Wilstermann Amy M, Bender Ryan P, Godfrey Murrell, Choi Sungjo, Anklin Clemens, Berkowitz David B, Osheroff Neil, Graves David E
Departments of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 2007 Jul 17;46(28):8217-25. doi: 10.1021/bi700272u. Epub 2007 Jun 20.
Etoposide is one of the most successful chemotherapeutic agents used for the treatment of human cancers. The drug kills cells by inhibiting the ability of topoisomerase II to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. Etoposide is composed of a polycyclic ring system (rings A-D), a glycosidic moiety at the C4 position, and a pendent ring (E-ring) at the C1 position. Although drug-enzyme contacts, as opposed to drug-DNA interactions, mediate the entry of etoposide into the topoisomerase II-drug-DNA complex, the substituents on etoposide that interact with the enzyme have not been identified. Therefore, saturation transfer difference [1H]-nuclear magnetic resonance spectroscopy and protein-drug competition binding assays were employed to define the groups on etoposide that associate with yeast topoisomerase II and human topoisomerase IIalpha. Results indicate that the geminal protons of the A-ring, the H5 and H8 protons of the B-ring, and the H2' and H6' protons and the 3'- and 5'-methoxyl protons of the pendent E-ring interact with both enzymes in the binary protein-ligand complexes. In contrast, no significant nuclear Overhauser enhancement signals arising from the C-ring, the D-ring, or the C4 glycosidic moiety were observed with either enzyme, suggesting that there is limited or no contact between these portions of etoposide and topoisomerase II in the binary complex. The functional importance of E-ring substituents was confirmed by topoisomerase II-mediated DNA cleavage assays.
依托泊苷是用于治疗人类癌症的最成功的化疗药物之一。该药物通过抑制拓扑异构酶II在双链DNA通过反应过程中连接其切割的核酸的能力来杀死细胞。依托泊苷由一个多环系统（A - D环）、C4位置的糖苷部分和C1位置的侧环（E环）组成。尽管与药物 - DNA相互作用相反，药物 - 酶接触介导了依托泊苷进入拓扑异构酶II - 药物 - DNA复合物，但尚未确定依托泊苷上与该酶相互作用的取代基。因此，采用饱和转移差[1H] - 核磁共振光谱和蛋白质 - 药物竞争结合试验来确定依托泊苷上与酵母拓扑异构酶II和人类拓扑异构酶IIα相关的基团。结果表明，A环的偕质子、B环的H5和H8质子、侧链E环的H2'和H6'质子以及3' - 和5' - 甲氧基质子在二元蛋白质 - 配体复合物中与两种酶相互作用。相比之下，用这两种酶均未观察到来自C环、D环或C4糖苷部分的显著核Overhauser增强信号，这表明在二元复合物中依托泊苷的这些部分与拓扑异构酶II之间存在有限或无接触。拓扑异构酶II介导的DNA切割试验证实了E环取代基的功能重要性。
