Network Analysis Reveals the Molecular Bases of Statin Pleiotropy That Vary with Genetic Background.

Many approved drugs are pleiotropic: for example, statins, whose main cholesterol-lowering activity is complemented by anticancer and prodiabetogenic mechanisms involving poorly characterized genetic interaction networks. We investigated these using the Saccharomyces cerevisiae genetic model, where most genetic interactions known are limited to the statin-sensitive S288C genetic background. We therefore broadened our approach by investigating gene interactions to include two statin-resistant genetic backgrounds: UWOPS87-2421 and Y55. Networks were functionally focused by selection of HMG1 and BTS1 mevalonate pathway genes for detection of genetic interactions. Networks, multilayered by genetic background, were analyzed for key genes using network centrality (degree, betweenness, and closeness), pathway enrichment, functional community modules, and Gene Ontology. Specifically, we found modification genes related to dysregulated endocytosis and autophagic cell death. To translate results to human cells, human orthologues were searched for other drug targets, thus identifying candidates for synergistic anticancer bioactivity. IMPORTANCE Atorvastatin is a highly successful drug prescribed to lower cholesterol and prevent cardiovascular disease in millions of people. Though much of its effect comes from inhibiting a key enzyme in the cholesterol biosynthetic pathway, genes in this pathway interact with genes in other pathways, resulting in 15% of patients suffering painful muscular side effects and 50% having inadequate responses. Such multigenic complexity may be unraveled using gene networks assembled from overlapping pairs of genes that complement each other. We used the unique power of yeast genetics to construct genome-wide networks specific to atorvastatin bioactivity in three genetic backgrounds to represent the genetic variation and varying response to atorvastatin in human individuals. We then used algorithms to identify key genes and their associated FDA-approved drugs in the networks, which resulted in the distinction of drugs that may synergistically enhance the known anticancer activity of atorvastatin.

Functional genomics and metabolomics advance the ethnobotany of the Samoan traditional medicine "matalafi".

The leaf homogenate of Psychotria insularum is widely used in Samoan traditional medicine to treat inflammation associated with fever, body aches, swellings, wounds, elephantiasis, incontinence, skin infections, vomiting, respiratory infections, and abdominal distress. However, the bioactive components and underlying mechanisms of action are unknown. We used chemical genomic analyses in the model organism Saccharomyces cerevisiae (baker's yeast) to identify and characterize an iron homeostasis mechanism of action in the traditional medicine as an unfractionated entity to emulate its traditional use. Bioactivity-guided fractionation of the homogenate identified two flavonol glycosides, rutin and nicotiflorin, each binding iron in an ion-dependent molecular networking metabolomics analysis. Translating results to mammalian immune cells and traditional application, the iron chelator activity of the P. insularum homogenate or rutin decreased proinflammatory and enhanced anti-inflammatory cytokine responses in immune cells. Together, the synergistic power of combining traditional knowledge with chemical genomics, metabolomics, and bioassay-guided fractionation provided molecular insight into a relatively understudied Samoan traditional medicine and developed methodology to advance ethnobotany.

Erratum: Publisher Correction: Genetic interaction networks mediate individual statin drug response in Saccharomyces cerevisiae.

[This corrects the article DOI: 10.1038/s41540-019-0112-5.].

Genetic interaction networks mediate individual statin drug response in Saccharomyces cerevisiae.

Eukaryotic genetic interaction networks (GINs) are extensively described in the Saccharomyces cerevisiae S288C model using deletion libraries, yet being limited to this one genetic background, not informative to individual drug response. Here we created deletion libraries in three additional genetic backgrounds. Statin response was probed with five queries against four genetic backgrounds. The 20 resultant GINs representing drug-gene and gene-gene interactions were not conserved by functional enrichment, hierarchical clustering, and topology-based community partitioning. An unfolded protein response (UPR) community exhibited genetic background variation including different betweenness genes that were network bottlenecks, and we experimentally validated this UPR community via measurements of the UPR that were differentially activated and regulated in statin-resistant strains relative to the statin-sensitive S288C background. These network analyses by topology and function provide insight into the complexity of drug response influenced by genetic background.

Characterisation of the biological response of Saccharomyces cerevisiae to the loss of an allele of the eukaryotic initiation factor 4A.

The translation initiation machinery is emerging as an important target for therapeutic intervention, with potential in the treatment of cancer, viral infections, and muscle wasting. Amongst the targets for pharmacological control of translation initiation is the eukaryotic initiation factor 4A (eIF4A), an RNA helicase that is essential for cap-dependent translation initiation. We set out to explore the system-wide impact of a reduction of functional eIF4A. To this end, we investigated the effect of deletion of TIF1, one of the duplicate genes that produce eIF4A in yeast, through synthetic genetic array interactions and system-wide changes in GFP-tagged protein abundances. We show that there is a biological response to deletion of the TIF1 gene that extends through the proteostasis network. Effects of the deletion are apparent in processes as distributed as chromatin remodelling, ribosome biogenesis, amino acid metabolism, and protein trafficking. The results from this study identify protein complexes and pathways that will make ideal targets for combination therapies with eIF4A inhibitors.

The genetic architecture in Saccharomyces cerevisiae that contributes to variation in drug response to the antifungals benomyl and ketoconazole.

The genetic basis of variation in drug response was investigated in individual Saccharomyces cerevisiae strains that exhibited different susceptibility to two antifungal agents: benomyl and ketoconazole. Following dose-response screening of 25 strains, 4 were selected on the basis of resistance or sensitivity relative to the standard laboratory strain BY. UWOPS87-2421 and L-1374 were respectively resistant and sensitive to benomyl; DBVPG6044 and Y12 were respectively resistant and sensitive to ketoconazole. We used advanced intercross lines and next generation sequencing-bulk segregant analysis to characterise the quantitative trait loci (QTL) underpinning drug responses after drug selection. Drug response was controlled by multiple QTL, ranging from a minimum of 5 to a maximum of 60 loci, almost all of which were not the primary drug target. For each drug, the resistant and the sensitive strain exhibited a number of shared loci, but also had strain-specific QTL. In our analysis, it was possible to estimate genetic effect of QTL, and a number of those shared between resistant and sensitive strains exhibited variable effect on the response phenotype. Thus, drug responses arise as a result of different genetic architectures, depending on the genetic background of the individual strain in question.

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.

Kinetochore genes are required to fully activate secretory pathway expansion in S. cerevisiae under induced ER stress.

Basal ER stress occurs when proteins misfold in normal physiological conditions and are corrected by the unfolded protein response (UPR). Elevated ER stress occurs when misfolding is refractory as found in numerous diseases such as atherosclerosis, Type II diabetes and some cancers. In elevated ER stress it is unclear whether cells utilise the same or different networks of genes as in basal levels of ER stress. To probe this question, we used secretory pathway reporters Yip3p-GFP, Erv29p-GFP, Orm2p-GFP and UPREpr-GFP placed on the yeast deletion mutant array (DMA) genetic background. The reporter's expression levels, measured by automated microscopy, at basal versus elevated ER stress induced by the over-expression of CPY* were compared. A novel group of kinetochore genes (CTF19 complex) were found to be uniquely required for full induction of all four ER stress reporters in elevated stress. A follow-up reporter screen was developed by mating the ctf19Δ kinetochore gene deletion strain into the genome-wide XXXp-GFP tagged library then testing with over-expressed CPY*. This screen identified Bcy1p and Bfr1p as possible signalling points that down-regulate the UPR and secretory pathway when kinetochore proteins are absent under elevated stress conditions. Bfr1p appears to be a checkpoint that monitors the integrity of kinetochores at increased levels of ER stress. This study concludes that functional kinetochores are required for full activation of the secretory pathway in elevated ER stress and that the responses to basal and elevated levels of ER stress require different networks of genes.

Networks of genes modulating the pleiotropic drug response in Saccharomyces cerevisiae.

The pleiotropic drug response (PDR) or multidrug resistance (MDR) are cellular defence mechanisms present in all species to deal with potential toxicity from environmental small molecule toxins or bioactives. The rapid induction of MDR by xenobiotics in mammalian cells and PDR in budding yeast (S. cerevisiae) has been well studied but how pathway specificity is achieved across different structural classes of xenobiotics is not well understood. As a novel approach to this problem we investigated the genome-wide network of genes modulating the yeast PDR. Fluorescently-tagged ABC pumps Pdr5p-GFP and Yor1p-GFP were used as real-time reporters for the Pdr1p/Pdr3p controlled response. Using the yeast non-essential gene deletion set fifty-four gene deletions that suppressed up-regulation of reporter fluorescence to the cell surface in the presence of atorvastatin were identified by high content confocal automated microscopy. Secondary validation using spot dilution assays to known PDR substrates and Western blot assays of Pdr5p expression confirmed 26 genes able to modulate the PDR phenotype. By analysis of network connectivity, an additional 10 genes that fell below the primary screen cut-off were predicted to be involved in PDR and confirmed as above. The PDR modulating genes taken together were enriched in signalling (Rho-GTPase, MAPK), Mediator complexes, and chromatin modification (subunits of ADA and SAGA complexes). Many of the gene deletions cause extra sensitivity in Δpdr1Δpdr3 strains strongly suggesting that there are alternative pathways to upregulate PDR, independently of Pdr1p/Pdr3p. We present here the first high-content microscopy screening for PDR modulators, and identify genes that are previously unsuspected regulators of PDR apparently contributing via network interactions.

Characterizing the laulimalide-peloruside binding site using site-directed mutagenesis of TUB2 in S. cerevisiae.

Baker's yeast, Saccharomyces cerevisiae, has significant sequence conservation with a core subset of mammalian proteins and can serve as a model for disease processes. The aim of this study was to determine whether yeast could be used as a model system to identify new agents that interact with the laulimalide-peloruside binding site on ß-tubulin. Agents that bind to this site cause stabilization of microtubules and interfere with cell division. Based on the location of the proposed laulimalide-peloruside binding site and of previously identified mutations shown to cause resistance in mammalian cells, we made the corresponding mutations in yeast and tested whether they conferred resistance to laulimalide and peloruside. Mutations A296T and R306H, which cause 6-fold and 40-fold increased resistance in human 1A9 ovarian carcinoma cells, respectively, also led to resistance in yeast to these compounds. Similarly, other mutations led to resistance or, in one case, increased sensitivity. Thus, we conclude that yeast is an appropriate model to screen for small molecule drugs that may be efficacious in cancer therapy in humans through the newly characterised laulimalide-peloruside binding site.

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.

The novel equisetin-like compound, TA-289, causes aberrant mitochondrial morphology which is independent of the production of reactive oxygen species in Saccharomyces cerevisiae.

Tetramic acids constitute a large class of natural products isolated from a variety of different fungal and bacterial species. While the presence of the distinctive 2,4-pyrrolidinedione ring system defines this class of compounds, these compounds are widely diverse both structurally and in the biological activities that they display. Equisetin-like compounds are tetramic acids that have been shown to be growth inhibitory towards bacteria, fungi, yeasts and mammalian cell lines; however, the mechanisms inhibiting prokaryotic and eukaryotic cell growth have not been fully explained. Here we report the isolation and biological characterisation of a novel equisetin-like tetramic acid named tetramic acid-289 (TA-289) produced by a Fusarium sp. fungus. This compound displayed pH- and carbon source-dependent cytotoxic effects in Saccharomyces cerevisiae and caused an irreversible cell cycle block via a microtubule independent mechanism. To fully elucidate a mechanism, we used an unbiased approach employing chemogenomic profiling of the yeast deletion library and demonstrated that TA-289 hypersensitive deletion strains are also sensitive to oxidants, respiratory inhibitors and have abnormal mitochondrial morphology. In support of the hypothesis that TA-289 perturbs mitochondrial function, we demonstrated the ability of this compound to generate reactive oxygen species only during fermentative growth, an effect reliant on an intact electron transport chain. In addition, mitochondrial morphological defects were detected upon exposure to TA-289 independent of the increase in oxidative stress. The generation of reactive oxygen species was not the sole cause of cell death by TA-289, as only partial amelioration of cell death was achieved by the deletion of genes encoding components of the electron transport chain, despite these deletions causing attenuation of the magnitude of oxidative stress. We propose that TA-289 induces cell death via the direct inhibition of a mitochondrially localised target or targets, and that the mitochondrial morphology defect and ROS production observed in this study is a direct consequence of the induction of cell death. This study highlights the complex interplay between mitochondrial function, cell death and the generation of reactive oxygen species when elucidating the mode-of-action of compounds that cause oxidative stress and cell death, and further deepens the mystery surrounding the molecular basis of the activity of equisetin-like compounds.

The cellular target specificity of pateamine A.

The natural product pateamine A (pateamine) from the sponge Mycale hentscheli is active against a wide range of dividing cells and has been shown to inhibit the functions of the eukaryotic initiation factor 4A (eIF4A). We have identified that pateamine is additionally able to modulate the formation of actin filaments and microtubules in vitro but at higher concentrations than required for inhibition of eIF4A. Cell cycle analysis confirmed that actin and tubulin are not major mediators of the cellular activity of pateamine. The range of targets identified demonstrates the value of multiple approaches to determining the mode of action of biologically active compounds.

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae.

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35µM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19µM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.

Molecular basis for fungicidal action of neothyonidioside, a triterpene glycoside from the sea cucumber, Australostichopus mollis.

Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.

Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis.

We developed a procedure for automated confocal microscopy to image the effect of the non-essential yeast gene deletion set on the localisation of the plasma membrane GFP-labelled protein Mrh1p-GFP. To achieve this it was necessary to devise an expression system expressing Redstar2 RFP-fluorescence specifically in the nucleus, mCherry RFP at a lower intensity in the cytoplasm and Mrh1p-GFP in the plasma membrane. This fluorescence labelling scheme utilising specifically designed image analysis scripts allowed automated segmentation of the cells into sub-regions comprising nuclei, cytoplasm and cell-surface. From this high-throughput high content screening approach we were able to determine that gene deletions including emc1Δ, emc2Δ, emc3Δ, emc4Δ, emc5Δ and emc6Δ, caused intracellular mislocalisation at the ER of a plasma membrane protein Mrh1p-GFP. CPY processing patterns were unaffected in these mutants and collectively our data suggest a transport role for the EMC genes within the early secretory pathway. HAC1 is central to the unfolded protein response (UPR) and in its absence, i.e. the absence of UPR, emc1Δ-, emc3Δ-, emc4Δ-, emc5Δ-hac1Δ double mutants were specifically hypersensitive to ER-stress (tunicamycin) lending credence to the usefulness of the high content microscope screening for discovery of functional effects of single mutants.