT抑制剂之间对RT结合的竞争，揭示了[3H]U - 88204与非核苷RT抑制剂BI - RG - 587之间相互排斥的竞争（科普，E.B.，米列塔，J.J.，施鲁特科夫斯基，A.G.，施，C.-K.，格罗布，P.M.和斯科格，M.T.（1991年）《核酸研究》19，3035 - 3039），表明两者共享相同的结合位点。浓度高达50 microM的膦甲酸单独或在模板 - 引物存在下与[3H]U - 88204竞争结合RT时均无竞争作用。当存在互补模板 - 引物时，双脱氧核苷酸RT抑制剂会影响[3H]U - 88204与RT的结合。[3H]U - 88204和双脱氧核苷酸ddGTP可同时结合RT，但一种配体的存在会降低RT对第二种配体的亲和力。由于ddGTP近似于RT的核苷酸底物，RT - 双脱氧核苷酸 - [3H]U - 88204复合物的直接证明验证了使用间接动力学方法来评估BHAP与RT相互作用的强度，并表明U - 88204对RT的抑制是通过影响核苷酸底物结合来实现的。
