| Project/Area Number |
23K23491
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| Project/Area Number (Other) |
22H02224 (2022-2023)
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Multi-year Fund (2024)
Single-year Grants (2022-2023) |
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| Section | 一般 |
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| Review Section |
Basic Section 37030:Chemical biology-related
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
BOONE CHARLES 国立研究開発法人理化学研究所, 環境資源科学研究センター, チームリーダー (70601342)
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| Co-Investigator(Kenkyū-buntansha) |
Pham LienThiKim 国立研究開発法人理化学研究所, 環境資源科学研究センター, 特別研究員 (50865300)
八代田 陽子 国立研究開発法人理化学研究所, 環境資源科学研究センター, 副チームリーダー (60360658)
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2024)
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| Budget Amount *help |
¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000)
Fiscal Year 2024: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2023: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000)
Fiscal Year 2022: ¥8,190,000 (Direct Cost: ¥6,300,000、Indirect Cost: ¥1,890,000)
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| Keywords | Target identification / Chemical genetics / CRISPR-Cas9 / Chemical genomics / target identification / chemical genetics |
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| Outline of Research at the Start |
Systematic chemical-genetic profiling with pools of barcoded mutant cells provides an unbiased strategy for linking compounds to their cellular target pathways. We propose to build and apply a genome-wide chemical-genetic interaction screening platform in a human cell line HAP1 to functionally characterize compounds provided from the RIKEN Natural Product Depository or the University of Tokyo Drug Discovery Initiative Core Library and elucidate their targets in human cells, which will be a valuable tool for drug development.
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| Outline of Annual Research Achievements |
We proposed to build and apply a genome-wide chemical genomic interaction screening platform in a human HAP1 cell line (a near-haploid cell line derived from a leukemic cell line), to functionally characterize compounds and elucidate their targets in human cells. Our unique and innovative approach will lead to the establishment of a reference human chemical-genetic interaction database for compounds with known modes of action, and the functional annotation of previously uncharacterized compounds from the RIKEN Natural Product Depository (NPDepo) or the University of Tokyo Drug Discovery Initiative (DDI) Core Library. We will biochemically validate roles of a collection of promising tool compounds that can be used to explore diverse aspects of cell biology and exploited as potential leads for drug development. In FY2022, we employed and set up a unique CRISPR-Cas9 gRNA library, the Toronto KnockOut library v3, covering ~18,000 human genes to enable genome-wide scale loss of function screens in mammalian cells. To select bioactive compounds to be examined in the human chemical genomics pipeline, we screened the RIKEN NPDepo and the University of Tokyo DDI Core Library for the compounds that possess strong and highly conserved target predictions based on our integrated yeast chemical-genetic platform.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In this FY2022, we completely established the human chemical genomics pipeline. We employed a unique CRISPR-Cas9 gRNA library, the Toronto KnockOut library v3, covering ~18,000 human genes to enable genome-wide scale loss of function screens in mammalian cells, which was developed by the Moffat Lab, University of Toronto. We are utilizing a haploid human cell line, HAP1, because it has a single copy of each gene, and this model cell performs well in our CRISPR-Cas9 assays. The analysis pipeline for NGS data and the database have been set up in collaboration with the Moffat Lab, University of Toronto, and the Chad Myers Lab, University of Minnesota.
We screened thereupon for chemical-genetic interactions of 10 “gold standard” compounds in that their chemical-genetic profiles clearly indicated the expected target pathway. We are still screening for other compounds with well-defined targets to build a set of reference chemical-genetic profiles derived from HAP1 cells.
Also, based on our yeast chemical-genomics database, we carefully selected a subset of potent and functionally diverse bioactive compounds from the RIKEN NPDepo and the University of Tokyo DDI Core Library with strong and highly conserved target predictions. We examined the bioactivity and dose-response of interesting bioactive compounds in our HAP1 model system. So far, 15 compounds were profiled for chemical-genetic interaction by using our established CRISPR-Cas9 screening platform.
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| Strategy for Future Research Activity |
We will screen the selected compounds for chemical-genetic interactions using the genome-wide CRISPR-Cas9 screening platform in HAP1 cells. The resultant chemical-genetic interactions n HAP1 cells will be compared with the ones in yeast cells, and then optimal methods for target validation will be considered and carried out. Especially, as to the several novel inhibitors for sphingolipid biosynthesis that we have been working on using yeast, we will generate chemical-genetic profiles for those inhibitors in HAP1 cells and will conduct metabolomic analysis upon treatment of those compounds to quantify levels of various sphingolipid metabolites, including sphingosines and ceramides, using liquid chromatography with tandem mass spectrometry (LC-MS).
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