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首页> 外文期刊>Molecular Cancer >Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico
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Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

机译：体外和计算机分析Glu710Gly拓扑异构酶IB突变体对喜树碱的分子机制

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Background DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear. Methods The three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation. Results The three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine. Conclusions These results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.
机译：背景DNA拓扑异构酶是通过断裂和重新结合DNA链来调节DNA拓扑状态的关键酶。人拓扑异构酶IB可被几种通过不同机制起作用的化合物抑制，包括临床使用的药物，例如天然化合物喜树碱的衍生物，该衍生物可逆地结合共价拓扑异构酶-DNA复合物，从而减慢了切割的DNA链的重新连接，因此诱导细胞死亡。最近鉴定出了三种在腺癌细胞系中赋予对伊立替康抗药性的酶突变，但抗药性的分子机制尚不清楚。方法在啤酒酵母模型系统中研究了这三种抗性突变体在喜树碱含量不断增加的情况下的活力。对其中一个突变体（Glu710Gly）进行了不同催化步骤的系统分析，并将其与经典分子动力学模拟研究的结构动力学性质相关联。结果在酵母细胞活力测定中，这三个突变体表现出不同程度的喜树碱抗性。对Glu710Gly突变体催化循环不同步骤的表征表明，其耐药性与高连接速率有关，而该连接速率几乎不受药物存在的影响。通过仿真分析动力学特性表明，该突变体在不同蛋白质结构域之间的运动中显示出低得多的相关性，并且该接头几乎完全失去了与包含活性位点酪氨酸的C末端结构域的相关性。结论这些结果表明赋予喜树碱对拓扑异构酶I敏感性的功能齐全的连接子是必需的，因为其结构动力学性质的不稳定与连接率和耐药性的增加有关。

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