CASPUR Inter-University Consortium for the Application of Super-Computing for Universities and Research, Rome, Italy.
PLoS One. 2010 Jun 3;5(6):e10934. doi: 10.1371/journal.pone.0010934.
Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death.
METHODOLOGY/PRINCIPAL FINDINGS: Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633-643 (the region connecting the linker to the core domain) that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed.
CONCLUSIONS/SIGNIFICANCE: Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in both short-range interactions and long-range protein domain communications.
人类拓扑异构酶 I 催化 DNA 超螺旋的松弛，这是转录、复制和染色体分离等基本细胞过程的关键。它是喜树碱类抗癌药物唯一的靶标。其中，拓扑替康已用于治疗肺癌和卵巢癌多年。喜树碱类药物可逆地与共价中间物 DNA-酶结合，稳定可切割复合物并降低重连接率。停滞的复合物随后与复制叉的推进碰撞，产生致命的双链 DNA 断裂，最终导致细胞死亡。
方法/主要发现：对 DNA-拓扑异构酶 I 二元复合物和 DNA-拓扑异构酶-拓扑替康三元复合物进行了长时间的分子动力学模拟，并进行了比较。药物的存在减少了二元复合物的构象空间，这可以通过主成分和聚类分析观察到。这种构象约束主要是由于连接区 633-643 残基（连接区与核心区的连接区）的灵活性降低，导致三元复合物连接区的整体移动性丧失。在模拟过程中，DNA/药物堆积相互作用得到完全维持，并且与酶形成氢键。拓扑替康使催化残基 Lys532 远离 DNA，使其无法参与重连接反应。观察到 Arg364 与拓扑替康的 B 和 E 环相互作用，形成两个稳定的直接氢键。还观察到蛋白质对拓扑替康的几何排列施加的有趣约束。
结论/意义：对拓扑替康与 DNA-酶复合物相互作用的原子尺度理解是解释其毒性作用和几种单个残基拓扑异构酶突变体中观察到的耐药性的基础。我们观察到由于拓扑替康的存在，在短程相互作用和远程蛋白质结构域通讯方面都发生了显著的变化。
