| 研究実績の概要 |
We conducted a yeast (Saccharomyces cerevisiae) chemical genomics (CG) approach for bioactive NPDepo compounds and compounds provided by domestic and foreign collaborators. In our S. cerevisiae CG pipeline, the set of ~1000 yeast essential genes was analyzed with three different strain collections including a set of temperature sensitive (TS) mutants, a set of heterozygous diploid (HET) deletion mutants, and a set of yeast strains each carrying an essential gene on a high-copy plasmid (MoBY; Molecular Barcoded Yeast). The nonessential gene set consisting of ~5000 viable haploid deletion mutants (WG; whole genome) was also analyzed. Based on our CG profiling, we have carried out the prediction of targets for several compounds (e.g., fatty acid synthase, aminoacyl-tRNA synthetase, cell wall synthesis, glycosylation, etc.). Especially, we focused on a clionamine analog which inhibits Mycobacterium tuberculosis (Mtb) survival in macrophages. Our CG analysis predicted a phosphatidylinositol 4-kinase (PI4K) as a target of the clionamine analog, which was supported by a pull-down assay and an in vivo inhibition assay. Finally, using mammalian cells, we validated PI4K as a viable host directed therapy cellular target against Mtb.
In collaboration with the Moffat Lab in University of Toronto, we developed a CRISPR-Cas9-based CG analysis method using human cell line HAP1 in suspension culture, which increased throughput. We launched to screen the compounds whose targets were successfully predicted by the yeast CG pipeline as well as our collaborators’ compounds.
|
