Meikle I, Cummings J, Macpherson J S, Hadfield J A, Smyth J F
Imperial Cancer Research Fund Medical Oncology Unit, Western General Hospital, Edinburgh, U.K.
Biochem Pharmacol. 1995 Jun 16;49(12):1747-57. doi: 10.1016/0006-2952(95)00086-f.
Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.
将蒽醌核与一系列在其C端经化学修饰的天然氨基酸进行单共轭，已被用作一种合成方法，用于合理设计新型拓扑异构酶（topo）抑制剂。对一系列16种新化合物（NU/ICRF 500 - 515）的拓扑异构酶I和II抑制的生物化学进行了研究，并对其构效关系进行了考察。仅三种化合物被证明能与DNA结合：两种丝氨酸衍生物（NU/ICRF 500和506）和一种精氨酸衍生物（NU/ICRF 510）。在使用纯化酶进行的解连环和松弛测定中，几种化合物被证明是拓扑异构酶II的有效催化抑制剂（在5微克/毫升（10 - 15微摩尔）或更低浓度下100%抑制），且不促进可裂解复合物的形成。这些化合物包括三种与DNA结合的物质（其中NU/ICRF 506活性最高）和一种二羟基苯丙氨酸类似物（NU/ICRF 513）。进一步研究表明，NU/ICRF 500和506均能拮抗安吖啶诱导的DNA裂解。只有NU/ICRF 506能明确抑制拓扑异构酶I的催化活性而不诱导DNA裂解，并且是唯一一种兼具拓扑异构酶I和II催化抑制作用的化合物。一种化合物NU/ICRF 505（酪氨酸共轭物）能稳定拓扑异构酶I的可裂解复合物，而不抑制拓扑异构酶I和II的催化活性。对NU/ICRF 505结构的修饰表明，酪氨酸环上存在未受阻的羟基以及氨基酸C端存在更具疏水性的乙酯都是必不可少的，这表明在三元复合物中药物、酶和DNA之间存在高度特异性的相互作用。分子建模研究表明，观察到的拓扑异构酶抑制差异是氨基酸取代基微小变化引起的主要构象改变的结果，并证实了诱导拓扑异构酶I裂解需要刚性结构要求，而拓扑异构酶II抑制的结构要求则相对不那么刚性。在拓扑异构酶抑制与对人卵巢癌细胞系A2780的体外细胞毒性（IC50范围为3.4 - 11.6微摩尔）之间观察到强烈相关性，这表明细胞杀伤机制至少部分涉及拓扑异构酶抑制。
