Development of Novel Antimalarials
| Project/Area Number |
05557019
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
寄生虫学(含医用動物学)
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| Research Institution | Osaka University |
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Principal Investigator |
HORII Toshihiro Osaka University, Res.Inst.Microbial Dis., Associate Professor, 微生物病研究所, 助教授 (80142305)
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| Co-Investigator(Kenkyū-buntansha) |
TAI Kumiko Osaka University, Res.Inst.Microbial Dis., Technical Officer, 微生物病研究所, 教務職員 (00187907)
MITAMURA Toshihide Osaka University, Res.Inst.Microbial Dis., Research Associate, 微生物病研究所, 助手 (80268846)
MORIMATSU Katsumi Osaka University, Res.Inst.Microbial Dis., Research Associate, 微生物病研究所, 助手 (70263308)
坂井 昭子 (板井 昭子) 東京大学, 薬学部, 教授 (60012647)
杉山 智彦 大阪大学, 微生物病研究所, 助手 (90252709)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1993: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | P.falciparum / Drug design / DHFR / Synthetic gene / Recombinant protein / Antifolate / 人口合成遺伝子 |
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| Research Abstract |
We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase (TS) complex of P.falciparum in E.coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages. The induced expression in an E.coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.
By using the recombinant Plasmodium falciparum DHFRs expressed in Escherichia coli, with amino acid alterations found in DHFR genes of the antifolate resistant strains, we screened 120 kinds of antifolate for possible antimalarials. The validity of the screen, as compared with the
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growth inhibition of the cultured parasite was verified by the subsequent examination of several substituted pyrrolo [2,3-d] pyrimidines for their antimalarial activity. The chemical compounds that were effective against the drug resistant enzymes correspondingly inhibited the growth of the cultured parasites. The test compounds gave 50% inhibitory concentration of the ranged 12-550 nM and 8-1800 nM respectively for the pfDHFRs with cycloguanil and pyrimethamine resistance. As compared to the sensitive enzyme, the fold decrease in sensitivity of the resistant enzymes to the pyrrolo-derivatives were 0.8-7.5 and 3.6-29 ; those for pyrimethamine and cycloguanil were 308 and 400. Similary, the growth inhibition of the cultured P.falciparum gave 50% inhibition concentration of 0.15-49 nM and 5.4-1500 nM respectively for the cycloguanil resistant (FCR3) , and pyrimethamine resistant (K1) strains. As contrasted with the sensitive strain (3D7) , the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds ; those for cycloguanil and pyrimethamine were 690 and 18640. Moreover, the pyrroloderivative code named T-49172 inhibited the growth of P.berghei in mice with 50% effective dose concentration that was comparable to that inhibited by cycloguanil. Less
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Report
(4 results)
Research Products
(5 results)
