title: Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae.
abstract: Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, USA. slokey@ucsc.edu.
authors.forename: Dustin A
authors.initials: DA
authors.lastname: Wride
authors.forename: Nader
authors.initials: N
authors.lastname: Pourmand
authors.forename: Walter M
authors.initials: WM
authors.lastname: Bray
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Kosarchuk
authors.forename: Sean C
authors.initials: SC
authors.lastname: Nisam
authors.forename: Tiffani K
authors.initials: TK
authors.lastname: Quan
authors.forename: Ray F
authors.initials: RF
authors.lastname: Berkeley
authors.forename: Sol
authors.initials: S
authors.lastname: Katzman
authors.forename: Grant A
authors.initials: GA
authors.lastname: Hartzog
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Dobkin
authors.forename: R
authors.initials: R
authors.lastname: Scott Lokey
mesh_terms: D001542:Benomyl / Q000494:pharmacology; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D026141:Drug Resistance, Multiple, Fungal / Q000235:genetics*; D017353:Gene Deletion; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C085480:PDR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C568590:Tub2 protein, S cerevisiae; D001542:Benomyl; D020123:Sirolimus