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Correlation between binding modes and inhibitory activities of ligands studied by molecular docking with topoisomerase I-DNA complex

Title
Correlation between binding modes and inhibitory activities of ligands studied by molecular docking with topoisomerase I-DNA complex
Authors
김지현
Issue Date
2004
Department/Major
대학원 약학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
김춘미
Abstract
DNA topoisomerase Ⅰ (topo Ⅰ)은 DNA double strand의 break나 rejoining에 관여함으로써 DNA의 replication, transcription 그리고 recombination을 조절한다. Topo Ⅰ의 active site 잔기인 Tyr723이 DNA의 cleavage strand의 3’-end와 공유 결합을 형성하면 topo Ⅰ-DNA cleavable complex가 생성되어 supercoiled DNA의 relaxation이 유도된다. 이 단계에 topo Ⅰ 저해제가 결합하여 안정한 ternary complex가 형성되면 DNA religation이 억제되어 double strand break가 발생하므로 세포가 사멸하게 된다. 암세포의 topo Ⅰ을 저해하여 항암 효과를 가지는 topo Ⅰ 저해제는 새로운 항암제의 후보 물질로서 계속해서 연구되고 있다. 대표적인 topo Ⅰ 저해제인 camptothecin(CPT)는 암세포의 topo Ⅰ-DNA cleavable complex에 intercalation되어 topo Ⅰ의 활성을 저해함으로써 항암 효과를 나타내는 것으로 알려져 있다. 이후 CPT를 lead compound로 하여 많은 topo I 저해제들이 합성 되었고, 근래에는 camptothecⅠn의 유도체들 뿐만 아니라 다양한 non-camptothecin DNA intercalator들이 새로운 class의 topo Ⅰ 저해제의 후보 물질로서 끊임없이 연구되고 있다. 본 연구에서는 topo Ⅰ의 저해제로 예상되는 두 종류의 화합물들을 part Ⅰ과 part Ⅱ로 나누어 Sybyl package(6.9.2)에 포함된 docking module인 FlexiDock을 사용하여 topo Ⅰ-DNA-topotecan ternary complex의 X-ray crystal 구조(1K4T)를 근거로 docking 연구를 수행하였다. Part Ⅰ에서는 topo Ⅰ inhibition assay를 통하여 topo Ⅰ의 저해 작용 여부가 밝혀진 Poria cocos(복령)의 methanol extracts에서 얻은 6개의 화합물, 즉 (S)-(+)-tumerone, ergosterol peroxide, polyporenic acid C, dehydropachymic acid, pachymic acid, 그리고 tumulosic acid를 ligands로 사용하여 topo Ⅰ-DNA cleavable complex에 docking한 결과, topo Ⅰ의 저해 효과가 높은 화합물 일수록 active site 잔기인 phosphotyrosine723 (Ptr723)과 매우 근접한 위치에서 DNA의 +1 site인 G11과 -1 site인 T10 사이에 intercalative mode로 결합되어 DNA base와 stacking interaction을 하였고 active site 잔기와 수소결합을 형성하는 것을 확인할 수 있었다. 반면에 topo Ⅰ의 저해 효과가 낮거나 없는 화합물의 경우 intercalative mode로 결합 되지 않거나 DNA의 +1/-1 site의 complement base인 C112와 A113 사이에 결합 되었으며 active site 잔기와 수소결합을 형성하지 않는 것을 발견하였다. Part Ⅱ에서는, part Ⅰ과 마찬가지로, topo Ⅰ inhibition assay 결과가 보고된 14개의 6-arylamino-7-chloro-quinzoline-5.8-dione의 유도체들을 ligand로 하여 docking 실험을 수행하였다. 그 결과 80% 이상의 topo Ⅰ 저해 효과를 가진 것으로 밝혀진 4개 화합물의 경우, topo Ⅰ의 active site 잔기나 DNA의 +1/ 1 site의 base와 수소결합을 형성하며 intercalative mode로 결합하여 안정한 ternary complex를 형성함을 확인하였다. Topo Ⅰ 저해 효과가 58%로 나타난 molecule 13의 경우 DNA 사이에 intercalation 되지 않았지만, 물분자를 경유하여 topo Ⅰ active site 잔기인 Ptr723과 수소결합을 형성하기 때문에 약한 topo Ⅰ 저해 활성을 가질 것으로 판단하였다. 반면에 35% 이하의 낮은 topo Ⅰ 저해 효과를 나타낸 9개 molecule의 경우 DNA의 base 사이에 intercalation 되지 않거나, intercalation 되더라도 quinazoline 모핵이 minor groove에 위치하기 때문에 DNA base와 stacking interaction을 하지 않는 F것으로 나타났다. 또한 topo Ⅰ의 active site 잔기와 수소결합을 형성하지 않아 topo Ⅰ-DNA cleavable complex에 안정하게 결합할 수 없을 것으로 판단하였다. 이상으로 part Ⅰ의 6개 화합물들과 part Ⅱ의 14개 화합물들의 docking 결과와 topo Ⅰ inhibition assay 결과를 비교함으로써 두 결과 사이에 상관관계가 성립함을 확인할 수 있었고, ligand들의 binding mode와 결합 위치를 규명함으로써 이 화합물들의 topo Ⅰ 저해제로서의 작용 기전을 설명할 수 있는 근거를 제시할 수 있었다.;DNA topoisomerase Ⅰ (topo Ⅰ) helps the control of DNA replication, transcription and recombination by assisting breaking and rejoining of DNA double strand. The active site residue, Tyr723, in topo Ⅰ becomes covalently linked to 3’-end of the DNA cleavage strand, then a topo Ⅰ-DNA cleavable complex is formed and supercoiled DNA is relaxed. In this step, topo I inhibitor binds the cleavage complex and stabilizes the ternary complex which eventually blocks DNA religation, resulting in double-strand breaks and ultimately cell death. Topo Ⅰ inhibitors are constantly studied as a novel candidate for anticancer drugs due to it’s inhibitory capacity of topo Ⅰ activity in tumor cells. Camptothecin (CPT), a representative topo Ⅰ inhibitor, have antitumor activity by intercalating into topo Ⅰ-DNA cleavable complex. Many CPT derivatives were developed as topo Ⅰ inhibitors and also various non-camptothecin DNA intercalators are recently synthesized for a new class of topo Ⅰ inhibitor. In this study, two kinds of compound expected to be topo Ⅰ inhibitors are divided into part Ⅰ and part Ⅱ and are docked into the X-ray crystal structure of human topo Ⅰ-DNA binding complex obtained from topo Ⅰ-DNA-topotecan ternary complex using the flexible docking program FlexiDock in Sybyl package (6.9.2). In part Ⅰ, the structures of six compounds extracted from Poria cocos, (S)-(+)-tumerone, ergosterol peroxide, polyporenic acid C, dehydropachymic acid, pachymic acid, and tumulosic acid which are suggested to have topo Ⅰ inhibiton activity were docked into topo Ⅰ-DNA cleavable complex. The results showed that the compounds which have potent topo Ⅰ inhibition activity were docked properly. They intercalated between the +1 and ?1 base pairs of DNA, located near the active site phosphotyrosine723 (Ptr723) and formed hydrogen bonds with active site residues. On the other hand, 2 compounds with no topo Ⅰ inhibition activity were not docked at all. They didn’t intercalate between +1/-1 site DNA base pairs and formed no hydrogen bonds. In part Ⅱ, fourteen analogs of 6-arylamono-7-chloro -quinazoline-5,8-dione which are expected to act as topo Ⅰ inhibitors were tested. Four compounds which showed over 80% of topo Ⅰ inhibition activity formed stable ternary complexes through hydrogen bonds with active site residues or +1/-1 site DNA bases and intercalated stably in between the DNA helices. Molecule 13 which has 58% inhibition activity was not intercalated but formed a hydrogen bond with Ptr723 via water, thus likely to show weak topo Ⅰ inhibition activity. On the other hand, 9 compounds which showed below 35% of inhibition activity were not docked. They did either not intercalate between +1/-1 site DNA base pairs or intercalate between the site but formed no hydrogen bond with active site residues. In conclusion, docking results of six compounds in part Ⅰ and fourteen compounds in part Ⅱ demonstrated the overall correlation between the inhibition activity and the binding affinity. These results suggest that the antitumor activity of well docked ligands may arise from the inhibition of topo Ⅰ by stabilizing the reversible topo Ⅰ-DNA cleavable complex.
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