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dc.contributor.author우선아-
dc.creator우선아-
dc.date.accessioned2016-08-26T03:08:14Z-
dc.date.available2016-08-26T03:08:14Z-
dc.date.issued2003-
dc.identifier.otherOAK-000000003612-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/194669-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000003612-
dc.description.abstractDNA topoisomerase I (top1)은 DNA strand의 breakage와 religation을 통해 DNA의 topology를 변화시키므로 암 화학요법제 개발의 새로운 target enzyme으로 연구되고 있다. 이 과정에서 사람 top1의 active site 잔기인 tyrosine은 DNA 이중나선의 잘려진 한 가닥의 3 -end와 공유결합을 형성한다. 이렇게 형성된 top1-DNA cleavable complex가 저해제에 의해 안정화되면 DNA의 religation이 일어나지 않아 세포가 사멸하게 된다. 본 연구에서는 top1-DNA complex와 저해제들간의 결합 상호작용을 예측하기 위해 genetic algorithm에 근거하여 개발된 FlexiDock docking program을 사용하였다. X-ray crystallography로 밝혀진 human top1-DNA covalent complex를 Protein Data Bank(PDB)로부터 얻고 이를 target enzyme으로 하여 5 가지의 top1 inhibitors, 즉 camptotecin(CPT), topotecan, 10,11-methylenedioxy-20(S)-CPT, NSC314622 그리고 saintopin을 docking하였다. 그 결과 저해제들은 DNA의 +1 base(G11)와 -1 base(T10) 사이에 intercalative mode로 결합하였으며, active site 잔기로 알려진 Arg364, Asp533 및 Asn722와 인접하여 수소결합과 hydrophobic interaction을 하여 religation이 일어나지 않도록 inhibitor-top1-DNA ternary complex의 구조를 안정화시켰다. CPT 및 그 유도체의 docking 결과, 공통적으로 E ring의 lactone 구조와 Arg364가 수소결합을 형성하여, lactone 구조가 약효를 나타내는데 필수적이라는 실험적인 근거를 뒷받침하였으며, 또한 20-hydroxyl기가 S형으로 존재할 때만 DNA의 +2 site base와 수소결합을 형성함으로써 S isomer 만이 활성을 나타낸다는 실험적 결과를 확인하였다. 이상의 결과로부터 top1 inhibitors의 정확한 결합위치와 binding mode를 예측하였으며, docking 결과가 strucuture-activity relationship에 잘 부합됨을 증명하였다. 이 연구를 통해 얻은 정보는 새로운 구조의 top1 inhibitor의 개발에 구조적인 근거를 제공할 수 있을 것이다 DNA topoisomerase I(top1)은 DNA의 replication, transcription및 repair에 관계하므로 임상적으로 쓰이고 있는 중요한 항암제나 항균제의 표적효소가 되고 있다. 이 효소는 DNA strand의 breakage와 religation을 통해 DNA의 topology를 변화시키며, 이 과정에서 사람 top1의 active site 잔기인 tyrosine은 DNA 이중나선의 잘려진 한 가닥의 3 -end와 공유결합을 형성한다. 이렇게 생성된 top1-DNA cleavable complex가 저해제에 의해 안정화되면 DNA의 religation이 일어나지 않아 세포가 사멸하게 된다. Benz(f)indole-4,9-dione 유도체는 1,4-naphthoquinone에 pyrrole핵이 결합되어 있는 화합물로서 항암, virus static, 항생, 항진균 및 항 혈액응고 등의 광범위한 생리작용이 있다고 보고되었다. Benz(f)indole-4,9-dione은 width가 7.161 인 planar tri-heterocyclic ring과 p-conjugated ketone group을 가진 DNA intercalator로서의 조건에 매우 잘 일치하는 분자로, benz(f)indole-4,9-dione을 구성하는 1,4-naphthoquinone 유도체들이 top1을 저해 한다는 사실이 밝혀지고 있어 benz(f)indole 유도체들이 DNA intercalation에 의해 top1을 저해할 것이라고 추정된다. Top1-DNA complex와 저해제들과의 상호작용을 규명하기 위해 이 실험에서 는 genetic algorithm에 근거하여 개발된 docking program인 FlexiDock을 사용하여 human top1-DNA covalent complex의 pdb 구조(1A31)에 benz(f)indole 모핵의 16개 유도체들을 docking하였다. 그 결과, IC50가 5 uM미만으로 약효가 좋은 5개의 유도체 중 4개는 모두 DNA 사이에 intercalative mode로 결합하면서 top1-DNA complex와 1~5개의 수소결합을 형성하였으나, 나머지 1개는 수소결합을 형성하지 않아 다른 4개와 그 결합양상에 차이가 있었다. IC50가 5-20 uM인 중간 정도의 약효를 가진 4개의 유도체는 모두 intercalative mode로 결합하였으나 2개는 active site 잔기와 수소결합을 형성하여 안정한 ternary complex를 이루었고 나머지 2개는 수소결합을 형성하지 못했다. IC50가 20 uM이상인 7개의 유도체 중 4개는 intercalative mode로 결합하지 않고 major 또는 minor groove로 빠져나갔으나 3개는 intercalative mode로 결합하면서 active site 잔기와 수소결합을 형성했다. Docking이 잘 된 경우의 구조는 top1 저해제인 camptothecin의 binding mode와 일치함으로써 이들 유도체의 항암활성이 top1-DNA cleavable complex의 trap에 의한 top1 저해에 기인할 가능성을 제시하였다. 열여섯개의 benz(f)indole 유도체 중 12개에서 activity와의 correlation을 발견하였다. ; DNA topoisomerse I(top1) has recently been investigated as a new target for cancer chemotherapy since it catalyzes changes in DNA topology through cycles of transient DNA strand breakage and religation. During this process, the active site tyrosine in human top1 becomes covalently linked to the 3 -ends of a single-stranded nick in the DNA duplex. Stabilization of the top1-DNA cleavable complex which prevents the religation is the common initial event leading to the cytotoxicity of top1 inhibitors. A computer program, FlexiDock with genetic algorithm, was used in this study to predict the binding interactions between the top1-DNA complex and inhibitors. Five inhibitors, camptothecin(CPT), topotecan, 10,11-methylenedioxy camptothecin, NSC314622 and saintopin, were docked into the X-ray crystal structure of the human top1-DNA cleavable complex. The ternary complexes formed show that the inhibitors intercalated between the -1 and +1 base pairs of DNA , and formed hydrogen bonds with active site residues, Arg364, Asp533 and Asn722. Top1-DNA cleavable complexes are stabilized by the inhibitors, thus unable to religate the DNA. Their interactions confirmed the experimental data that the lactone structure of E ring was essential for the activity by forming hydrogen bonds with Arg364 and only S isomer of 20-hydroxyl group showed activity by forming a hydrogen bond with +2 site base of DNA in CPT and their derivatives. The results presented the potential sites of interaction for each compound and agreed well with structure-activity relationship. The information obtained from this work may provide a rational basis for the design of novel top1 inhibitors. DNA topoisomerases have been a target enzymes for anticancer and antibacterial drugs since they take part in the mechanism of replication, transcription and repair of DNA. They catalyze changes in DNA topology through cycles of transient DNA strand breakage and religation. During this process, the active site tyrosine in human DNA topoisomerase I(top1) becomes covalently linked to the 3 -ends of a single-stranded nick in the DNA duplex. Stabilization of the top1-DNA cleavable complex is the common initial event leading to the cytotoxicity of top1 inhibitors. It is known that benz(f)indole derivatives posses versatile pharmacological activities such as anticancer, virus-static, antibacterial, fungicidic, and anticoagulant actions. The antitumor activity of quinone derivatives have been thoroughly studied and it is known that they act as topoisomerase inhibitors via DNA-intercalation. Benz(f)indole is a compound which has a planar tri-heterocyclic ring of width with 7.161 A and a p-conjugated ketone group. Therefore, the compounds are fully consistent with the conditions of intercalators. Using the flexible docking program FlexiDock, novel antitumor agents with benz(f)indole structures were docked into the human top1-DNA complex. Sixteen agents were tested. Out of five agents with IC50 between 0.1 and 5uM, four were docked well, intercalating DNA and forming up to 5 H-bonding to top I-DNA complex, while one was not. Out of four agents with moderate activity with IC50 between 5 and 20uM, two were docked well while two were not. Among remaining seven agents with IC50 over 20uM, four were not docked while three were. The well docked structures showed similar intercalative binding modes with the known top1 inhibitors, such as camptothecin. These results suggest that the antitumor activity of well docked benz(f)indole derivatives may arise from the inhibition of top1 by trapping reversible top1-DNA cleavable complex. On the other hand, the activity of agents not well docked may come from different mechanisms.-
dc.description.tableofcontents표목차 = iv 그림목차 = vii Part I. Docking of topoisomerase I inhibitors into human top1-DNA complex = 1 논문개요 = 2 I. 서론 = 4 II. 실험방법 = 9 1. Preparation of the Top1-DNA cleavable complex = 9 2. Ligand preparation = 11 3. Ligand docking into the Top1-DNA cleavable complex = 13 3.1 FlexiDock pocket = 14 3.2 Flexible bond = 14 3.3 H-bond donor/acceptor = 14 3.3.1 Donors in protein = 15 3.3.2 Acceptors in proein = 15 3.3.3 Donors in ligand = 15 3.3.4 Acceptors in ligand = 15 III. 결과 및 고찰 = 16 1. Docking of Camptothecin(CPT) = 16 2. Docking of Topotecan = 23 3. Docking of 10, 11-methylenedioxy-20(S)-camptothecin (MDO-CPT) = 28 4. Docking of NSC314622 = 33 5. Docking of Saintopin = 38 IV. 결론 = 42 V. 참고문헌 = 44 Abstract = 48 Part II. Docking of benz(f)indole deriavatives into human top1-DNA complex. = 50 논문개요 = 51 I. 서론 = 53 II. 실험방법 = 56 1. Preparation of the Top1-DNA cleavable complex = 56 2. Ligand preparation = 56 3. Ligand docking into the Top1-DNA cleavable complex = 56 4. Docking 여부의 판단 = 61 III. 결과 및 고찰 = 62 1. IC50가 5 uM 미만인 molecule의 docking = 62 1.1 Docking of Molecule 4 = 64 1.2 Docking of Molecule 6 = 66 1.3 Docking of Molecule 8 = 67 1.4 Docking of Molecule 11 = 68 1.5 Docking of Molecule 13 = 69 2. IC50가 20 uM 미만인 Molecule의 docking = 71 2.1 Docking of Molecule 1 = 74 2.2 Docking of Molecule 3 = 75 2.3 Docking of Molecule 14 = 76 2.4 Docking of Molecule 16 = 78 3. IC50가 20 uM 이상인 molecule의 docking = 80 3.1 Docking of Molecule 2 = 83 3.2 Docking of Molecule 5 = 83 3.3 Docking of Molecule 7 = 83 3.4 Docking of Molecule 9 = 84 3.5 Docking of Molecule 10 = 85 3.6 Docking of Molecule 12 = 85 3.7 Docking of Molecule 15 = 86 IV. 결론 = 88 V. 참고문헌 = 90 Abstract = 94-
dc.formatapplication/pdf-
dc.format.extent6480767 bytes-
dc.languagekor-
dc.publisher이화여자대학교 대학원-
dc.titleStudy of binding modes and their correlationship with activity between human topoisomerase I and its inhibitors by computer : aided molecular docking-
dc.typeMaster's Thesis-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 약학과-
dc.date.awarded2003. 2-
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