A Functional Variomics Tool for Discovering Drug-Resistance Genes and Drug Targets

Abstract

Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible novel target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems such as human cell lines will also be useful.

The Role of Signaling via Aqueous Pore Formation in Resistance Responses to Amphotericin B

“…For both Hsp90 and Hsp70, there is evidence of the ability to interact preferentially with lipid raft domains (69,70). It has also been reported that the sphingolipid-binding protein Pmp3, which is highly conserved in fungi, is a potent AmB resistance factor (71,72). Such resistance can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate (73).…”

The yeast Pmp3p has a significant role in plasma membrane organization

“…The deletion of PMP3 induces sensitivity to NaCl, hygromycin B, tetramethylammonium (Navarre and Goffeau, 2000) and amphotericin B (Huang et al, 2013). The NaCl phenotype cannot be explained through an effect on the two major Na + expelling pumps, because pmr2Δ nha1Δ Na + sensitivity is exacerbated when PMP3 is deleted (Navarre and Goffeau, 2000).…”

“…Amphotericin B is a polyene that binds to ergosterol and forms transmembrane pores on the cell membrane, leading to leakage of the cellular contents and cell death (Dennis et al, 1970;Bolard, 1986). Huang et al suggest that Pmp3p antagonizes the ergosterol-binding effect of amphotericin B, probably by preventing the insertion of amphotericin B into the plasma membrane (Huang et al, 2013). A recent study has shown that the amphotericin-B-treated phenotype of pmp3Δ can be modulated through the sphingolipid pathway (Bari et al, 2015).…”

Alzheimer’s Disease: Molecular Hallmarks and Yeast Models

“…Despite nearly a billion years of evolutionary divergence, recent estimates showed that a fifth of yeast genes have human disease orthologs lending support to functional discovery investigations using this model [92]. Moreover, thanks to amenability of S. cerevisiae to both classical and advanced molecular genetic techniques, to relatively simple, cheap and quick genetic and environmental manipulations, to the large knowledge base and data collections, high-throughput screening technologies and functional genomics that are not possible in humans [93][94][95], this organism has become a valuable and prevalent eukaryotic model organism to unravel complex and fundamental intracellular mechanisms underlying neurodegeneration [96][97][98][99][100][101][102][103][104].…”