Search for Novel Anti-cancer Drugs Targetting DNA Replication and Repair

寻找针对 DNA 复制和修复的新型抗癌药物

• 批准号: 08557131
• 负责人: HANAOKA Fumio
• 金额: $ 9.15万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557131/
• 关键词: DNA replication; DNA repair; cell-free system; inhibitors; anti-cancer drugs; saponin; DNA helicase; SV40T antigen

We have screened more than one hundred of culture supernatants of Actinomycetes and Eumycetes using cell-free systems for SV40 DNA replication and the nucleotide excision repair (NER) with UV-irradiated SV40 minichromosomes to obtain either inhibitors or stimulators for DNA replication and repair. As s result of these screening, we obtained a couple of samples for replication inhibitors and NER inhibitors.One of the replication inhibitors (RK606) was purified, and its structure was identified as a kind of soybean saponin (soyasaponin II). RK606 also inhibited SV40 DNA replication with purified proteins, therefore, we examined the effect of RK606 on individual enzymes and proteins. It turned out that RK606 specifically inhibited DNA helicase activity of SV40 T antigen. Interestingly, a cellular DNA helicase was not inhibited by this compound, indicating that RK606 might be a specific inhibitor of T antigen helicase. To our knowledge, this is the first inhibitor besides ATP analogues for any DNA helicase.One of the repair inhibitors (RK679) was examined for its stability at different pH and for its extractability by several solvents. The behaviors of the repair inhibitor were similar to those of some kinds of tyrosine kinase inhibitors, genistein and daizein, therefore, we checked the effect of these tyrosine kinase inhibitors on cell-free NER assay. However, we could not find any effect of these compounds on NER,suggesting that RK679 is not either genistein or daizein but is rather different compound.

我们用无细胞系统筛选了100多株放线菌和真菌培养上清中的SV40 DNA复制和核苷酸切除修复（NER）的抑制剂和刺激剂，以获得DNA复制和修复的抑制剂和刺激剂。通过筛选，我们获得了两个复制抑制剂和NER抑制剂的样品，其中一个复制抑制剂（RK606）被纯化，其结构被鉴定为一种大豆皂苷（soyasaponin II）。RK606也抑制SV40 DNA复制与纯化的蛋白质，因此，我们检查了RK606对个别酶和蛋白质的影响。结果表明，RK606特异性抑制SV40 T抗原的DNA解旋酶活性。有趣的是，细胞DNA解旋酶不被该化合物抑制，表明RK606可能是T抗原解旋酶的特异性抑制剂。据我们所知，这是除ATP类似物之外的第一个DNA解旋酶抑制剂，研究了其中一种修复抑制剂（RK679）在不同pH下的稳定性和几种溶剂的萃取性。该修复抑制剂的行为与染料木黄酮和大豆苷元等酪氨酸激酶抑制剂的行为相似，因此，我们检查了这些酪氨酸激酶抑制剂对无细胞NER测定的影响。然而，我们没有发现这些化合物对NER的任何影响，这表明RK679既不是染料木黄酮也不是大豆苷元，而是完全不同的化合物。

项目成果

van der Spek, P.J., Visser, C.E., Hanaoka, F., Smit, B., Hagemeijer, A., Bootsma, D., and Hoeijmakers, J.H.J.: "Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

van der Spek, P.J.、Visser, C.E.、Hanaoka, F.、Smit, B.、Hagemeijer, A.、Bootsma, D. 和 Hoeijmakers, J.H.J.：“小鼠同系物的克隆、比较作图和 RNA 表达

Withers-Ward,E.S.et al.: "Human immunodeficiancy virus type 1 Vpr interacts with HHR23A,acellular protein implicated in nucloocide excision DNA repair" J.Virol.71. 9732-9742 (1997)

Withers-Ward，E.S.等人：“人类免疫缺陷病毒 1 型 Vpr 与 HHR23A 相互作用，HHR23A 是一种参与核苷酸切除 DNA 修复的非细胞蛋白”J.Virol.71。

Yokoi,M.et al.: "Molecular cloning of the cDNA for the catalytxc subunit of plane DNA polymarased and its celd-cycle dependent oxpression." Genes to Cells. 2. 695-710 (1997)

Yokoi,M.et al.：“平面 DNA 聚合酶催化亚基 cDNA 的分子克隆及其 celd 循环依赖性氧化作用。”

Ohkuma,Y.: "Multiple furctions of general tronscription factors TFIIE and TFIIH in transcription : Possible points of regulation by trans-acting factors." J.Biochem.122. 481-489 (1997)

Ohkuma,Y.：“转录中一般转录因子 TFIIE 和 TFIIH 的多个分支：反式作用因子的可能调控点。”

Ito,K.et al.: "c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T artigen." EMBO J.15. 5636-5646 (1996)

Ito,K.等人：“c-Jun 通过多瘤病毒大 T 抗原刺激起源依赖性 DNA 解旋。”