薬剤の標的となる分子の探索と新規化学療法剤の開発

1996 年度 実績報告書

• 研究課題/領域番号: 08281105
• 研究種目: 重点領域研究
• 研究機関: 岡山大学
• 研究代表者: 綿矢 有佑 岡山大学, 薬学部, 教授 (90127598)
• 研究分担者: 木村 政継 大阪市立大学, 医学部, 助手 (60195378) ; 北 潔 東京大学, 医科学研究所, 助教授 (90134444) ; 板井 昭子 北里研究所, 部長研究員 (60012647) ; 松田 彰 北海道大学, 薬学部, 教授 (90157313)
• キーワード: マラリア / 抗マラリア薬 / マラリア治療薬 / 標的分子 / スクリーニング / インターネット / 原虫 / 熱帯熱マラリア

研究概要

本研究はランダムスクリーニングと薬剤標的分子の探索結果より抗マラリア薬をデザインし抗マラリア作用を示す新規薬剤の開発を行う。研究代表者・綿矢は熱帯熱マラリア原虫の培養でヒト血清を用いる培地に起因するデータの不安定性を解決すべく、無血清GIT培地に脂質成分を添加したin vitro抗マラリア活性スクリーニング系を確立した。更に、公募で26研究機関より864種類の化合物の提供を受け一次スクリーニングを実施し38サンプルに有用な活性を見出した。最も効果の高かった物質のIC_<50>は39nMであり、その選択毒性は410倍であった。また、インターネット上に本重点研究のホームページ(http://eagle.pharm.okayama-u.ac.jp)を開設した。松田は強力なメチル化阻害剤であるネプラノシンAの構造変換を行い細胞毒性を減弱した誘導体を各種合成し抗マラリア作用を検討した。その結果6′-ホモネプラノシンA,6′-R-メチルネプラノシンA,及び2-フルオロネプラノシンAにin vitroでの強力な抗マラリア作用を見い出した。北は三日熱マラリア原虫シトクロム酸化酵素のサブユニットIIIの遺伝子のクローニングを行いその一次構造について宿圭であるヒトとの相違点を見い出した。また本遺伝子が実際に赤血球内で発現されている事を明らかにした。板井はマラリア治療薬の開発を目指して、コンピュータを用いた手法によりマラリア原虫のジヒドロ葉酸還元酵素の新規阻害剤を化合物ライブラリーから探索することに成功し、さらに活性や選択性(マラリア対ヒト)を改良した阻害剤の分子設計合成を行った。木村はP.yoeliiの新しいP-type陽イオン輸送性ATPase遺伝子の全塩基配列を決定し、蛋白質(1877aa)の構造特異性を解析した。さらに、この蛋白質はオルガネラ膜上で、ガメトサイト特異的に発現することを見い出した。

研究成果

• [文献書誌] F.Kawamoto: "Sequence variation in the 18S rRNA gene,a Target for PCR-based Malaria Diagnosis,in Plasmodium Ovale from Southern Vietnam" J.Clinical Microbiol.34. 2287-2289 (1996)
• [文献書誌] O.Kaneko: "Plasmodium falciparum : Variation in Block 4 of the Precursor to the Major Merozoite Surface Proteins (MSP1) in Natural Populations" Exp.Parasitol. 84. 92-95 (1996)
• [文献書誌] R.K.B.Brobey: "Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials" Mol.Biochem.Parasitol.84. 225-237 (1996)
• [文献書誌] A.Inoue: "Molecular cloning of a RNA binding protein,S1-1." Nucleic.Acid Res.15. 2990-2997 (1996)
• [文献書誌] K.Kageyama: "Promotive action of acylated ascorbate on cellular DNA synthesis and growth at low doses in contrast to inhibitory action at high doses or upon combination with hyperthermia" J.Cancer Res.& Clinical Oncol.122(1). 41-44 (1996)
• [文献書誌] O.Kaneko: "Plasmodium falciparum : Allelic variation in the Merozoite Surface Protein 1 gene in wild isolates from southern Vietnam" Exp.Parasitol.(in press). (1996)
• [文献書誌] W.Wang: "Inactivation of inosine 5′-monophosphate dehydrogenase by the antiviral agent 5-ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide 5′-monophosphate" Biochemistry. 35. 95-101 (1996)
• [文献書誌] N.Minakawa: "Synthesis of 3-halo-3-deazainosines : Conformational lock with the halogen at the 3-position of the 3-deazainosine in anti-conformation" Heterocycles. 42. 149-154 (1996)
• [文献書誌] S.Shuto: "Synthesis of 2′-deoxy and 5′-phosphate derivatives of bredinin. A photochemical imidazole-ring cleavage and subsequent reconstruction of the base moiety" Tetrahedron Lett.37. 187-190 (1996)
• [文献書誌] S.Ono: "Highly stereoselective nucleophilic addition to cyclopropyl carbonyls : the facial selectivity in the cyclopropyl ketones is opposite to that in the corresponding aldehyde" Tetrahedron Lett.37. 221-224 (1996)
• [文献書誌] N.Minakawa: "Synthesis of 5-amino-4-imidazolecarboxamide (AICA) deoxyribosides from deoxyinosines and their conversion into 3-deazapurine derivatives" Chem.Pharm.Bull.44. 288-295 (1996)
• [文献書誌] S.Shuto: "Synthesis of (+) and (-)-milnaciprans and their conformationally restricted analogs" Tetrahedron Lett.37. 641-644 (1996)
• [文献書誌] T.Iino: "Stereoselective synthesis of (2′S)-2′C-alkyl-2′-deoxyuridine." Nucleosides Nucleotides. 15. 169-181 (1996)
• [文献書誌] N.Minakawa: "Synthesis and antileukemic activity of 5-carbon-substituted l-b-D-ribofuranosyl-imidazole-4-carboxamides" Nucleosides Nucleotides. 15. 251-263 (1996)
• [文献書誌] Y.Yoshimura: "A novel synthesis of new antineoplastic 2′-deoxy-2′-substituted-4′-thiocytidines" J.Org.Chem.61. 822-823 (1996)
• [文献書誌] S.Shuto: "Conformational restriction by repulsion between adjacent substituents on a cyclopropane ring : Design and enantioselective synthesis of 1-phenyl-2-(1-aminoalkyl)-cyclopropane-N,N-diethylcarboxamides as potent NMDA receptor antagonists" J.Org.Chem.61. 915-923 (1996)
• [文献書誌] A.Kakefuda: "Synthesis of DNA containing oxetanocin A and (2R,3R)-2-C-(adenin-9-yl)-1,4-anhydro-2,3-dideoxy-3-C-hudroxymethyl-D-arabitol" Tetrahedron. 52. 2863-2876 (1996)
• [文献書誌] H.Awano: "Synthesis and antiviral activity of 5-substituted (2′S)-2′-dexy-2′-C-methylcytidinesand -uridines" Arch.Pharm.Pharm.Med.Chem.329. 66-72 (1996)
• [文献書誌] A.Matsuda: "Polydeoxyaminohexopyranosylnucleosides. Synthesis of 1-(2,3,4-trideoxy-3-nitro-b-D-erythro- and threo-hexopyranosyl)-uracils from uridine" Nucleosides Nucleotides. 15. 205-217 (1996)
• [文献書誌] X.Gu: "Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA" Nucleic Acids Res.24. 1059-1064 (1996)
• [文献書誌] S.Shuto: "Enzymatic Synthesis of 5′-phosphatidyl derivaties of 1-(2-C-cyano-2-deoxy-b-D-arabino-pentofuranosyl)cytosine (CNDAC) and their notable antiumor effects in mice" Bioorg.Med.Chem.Lett.6. 1021-1024 (1996)
• [文献書誌] 松田 彰: "固形がんに有効な新規ヌクレオシド、CNDACの分子設計" Jpn.J.Cancer Chemother.23. 202-210 (1996)
• [文献書誌] 松田 彰: "リレー連載「化学はどこまで生命に近づけるか」第10回抗がん剤「がんと戦うために化学を使おう」" 化学. 51. 180-183 (1996)
• [文献書誌] 周東 智: "リン脂質の経口吸収特性を利用する制癌剤のリンパ系ターゲテイング-制癌疑似リン脂質5′-ホスファチジル-FURの設計・合成および生物活性-" Drug Delivery System. 11. 81-88 (1996)
• [文献書誌] S.Shuto: "Synthesis and Potent Antiviral Activity of 6′-Homoneplanocin A (HNPA)" J.Med.Chem.39. 2392-2399 (1996)
• [文献書誌] 松田 彰: "環状ADPリボース-Ca2_+動員に関与する新しいセカンドメッセンジャー-" ファルマシア. 32. 810-816 (1996)
• [文献書誌] M.Aoyagi: "Effects of 2′-a-and 2′-b-bromo-2′-deoxyadenosine on oligonucleotide hybridization and nuclease stability" Bioorg.Med.Chem.Lett.6. 1573-1576 (1996)
• [文献書誌] T.Obara: "A potent adenosylhomocysteine hydrolase inhibitor lacking antiviral activity. Synthesis and antiviral activity of 6′-carboxylic acid derivatives of neplanocin A" Nucleosides Nucleotides. 15. 1157-1167 (1996)
• [文献書誌] T.Kira: "The FIAU derivative (2′S)-2′-deoxy-2′-C-methyl-5-iodouridine (SMIU) is a novel,less cytotoxic and potent anti-HSV and anti-VZV agent" Antiviral Chem.Chemother.7. 209-212 (1996)
• [文献書誌] A.Matsuda: "1-(3-C-Ethynyl-b-D-ribo-pentofuranosyl)uracil as a potential broad spectrum multifunctional antitumor nucleoside" Bioorg.Med.Chem.Lett.6. 1887-1892 (1996)
• [文献書誌] N.Minakawaw: "Synthesis of [2-2H]-5-ethynyl-1-(b-D-ribofuranosyl)imidazole-4-carboxamide and 5-ethynyl-1-([5-3H]-b-D-ribofuranosyl)imidazole-4-carboxamide (EICAR)" J.Labelled Compd.Radiopharm.38. 809-824 (1996)
• [文献書誌] A.E.A.Hassan: "Conversion of (Z)-2′-cyanomethylene-2′-deoxyuridines into their (E)-isomers via addition of thiophenol to the cyanomethylene moiety followed by oxidative syn-elimination reactions" J.Org.Chem.61. 6261-6267 (1996)
• [文献書誌] S.Shuto: "Synthesis,antitumor effects,and possible enzymatic activation mechanism of 5′-phosphatidyl-2′-deoxy-2′-methylenecytidine (DMDC)" Bioorg.Med.Chem.Lett.6. 2177-2182 (1996)
• [文献書誌] T.Ono: "Cell kill kinetics of an antineoplastic nucleoside,1-(2-deoxy-2-methylene-b-D-erythoro-pentofuranosyl)cytosine" Biochem.Pharmacol.52. 1279-1285 (1996)
• [文献書誌] T.Obara: "Synthesis and antiviral activity of 2-halo derivatives of neplanocin A" J.Med.Chem.19. 3847-3852 (1996)
• [文献書誌] S.Miura: "In vitro and in vivo antitumor activity of a novel mucleoside,4′-thio-2′-deoxy-2′-methylidnecytidine" Bio.Pharm.Bull.
• [文献書誌] Y.I.Pavlov: "Base analog N6-hydroxylaminopurine mutagenesis in Escherichia coli : molecular specificity and genetic control" Mutation Res.357. 1-15 (1996)
• [文献書誌] S.Tabata: "Antitumor effect of a novel multifunctional antitumor nucleoside,3′-ethynylcytidine,on human cancers" Oncology Reports. 3. 1029-1034 (1996)
• [文献書誌] N.Nishizono: "Synthesis of thietane nucleosides via the Pummerer reaction as a key step" Tetrahedron Lett.37. 7569-7572 (1996)
• [文献書誌] S.Shuto: "Synthesis and biological activity of conformationally restricted analogs of milnacipran : (1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]cyclopropane-N,N-diethylcarboxamide,an efficient noncompetitive N-methyl-D-aspartic acid receptor antagonist" J.Med.Chem.39. 4844-4852 (1996)
• [文献書誌] H.Hattori: "1-(3-C-Ethynyl-b-D-ribo-pentofuranosyl)cytosine (ECyd),1-(3-C-Ethynyl-b-D-ribo-pentofuranosyl)uracil (EUrd),and their Nucleobase Analogues as New Potential Multifunctional Antitumor Nucleosides with a Broad Spectrum of Activity" J.Med.Chem.39. 5005-5011 (1996)
• [文献書誌] H.Ohshima: "Damage induced by hydroxyl radicals generated in the hydration layer of g-irradiated frozen aqueous solution of DNA" J.Radiat.Res.37. 199-207 (1996)
• [文献書誌] N.Nishizono: "Synthesis of novel nucleosides with a thietane ring" Nucleic Acids Sympo.Series. 35. 59-60 (1996)
• [文献書誌] S.Takatori: "Antitumor mechanism of 3′-ethynylcytidine (ECyd) and 3′-ethynyluridine (EUrd)" Nucleic Acids Sympo.Series. 35. 209-210 (1996)
• [文献書誌] F.Saruta: "Cloning of a cDNA ecoding the small subunit of cytochrome b558 (cybS) of mitochondrial fumarate reductase (Complex II) from adult Ascaris suum" Biochim.Biophys.Acta. 276. 1-5 (1996)
• [文献書誌] Y.Ogawa: "Dnak heat shock protein of Escherichia coli maintains the negative supercoiling of DNA against thermal stress" J.Biol.Chem.271. 29407-29414 (1996)
• [文献書誌] N.Minagawa: "An antibiotic,Ascofuranone,specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei" Mol.Biochem.Parasitol.81. 127-126 (1996)
• [文献書誌] P.K.C.Lim: "Cytochrome c oxidase III (COIII) gene of Plasmodium vivax : Complete coding sequence and its expression in the erythrocytic stage of the parasite" Jpn.J.Parasitol.45. 373-383 (1996)
• [文献書誌] Y.Nomura: "Incorporation of 5-(N-aminoalkyl)carbamoyl-2′-deoxycytidines into oligodeoxyribonucleotides by a convenient post-synthetic modification method" Bioorg.Med.Chem.Lett.6. 2811-2816 (1996)
• [文献書誌] Y.Ueno: "Facile synthesis of 5′-5′-linked oligodeoxyribonucleotides with the potential for triple-helix formation" Bioorg.Med.Chem.Lett.6. 2817-2822 (1996)
• [文献書誌] S.Ichikawa: "Synthesis of 3′-b-branched-chain ribonucleosides via samarium diiodide-mediated intramolecular Reformatsky type reaction and their antitumor activities" Nucleic Acids Sympo.Series. 35. 13-14 (1996)
• [文献書誌] Y.Yoshimura: "Synthesis of 2′-deoxy-2′-fluoro-4′-thioarabinonucleosides as potential antitumor and antiviral agents from D-glucose" Nucleic Acids Sympo.Series.35. 15-16 (1996)
• [文献書誌] E.Nozawa: "Synthesis and antitumor activity of 1-(3-C-ethynyl-b-D-ribo-pentfuranosyl)cytosine,-uracil and their surag modified derivatives" Nucleic Acids Sympo.Series. 35. 31-32 (1996)
• [文献書誌] Y.Yoshimura: "Synthesis of iso-4′-thionucleosides using the Mitsunobu reaction" Nucleic Acids Sympo.Series. 35. 55-56 (1996)
• [文献書誌] T.Kakutani: "Activation of c-fos and c-jun Genes and Protease Association in Cell Death Induced with 5-Fluoro-2′-deoxyuridine in Mouse Mammary Tumor FM3A Cell Line" Nucleic Acids Symp.Ser.35. 269-270 (1996)
• [文献書誌] S.Kankawa: "The Endonuclase S Relative to dNTP Imbalance Death" Nucleic Acids Symp.Ser.35. 271-272 (1996)
• [文献書誌] M.Arai: "A Colourimetric DNA Diagnotic method for Falciparum Malaria and Vivax Malaria : a Field Trial in the Solomon Islands" Nucleosides Nucleotides. 15. 719-731 (1996)
• [文献書誌] M.Kimura: "Cases of Ovale Malaria and Malariae Malaria Successfully Treated with Mefloquine" Jpn.J.Trop.Med.Hyg.24. 171-175 (1996)
• [文献書誌] M.Miyake: "PCR Diagnosis of Malaria at Convalescent Stage Post-Treatment" J.Travel.Med.3. 119-121 (1996)
• [文献書誌] T.Toyoda: "New Inhibitors based on Dihydrofolate Reductase Domain of Plasmodium falciparum" Biochem.Biophys.Res.Comm.(in press). (1996)