Confirmation Of The Cellular Targets Of Benomyl And Rapamycin And Studies Into The Mode Of Action Of Cationic Amphiphilic Drugs (CADs)

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 pdr1 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. Cationic amphiphilic drugs (CADs) are a broad class of chemicals that can cause adverse effects in humans, and particularly liver toxicity; the origin of this toxicity is not well understood. The CAD haloperidol, for example, is known to cause drug induced liver cholestasis and phospholipidosis (DIPL), severe side effects similar to those seen in familial intrahepatic cholestasis (FIC). In a high throughput screen in yeast, we discovered that several known CADs, as well as a number of novel related compounds, exhibit synthetic lethality with RCY1, a gene encoding an F-box protein involved in endocytic recycling of membrane proteins. To investigate the origin of this synthetic lethality, we isolated S. cerevisiae mutants resistant to the CADs haloperidol and a novel CAD identified in this study, 3346-2086. Whole genome sequencing revealed that resistance is often associated with mutations in the yeast phospholipid transporters, DRS2, DNF1 and DNF2. Further investigations showed that in the absence of these mutations, Rcy1- cells treated with 3346-2086 rapidly lose the ability to transport fluorescent analogues of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), an effect that is largely blocked in the mutants. Our findings provide insight into the functional relationship between Rcy1p and phospholipid transporters and the conditions under which administration of CADs can give rise to drug-induced liver cholestasis and phospholipidosis