2021 Fiscal Year Annual Research Report
植物内生真菌を用いたケミカルバイオロジーによる抗トキソプラズマ薬の探索
| Project/Area Number |
20F20402
|
|---|
| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
|---|
Principal Investigator |
西川 義文 帯広畜産大学, 原虫病研究センター, 教授 (90431395)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ARIEFTA NANANG 帯広畜産大学, 原虫病研究センター, 外国人特別研究員
|
|---|
| Project Period (FY) |
2020-11-13 – 2023-03-31
|
| Keywords | 内生菌 / カビ / トキソプラズマ / マラリア / 細胞毒性 |
|---|
| Outline of Annual Research Achievements |
This study aims to find effective anti-Toxoplasma and antiplasmodial agents from endophytic fungi. Sixteen plants were used as sources of the fungi isolation, resulting in the isolation of 130 endophytic fungi. The 130 methanolic extracts of these fungi were in vitro screened against Plasmodium falciparum 3D7 and Toxoplasma gondii RH-GFP. Seventeen fungi were identified as potential candidates. Fungus 16 (AL-9.1), which inhibited the growth of T. gondii RH-GFP and had low cytotoxicity against human foreskin fibroblast (HFF) cells, was chosen for further study. This fungus was subsequently identified as Fusarium petersiae by sequencing analysis of the ITS region of its rDNA. The methanol, hexane, chloroform, and ethyl acetate fractions derived from this fungus were exhibited IC50 values (μg/ml) of 67.22 ± 8.95, 17.43 ± 5.40, 86.08 ± 19.69, and 36.67 ± 9.72, respectively, against T. gondii RH-GFP. Additionally, treatment of experimental mice with the methanolic extract for 7 days after infection with 5,000 tachyzoites of the T. gondii PRU HX-KU- strain increased their survival rate (100% in 100 mg/Kg, 66.67% in untreated mice). The liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was then used to identify the main components of the active fractions, resulting in the identification of four main components. Subsequently, these compounds exhibited >70% growth inhibition against T. gondii RH-GFP at a concentration of 10 μM.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
This study showed the in vitro anti-Toxoplasma and antiplasmodial activities of extracts derived from fungal endophytes. Bioactivity-guided isolation is an essential primary method of screening for active compounds. From the screening processes, we found that the methanolic extract of fungus 16 (F. petersiae) exhibited inhibition efficacy against T. gondii in vitro and in vivo. However, it is challenging to determine compounds or parts of the compound responsible for a particular activity in the screening process. Often, compounds with high activity are exceeded and do not show any action due to various conditions such as the concentration and the effect of other matrices. Therefore, we utilized LC-MS/MS analysis to detect the main components of the active sub-fractions, identifying four main components. From the inhibition test, these compounds exhibited >70% growth inhibition against T. gondii RH-GFP at a concentration of 10 μM.
|
| Strategy for Future Research Activity |
We will continue to do isolation guided by anti-Toxoplasma and antimalaria activities of the hit fungi from screening processes. In addition, we will be more focused on the bioactivity of identified compounds. In the meantime, the isolation process of the extract from the fungal fermentation process will be continued to increase the compound depository, which probably has a related structure to the identified compounds from LC-MS/MS. The isolation of these compound derivatives will be valuable in the structure-activity relationship study and the development of more potent candidates. The structures of isolated active compounds will be determined by spectroscopic methods, including nuclear magnetic resonance (NMR), high-resolution mass spectroscopy (HR-MS), time-dependent density-functional theory (TD-DFT), and chemical derivatization.
Additionally, the identified compounds will be further screened in silico for the protein target and subjected to molecular docking and molecular dynamic simulations. Finally, based on the in-silico study results, protein inhibition activity will be confirmed in vitro by producing recombinant protein and protein kinetic study. Furthermore, prolonged exposure to parasites with low doses of isolated active compounds will generate resistant strains. The compound-resistant genome analysis will be investigated to identify the gene mutation. These approaches will clarify the potential mode of action of active compounds and support the development of new drugs.
|
