Biomolecules & Therapeutics
Systematic Target Screening Revealed That Tif302 Could Be an Off-Target of the Antifungal Terbinafine in Fission Yeast
Sol Lee1, Miyoung Nam1, Ah-Reum Lee1, Jaewoong Lee1, Jihye Woo1, Nam Sook Kang1, Anand Balupuri1, Minho Lee2, Seon-Young Kim3, Hyunju Ro4, Youn-Woong Choi5, Dong-Uk Kim6,* and Kwang-Lae Hoe1,*
1Department of New Drug Development, Chungnam National University, Daejeon 34134,
2Department of Life Science, Dongguk University-Seoul, Goyang 10326,
3Personalized Genomic Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141
4Department of Biological Science, College of Bioscience & Biotechnology, Chungnam National University, Daejeon 34134,
5Korea United Pharm. Inc., Seoul 06116,
6Rare Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
*E-mail: (Hoe KL), (Kim DU)
Tel: +82-42-821-8627 (Hoe KL), +82-42-860-4159 (Kim DU)
Fax: +82-42-821-8927 (Hoe KL), +82-42-860-4149 (Kim DU)
Received: September 25, 2020; Revised: October 12, 2020; Accepted: October 13, 2020; Published online: November 23, 2020.
© The Korean Society of Applied Pharmacology. All rights reserved.

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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, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 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 erg1 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 erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 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 tif302 heterozygous strain was attributed to both decreased erg1 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.
Keywords: Antifungal, Terbinafine, Drug target, Ribosomal protein, Sterol, Translation

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