DNAポリメラーゼβおよびDNAトポイソメラーゼII新規特異的阻害物質の研究

• Project/Area Number: 14780466
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Bioorganic chemistry
• Research Institution: Kobe Gakuin University
• Principal Investigator: 水品 善之 神戸学院大学, 栄養学部, 講師 (20307705)
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000); Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
• Keywords: DNAポリメラーゼβ / DNAトポイソメラーゼII / 酵素阻害剤 / 蛋白質立体構造 / 抗癌剤 / コンピュータ・シミュレーション / タンパク質立体構造

Research Abstract

(1)天然材料からDNAポリメラーゼおよびDNAトポイソメラーゼII阻害物質の探索
天然からDNAポリメラーゼβおよびDNAトポイソメラーゼIIを選択的に阻害する物質を探索した。薬用植物であるナンテンの葉から細胞を脱分化させて、大量培養した細胞の脂溶性抽出液に両酵素の阻害活性が見られたので、阻害物質を単離・精製した。化学構造を^1H-および^<13>C-NMR,FAB-MSで解析した結果、トリテルペンであった。そこで、この細胞に存在する各種トリテルペン類を単離・精製、化学構造を決定してから、トリテルペンの構造とDNAポリメラーゼβおよびDNAトポイソメラーゼIIの阻害活性の相関を調査した。カルボキシル基(COOH基)を有すること、水酸基(OH基)が少ないことが両酵素の阻害に必須であることをが示唆されたDNAポリメラーゼβとDNAトポイソメラーゼIIの共通阻害にはトリテルペンのステロール骨格の構造が関わっており、olide型やlupane型よりも、ursane型が阻害に重要であることがわかった。
(2)酵素-阻害剤の分子間相互作用の解析
DNAトポイソメラーゼII阻害剤として報告されているHoppospongic acid A (HA-A)がDNAポリメラーゼβを阻害することを見出した。DNAポリメラーゼβ (39kDa)はトリプシンで加水分解してdNTP結合活性を持つ31kDaドメイン断片とDNA結合活性を持つ8kDaドメイン断片に分けることができる。これら酵素や断片を用いてHAAとの分子間相互作用測定を実施した。その結果、HA-Aは8kDaドメインのssDNA結合活性に必須なアミノ酸群と結合することが解明された。そして、そのアミノ酸群を鋳型にしてDNAトポイソメラーゼIIのssDNA結合活性領域におけるHA-A結合シミュレーションに成功した。

Research Products

• [Publications] Yoshiyuki Mizushina et al.: "Cyanogenic glycoside from Taraxacum offcinale"Natural Med.. 57・1. 36 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Kohamaic acid A, a novel sesterterpenic acid, inhibits activities of DNA polymerases from deuterostomes"Biochim.Biophys.Acta. 1648・1-2. 55-61 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Molecular action mode of hippospogic acid A, an inhibitor of gastrulation of starfish embryos"J.Biochem.. 133・4. 541-552 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "The inhibitory action of pyrrolidine alkaloid, 1,4-dideoxy-1,4-imino-D-ribitol, on eukaryotic DNA polymerases"Biochem.Biophys.Res.Commun.. 304・1. 78-85 (2003)
• [Publications] Kiminori Matsubara et al.: "Inhibitory effect of pyridoxal 5'-phosphate on endothelial cell proliferation, replicative DNA polymerase and DNA topoisomerase"Int.J.Mol.Med.. 12・1. 51-55 (2003)
• [Publications] Chikako Murakami et al.: "Analysis of cell cycle regulation by l-mono-0-acyl-3-0-(α-D-sulfoquinovosyl)-glyceride (SQMG),an inhibitor of eukaryotic DNA polymerases"Biochem.Pharmacol.. 66・4. 541-550 (2003)
• [Publications] Hiromi Yoshida et al.: "Variation in the content of tocopherols and istribution of fatty acids within soya bean seeds (Glycine max L.)"J.Food Comp.Anal.. 16. 429-440 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Inhibition of DNA polymerases and DNA topoisomerase II by triterpenes produced by plant callus"Biochem.Biophys.Res.Commun.. 305・2. 365-373 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Selective inhibition of mammalian DNA polymerase alpha by vitamin D2 and D3"J.Pharmacol.Sci.. 92・3. 283-290 (2003)
• [Publications] Hiromi Yoshida et al.: "Effects of microwave treatment on the oxidative stability of peanut (Arachis hypogaea) oils and the molecular species of their triacylglycerols"Eur.J.Lipid.Sci.Technol.. 105. 351-358 (2003)
• [Publications] Hiromi Yoshida et al.: "Roasting effects on the distribution of tocopherols and phospholipids within each structural part and section of soybeans"J.Am.Oil Chem.Soc.. 80・7. 665-674 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Some anti-chronic inflammatory compounds are DNA polymerase λ-specific inhibitors"Biochem.Pharmacol.. 66・10. 1935-1944 (2003)
• [Publications] Akira Iida et al.: "Phenylethanoid glycosides targeting mammalian DNA polymerases"Natural Med.. 57・4. 146-149 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Inhibitory action of emulsified sulfoquinovosyl acylglycerol on mammalian DNA polymerases"Lipids. 38・10. 1065-1074 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase α and ε"Biochem.Biophys.Res.Commun.. 312・4. 1025-1032 (2003)
• [Publications] Shim-ichi Matsuura et al.: "Novel immobilization technique of single-stranded DNA molecules on a glass surface for direct analysis of DNA polymerase activity"ISBC2003. 2003. 322-325 (2003)
• [Publications] Kiminori Matsubara et al.: "Petasiphenol which inhibits DNA polymerase λ activity is an inhibitor of in vitro angiogenesis"Oncol.Rep.. 11・2. 447-451 (2004)
• [Publications] Toshihiro Akihisa et al.: "Dehydrotrametenonic Acid and Dehydroeburiconic Acid from Poria cocos and Their Inhibitory Effects on Eukaryotic DNA Polymerase α and β"Biosci.Biotechnol.Biochem.. 68・2. 448-450 (2004)
• [Publications] Isoko Kuriyama et al.: "L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase ε from a plant cultured cells, Nicotina tabacum L."Bioorg.Med.Chem.. 12・5. 957-962 (2004)
• [Publications] Hiromi Yoshida et al.: "Lipid classes, fatty acid composition and triacylglycerol molecular species in the kernels of pumpkin seeds (Cucurbita spp.)"J.Sci.Food.Agric.. 84. 158-163 (2004)
• [Publications] Chikako Murakami et al.: "Mechanism of cell cycle arrest by sulfoquinovosyl monoacylglycerol with a C18-saturated fatty acid (C18-SQMG)"Biochem.Pharmacol.. 67・7. 1373-1380 (2004)
• [Publications] Hiromi Yoshida et al.: "Variations in the composition of acyl lipids and triacylglycerol molecular species of pumpkin (Cucurbita spp.) seeds"Eur.J.Lipid Sci.Technol.. 106. 101-109 (2004)
• [Publications] Kouji Kuramochi et al.: "Structure-activity relationships of epolactaene analogs as DNA polymerase inhibitors"Bioorg.Med.Chem.. (in press). (2004)
• [Publications] Hiromi Yoshida et al.: "Fatty acid distributions of triacylglycerols and phospholipids in peanuts seeds (Arachis hypogaea L.) following microwave treatment"J.Food.Comp.Anal.. (in press). (2004)
• [Publications] Yoshiyuki Mizushina et al.: "Selective inhibition of the activities of both DNA polymerases and DNA topoisomerase II by lanostane-type triterpene acids from Poria cocos"Cancer Sci.. (in press). (2004)
• [Publications] Yoshiyuki Mizushina et al.: "A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase β and λ"J. Biol. Chem.. 277・1. 630-638 (2002)
• [Publications] Hiroeki Sahara et al.: "Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmonoacylglycerols"Jpn. J. Cancer Res.. 93・1. 85-92 (2002)
• [Publications] Madoka Watanabe et al.: "Biotinylated lithocholic acids for affinity chromatography of mammalian DNA polymerases α and β"Bioorg. Med. Chem. Lett.. 12・3. 287-290 (2002)
• [Publications] Yoshiyuki Mizushina et al.: "Selective inhibition of the activities of both eukaryotic DNA polymerases and DNA topoisomerases by elenic acid"Biochem. Pharmacol.. 63・3. 399-407 (2002)
• [Publications] Shinji Kamisuki et al.: "Actions of derivatives of dehydroaltenusin, a new mammalian DNA polymerase α-specific inhibitor"Biochem. Pharmacol.. 63・3. 421-427 (2002)
• [Publications] Chikako Murakami et al.: "Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases"Biochim. Biophys. Acta. 1574・1. 85-92 (2002)
• [Publications] Chikako Murakami et al.: "Novel anti-inflammatory compounds from Rubus sieboldli, terpenoids, are inhibitors of mammalian DNA polymerases"Biochim. Biophys. Acta. 1596・2. 193-200 (2002)
• [Publications] Chikako Murakami et al.: "Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as a mammalian DNA polymerases inhibitors"Arch. Biochem. Biophys.. 403・2. 229-236 (2002)
• [Publications] Yoshitaka Matsumoto et al.: "An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin"Transplantation. 74・2. 261-267 (2002)
• [Publications] Masako Oda et al.: "Inhibition of telomerase by linear-chain fatty acids : A structural analysis"Biochem. J.. 367・2. 329-334 (2002)
• [Publications] Nobuyuki Kasai et al.: "Three-dimensional structural model analysis of binding site of an inhibitor, nervonic acid, of both DNA polymerase β and HIV-1 reverse transcriptase"J. Biochem.. 132・5. 819-828 (2002)
• [Publications] Yoshiyuki Mizushina et al.: "Petasiphenol : a DNA polymerase λ inhibitor"Biochemistry. 41・49. 14463-14471 (2002)
• [Publications] 坂口謙吾, 菅原二三男, 水品善之: "真核生物DNAポリメラーゼの阻害剤:ケミカルノックアウト剤およびサプレッサー様天然物"生化学(日本生化学会). 47・3. 29-32 (2002)
• [Publications] 水品善之 他: "分子プローブを用いたDNAポリメラーゼβとDNAトポイソメラーゼIIの立体構造相関の解析"蛋白質 核酸 酵素(共立出版). 47・6. 708-714 (2002)
• [Publications] Chikako Murakami et al.: "Effects of glycolipids from spinach on mammalian DNA polymerases"Biochem. Pharmacol.. 65・2. 259-267 (2003)
• [Publications] Chikako Murakami et al.: "A novel DNA polyrnerase inhibitor and a potent apoptosis inducer : 2-mono-0-acy1-3-0-(α-D-sulfoquinovosyl)-glyceride with one stearic acid"Biochim. Biophys. Acta. 1645・1. 72-80 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Flavonoid glycoside : a new inhibitor of eukaryotic DNA polymerase α, and a new carrier for inhibitor-affinity chromatography"Biochem. Biophys. Res. Commun.. 301・2. 480-487 (2003)
• [Publications] Shunya Takahashi et al.: "Total synthesis of dehydroaltenusin"Tetrahedron Lett.. 44・9. 1875-1877 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "A sulfoquinovosyl diacylglycerol is a DNA polymerase ε inhibitor"Biochem. J.. 370・1. 299-305 (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Kohamaic acid A, a novel sesterterpenic acid, inhibits activities of DNA polymerases from deuterostomes"Biochim. Biophys. Acta. (in press). (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Cyanogenic glycoside from Taraxacum officinale"Natural Med.. (in press). (2003)
• [Publications] Yoshiyuki Mizushina et al.: "Molecular action mode of hippospogic acid A, an inhibitor of gastrulation of starfish embryos"J. Biochem.. (in press). (2003)
• [Publications] Chikako Murakami et al.: "Analysis of cell cycle regulation by 1-mono-0-acy1-3-0-(α-D-sulfoquinovosyl)-glyceride (SQMG), an inhibitor of eukaryotic DNA polymerases"Biochem. Pharmacol.. (in press). (2003)