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

• 研究課題/領域番号: 14780466
• 研究種目: 若手研究(B)
• 配分区分: 補助金
• 研究分野: 生物有機科学
• 研究機関: 神戸学院大学
• 研究代表者: 水品 善之 神戸学院大学, 栄養学部, 講師 (20307705)
• 研究期間 (年度): 2002 – 2003
• 研究課題ステータス: 完了 (2003年度)
• 配分額: 3,500千円 (直接経費: 3,500千円); 2003年度: 1,600千円 (直接経費: 1,600千円); 2002年度: 1,900千円 (直接経費: 1,900千円)
• キーワード: 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

研究概要

(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。

研究成果

• [文献書誌] Yoshiyuki Mizushina et al.: "Cyanogenic glycoside from Taraxacum offcinale"Natural Med.. 57・1. 36 (2003)
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• [文献書誌] 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)
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• [文献書誌] 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)
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• [文献書誌] Shunya Takahashi et al.: "Total synthesis of dehydroaltenusin"Tetrahedron Lett.. 44・9. 1875-1877 (2003)
• [文献書誌] Yoshiyuki Mizushina et al.: "A sulfoquinovosyl diacylglycerol is a DNA polymerase ε inhibitor"Biochem. J.. 370・1. 299-305 (2003)
• [文献書誌] Yoshiyuki Mizushina et al.: "Kohamaic acid A, a novel sesterterpenic acid, inhibits activities of DNA polymerases from deuterostomes"Biochim. Biophys. Acta. (in press). (2003)
• [文献書誌] Yoshiyuki Mizushina et al.: "Cyanogenic glycoside from Taraxacum officinale"Natural Med.. (in press). (2003)
• [文献書誌] Yoshiyuki Mizushina et al.: "Molecular action mode of hippospogic acid A, an inhibitor of gastrulation of starfish embryos"J. Biochem.. (in press). (2003)
• [文献書誌] 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)