Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms.

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA). However, the biological activities of many xenobiotics are masked by the pleiotropic drug resistance (PDR) network which effluxes xenobiotics that are PDR substrates. The PDR network in S. cerevisiae is almost entirely under the control of two functionally homologous transcription factors Pdr1p and Pdr3p. Herein we report the construction of a PDR-attenuated haploid non-essential DMA (PA-DMA), lacking PDR1 and PDR3, which permits the MoA elucidation of xenobiotics that are PDR substrates at low concentrations. The functionality of four key cellular processes commonly activated in response to xenobiotic stress: oxidative stress response, general stress response, unfolded stress response and calcium signalling pathways were assessed in the absence of PDR1 and PDR3 genes and were found to unaltered, therefore, these key chemogenomic signatures are not lost when using the PA-DMA. Efficacy of the PA-DMA was demonstrated using cycloheximide and latrunculin A at low nanomolar concentrations to attain chemical genetic profiles that were more specific to their known main mechanisms. We also found a two-fold increase in the number of compounds that are bioactive in the pdr1Δpdr3Δ compared to the wild type strain in screening the commercially available LOPAC(1280) library. The PA-DMA should be particularly applicable to mechanism determination of xenobiotics that have limited availability, such as natural products.

Chemical genetic and chemogenomic analysis in yeast.

Chemogenomics is the systematic genome-wide study of the cellular response to small molecule agents. Modern high-throughput genetic techniques allow massively parallel examination of the genetic effects of such biologically active small molecules (BASM). Here we present methodology for the identification and characterization of potentially bioactive compounds using the budding yeast Saccharomyces cerevisiae as a model organism. First, we present a method for screening libraries of compounds for growth inhibition in solid or liquid phase, followed by techniques for potency determination using a half-log dose response. Then the Deletion Mutant Array (DMA), a genome-wide library of single gene deletion strains, is used to probe the chemical genetic interactions of individual BASMs on genetic networks-a process that can be achieved with a solid phase pinning assay or a pooled liquid assay utilizing barcode microarray techniques. Finally, we offer some considerations for optimizing these protocols.

Laulimalide and peloruside A inhibit mitosis of Saccharomyces cerevisiae by preventing microtubule depolymerisation-dependent steps in chromosome separation and nuclear positioning.

The activity and mechanism of action of two microtubule-stabilising agents, laulimalide and peloruside A, were investigated in Saccharomyces cerevisiae. In contrast to paclitaxel, both compounds displayed growth inhibitory activity in yeast with wild type TUB2 and were susceptible to the yeast pleiotropic drug efflux pumps, as evidenced by the increased sensitivity of a pump transcription factor knockout strain, pdr1Δpdr3Δ. Laulimalide (IC50=3.7 µM) was 5-fold more potent than peloruside A (IC50=19 µM) in this knockout strain. Bud index assays and flow cytometry revealed a G2/M block as seen in mammalian cells subsequent to treatment with these compounds. Furthermore, peloruside A treatment caused an increase in the number of cells with polymerised spindle microtubules. These results indicate an anti-mitotic action of both compounds with tubulin the likely target. This conclusion was supported by laulimalide and peloruside chemogenomic profiling using a yeast deletion library in the pdr1Δpdr3Δ background. The chemogenomic profiles of these compounds indicate that, in contrast to microtubule destabilising agents like nocodazole and benomyl, laulimalide and peloruside A inhibit mitotic processes that are reliant on microtubule depolymerisation, consistent with their ability to stabilise microtubules. Gene deletion strains hypersensitive to laulimalide and peloruside A represent possible targets for drugs that can synergize with microtubule stabilising agent and be of potential use in combination therapy for the treatment of cancer or other diseases.