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.a

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

“…However, its use has mostly relied on the availability of rapid methods for mapping the resistance locus to a given chromosomal position. The advent of whole-genome tiling arrays and whole-genome sequencing has made the method more practical in other single-celled organisms such as Mycobacterium tuberculosis bacilli 35 , malaria parasites 36 , trypanosomes 37 , and yeast 38 39 40 . Unlike other essential gene products that are often described as possible drug targets, drug targets discovered through directed evolution are known from the outset to be druggable and are considered “chemically validated.” The method has been successfully used to identify the targets of several antimalarial compounds initially found through phenotypic screens, as well as to identify genes contributing to the resistome 2 41 42 .…”

Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe

“…Here, we describe how coupling of chemical mutagenesis, phenotypic selection, suppression analysis, and genomic sequencing-based mutational mapping, can be applied to rapidly derive strong phenotype-genotype correlations in a microbe with no preestablished molecular genetic tools, leading to a functional annotation of previously uncharacterized genes. A similar conceptual framework has been proposed to explore gene function in the obligate intracellular pathogen Chlamydia trachomatis (38), for conservation in protein function in bacteriophages (39), and to identify genes required for magnetosome formation (40) and microbial drug resistance (41,42). By extending the application of genomic sequencing to the largescale analysis of suppressor mutations, we genetically confirmed the contribution of specific genes to a complex trait and further revealed functions for new proteins whose activity could not be inferred solely from sequence homologies.…”

Reverse Chemical Genetics: Comprehensive Fitness Profiling Reveals the Spectrum of Drug Target Interactions

“…In previous work, yeast has been employed to study genome-wide gene-drug interactions [19–23], and is a well-established model for anticancer drug research [6,17,24,25]. Methotrexate acts as an antimetabolite that targets the enzyme dihydrofolate reductase, which functions to maintain folate homeostasis in nucleus and mitochondria by reducing dihydrofolate into tetrahydrofolate as a key element of thymidylate and protein synthesis [15].…”

“…Yeast dihydrofolate reductase ( DFR1 ) is a validated functional orthologue of human dihydrofolate reductase (hDHFR), which is commonly used to study the MTX mechanism of action and enzymology [ 15 , 17 , 18 ]. In previous work, yeast has been employed to study genome-wide gene-drug interactions [ 19 – 23 ], and is a well-established model for anticancer drug research [ 6 , 17 , 24 , 25 ]. Methotrexate acts as an antimetabolite that targets the enzyme dihydrofolate reductase, which functions to maintain folate homeostasis in nucleus and mitochondria by reducing dihydrofolate into tetrahydrofolate as a key element of thymidylate and protein synthesis [ 15 ].…”