Multidrug Sensitive Yeast Strains, Useful Tools for Chemical Genetics

Abstract

The budding yeast Saccharomyces cerevisiae is a useful eukaryote model organism for application to chemical biology studies, for example, drug screening, drug evaluation, and target identification. To use yeast for chemical biology research, however, it has been necessary to construct yeast strains suitable for various compounds because of their high drug resistance. Hence, the deletion of all multidrug resistance genes except for those that are important for viability and for genetic experiments/manipulation could increase the drug sensitivity without influencing the transformation, mating, or sporulation efficiency. There are two major factors conferring multidrug resistance in S. cerevisiae: one is the drug efflux system and the other is the permeability barrier. We therefore constructed a strain which shows high sensitivity to multiple drugs by disrupting the drug efflux system using ATP-binding cassette transporters and suppressing the membrane barrier system by introducing an ERG6-inducible system. In this review, we discuss the construction of our multidrug-sensitive yeast strains and their application in chemical biology.

Systematic analyses of the resistance potential of drugs targeting SARS-CoV-2 main protease

“…To systematically identify potential resistance mutations, we performed deep mutational scanning experiments in the presence of nirmatrelvir and ensitrelvir. Because yeast possess numerous drug efflux pumps and are protected by a cell wall, they often require genetic engineering to make them accessible to drugs (Chinen et al 2016; Suzuki et al 2011; Rogers et al 2001). To increase the druggability of the yeast in our assays, we used a strain called Δ4 with four efflux pumps deleted (Δsnq2 Δpdr5 Δpdr1 Δyap1) and added a low concentration of SDS to increase permeability.…”

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

“…There are technical hurdles to overcome in using our yeast-based screens to investigate resistance because many small-molecules are ineffective due to poor permeability and/or export from yeast. We are assessing strategies to both increase the druggability of yeast and porting our assays to mammalian cells (Chinen, Hamada et al 2017). The results from our current work on M pro in yeast as well as previous studies using fitness landscapes to analyze drug resistance in other proteins (Deng, Huang et al 2012, Choi, Landrette et al 2014, Firnberg, Labonte et al 2014, Ma, Boucher et al 2017) indicates a strong potential of these approaches to improve our understanding and ability to combat resistance evolution.…”

Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease

“…To systematically identify potential resistance mutations, we performed deep mutational scanning experiments in the presence of nirmatrelvir and ensitrelvir. Because yeast possess numerous drug efflux pumps and are protected by a cell wall, they often require genetic engineering to make them accessible to drugs. − To increase the druggability of the yeast in our assays, we used a strain called Δ4 with four efflux pumps deleted (Δsnq2 Δpdr5 Δpdr1 Δyap1) and added a low concentration of SDS to increase permeability. Under these conditions, we found that the addition of either nirmatrelvir or ensitrelvir restored the yeast growth rate that was retarded due to M pro expression (Figure S1).…”