Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies.
Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies.
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DOI：
10.1016/j.bmc.2004.06.046
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发表时间：
2004-10
影响因子：
3.5
通讯作者：
G. Laco;W. Du;G. Kohlhagen;J. M. Sayer;D. Jerina;T. Burke;D. Curran;Y. Pommier
中科院分区：
文献类型：
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作者：
G. Laco;W. Du;G. Kohlhagen;J. M. Sayer;D. Jerina;T. Burke;D. Curran;Y. Pommier
人类拓扑异构酶I （Top1）在细胞复制和转录中起着关键作用，因此是一个重要的抗癌靶点。Homocamptothecin是抑制Top1的先导化合物，由5个共轭平面环（a - e）组成。同角樟碱e环β-羟内酯在pH值为bbb7.0时缓慢打开为羧酸盐。我们从以下几个方面分析了哪一种形式的同星星碱具有生物化学相关性：(1)体外活性测试了同星星碱羧酸酯，发现其无活性；(2)与Top1和dsDNA孵育，发现同角星酸β-羟内酯形态稳定；(3)对同樟碱e环β-羟内酯进行了防开孔修饰，衍生物要么无活性，要么活性较低。这些结果表明，同角星碱β-羟内酯是其活性形式，其充分活性需要一个e环羰基氧和相邻的未取代/未质子化的环原子。通过将+1脱氧鸟苷旋出螺旋，将同角星碱及其衍生物对接到Top1/DNA活性位点模型中，比较配体与Top1/DNA活性位点的相互作用能和配体的体外活性。发现配体相互作用能与体外活性相关，而配体在Top1/DNA活性位点的取向解释了e环β-羟内酯的重要性，而不依赖于e环开口。Top1/DNA活性位点模型的一个重要组成部分是旋转的+1脱氧鸟苷，体外实验和分子模型研究支持+1脱氧鸟苷从螺旋中旋转出来。这些结果允许通过与尚未利用的Top1活性位点残基（包括Glu356， Asn430和Lys751）的工程相互作用来合理设计更有效的Top1抑制剂。
Human topoisomerase I (Top1) plays a pivotal role in cell replication and transcription, and therefore is an important anti-cancer target. Homocamptothecin is a lead compound for inhibiting Top1, and is composed of five conjugated planar rings (A–E). The homocamptothecin E-ring β-hydroxylactone opens slowly to a carboxylate at pH>7.0. We analyzed, which form of homocamptothecin was biochemically relevant in the following ways: (1) the homocamptothecin carboxylate was tested for activity in vitro and found to be inactive; (2) homocamptothecin was incubated with Top1 and dsDNA, and we found that the homocamptothecin β-hydroxylactone form was stabilized; (3) the homocamptothecin E-ring β-hydroxylactone was modified to prevent opening, and the derivatives were either inactive or had low activity. These results indicated that the homocamptothecin β-hydroxylactone was the active form, and that an E-ring carbonyl oxygen and adjacent unsubstituted/unprotonated ring atom were required for full activity. Homocamptothecin and derivatives were docked into a Top1/DNA active site model, in which the +1 deoxyguanosine was rotated out of the helix, in order to compare the interaction energies between the ligands and the Top1/DNA active site with the in vitro activities of the ligands. It was found that the ligand interaction energies and in vitro activities were correlated, while the orientations of the ligands in the Top1/DNA active site explained the importance of the E-ring β-hydroxylactone independently of E-ring opening. An essential component of this Top1/DNA active site model is the rotated +1 deoxyguanosine, and in vitro experiments and molecular modeling studies supported rotation of the +1 deoxyguanosine out of the helix. These results allow for the rational design of more potent Top1 inhibitors through engineered interactions with as yet unutilized Top1 active-site residues including: Glu356, Asn430, and Lys751.