Lee Sol, Nam Miyoung, Lee Ah-Reum, Lee Jaewoong, Woo Jihye, Kang Nam Sook, Balupuri Anand, Lee Minho, Kim Seon-Young, Ro Hyunju, Choi Youn-Woong, Kim Dong-Uk, Hoe Kwang-Lae
Department of New Drug Development, Chungnam National University, Daejeon 34134, Republic of Korea.
Department of Life Science, Dongguk University-Seoul, Goyang 10326, Republic of Korea.
Biomol Ther (Seoul). 2021 Mar 1;29(2):234-247. doi: 10.4062/biomolther.2020.166.
We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, , , and ] and 4 essential genes (, , , and ). Expectedly, their mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the heterozygous strain was attributed to both decreased mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.
我们使用了裂殖酵母的杂合基因缺失文库，该文库包含所有必需和非必需基因，用于对抗真菌药特比萘芬的靶基因进行微阵列筛选，特比萘芬通过Erg1酶抑制麦角固醇合成。我们鉴定出了14个杂合菌株，它们对应于10个非必需基因（7个核糖体蛋白编码基因， ， ， ， ， ， ）和4个必需基因（ ， ， ， ）。不出所料，与对照菌株SP286相比，它们的mRNA和蛋白质水平有所下降。当我们使用同源单倍体缺失菌株研究非必需靶基因的作用机制时，与对照菌株ED668相比，SAGA亚基基因的敲除导致转录水平下调。然而，核糖体蛋白基因的敲除并未使细胞对靶向麦角固醇的抗真菌药敏感。令人惊讶的是，如在mRNA实验比例的比较分析中所观察到的，核糖体蛋白基因作为阻遏物复合物的组成部分参与了转录过程。为了了解药物与新的必需靶基因之间相互作用的作用机制，我们用特比萘芬和益康唑（或环己酰亚胺）进行了等效线图分析。杂合菌株对特比萘芬的敏感性归因于mRNA水平的降低和翻译的抑制。此外，Tif302是特比萘芬和环己酰亚胺发挥药效所必需的。基于分子模拟分析，特比萘芬可以直接与酵母中的Tif302结合，这表明Tif302是特比萘芬潜在的脱靶位点。总之，这种全基因组筛选系统可用于在任何感兴趣的条件下鉴定和表征靶基因。
