Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 FEMS Yeast Research, 18, 2018, foy020 doi: 10.1093/femsyr/foy020 Advance Access Publication Date: 4 March 2018 Minireview MINIREVIEW 1 1 1 1 Andreas Zimmermann , Sebastian Hofer , Tobias Pendl , Katharina Kainz , 1,2,∗ 1,∗ Frank Madeo and Didac Carmona-Gutierrez 1 2 Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, 8010, Austria and BioTechMed Graz, Graz, 8010, Austria Corresponding authors: Frank Madeo, Institute of Molecular Biosciences, Humboldtstrasse 50/EG, 8010, Graz. Tel.: +433163808878; E-mail: firstname.lastname@example.org; Didac Carmona-Gutierrez, Institute of Molecular Biosciences, Humboldtstrasse 50/EG, 8010, Graz. Tel.: +433163801510; E-mail: email@example.com One sentence summary: The availability of various genetic mutant libraries combined with a high degree of mechanistic conservation relevant to aging make yeast a powerful screening platform for the identification and mechanistic study of novel anti-aging compounds. Editor: Sergio Giannattasio ABSTRACT In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae,has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification. Keywords: pharmacological screen; anti-aging; age-related disease; yeast; chemogenomics; drug discovery INTRODUCTION Abbreviations: Aging describes the multifaceted decline of cellular and organ- RLS: Replicative lifespan ismal function over time, and represents a major risk factor for CLS: Chronological lifespan FCG: Forward chemical genetics the susceptibility to diseases. Indeed, there is a profound over- lap between cellular pathways that influence aging and those RCG: Reverse chemical genetics ROS: Reactive oxygen species linked to cancer, neurodegeneration and cardiovascular disor- ders as well as metabolic syndrome (de Cabo et al. 2014). Hence, HTS: High-throughput screen HIP: Haploinsufficiency profiling recent efforts have aimed at the identification of molecules that decelerate the aging process per se and thus may act as a pre- HOP: Homozygous profiling MSP: Multicopy suppression profiling ventive measure that collectively ameliorates age-related dis- eases. Vice versa, therapeutics for such diseases may also prolong RMS: Random mutation suppression overall healthspan or lifespan. In the search for anti-aging in- CR: Caloric restriction terventions, biological screening platforms are important tools for drug discovery. Cell-based assays are of particular relevance Received: 5 December 2017; Accepted: 27 February 2018 FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the orig- inal work is properly cited. 1 Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 2 FEMS Yeast Research, 2018, Vol. 18, No. 6 as they have several advantages compared to in vitro screens. Upon nutrient scarcity (e.g. in nutrient-limited batch cultures) First, they enable a drug to be studied in the cellular context, yeast enter a stationary phase and stop dividing. Depending on including the ability to pass cellular membranes and to with- the culture conditions and strain, yeast lifespan in this post- stand immediate export by multidrug transporters. Second, drug mitotic phase ranges from a few days up to several weeks. This candidates that exhibit cytotoxicity by off-target effects, or a dif- comparatively short lifespan is particularly convenient in large- ferent response of the desired target in vivo upon ligand binding, scale aging studies, which in other typically used aging model can easily be ruled out by monitoring cell viability. Third, cellu- organisms can take between 20 days (nematodes; Riddle et al. lar platforms are highly compatible with unbiased phenotypical 1997), over ∼3 months (fruit flies; Linford et al. 2013)and up to screens, as the desired phenotype (e.g. improved survival dur- ∼3 years (mice; Flurkey, Currer and Harrison 2007). ing aging) can be selected for, irrespective of the molecular tar- The two stages of yeast aging, mitotic and post-mitotic ag- get. The requirements for such screening platforms include a ing, can be monitored using two different models, replicative high degree of conservation, an economic workflow, and simple lifespan (RLS), and chronological lifespan (CLS), respectively. RLS and reliable readouts. One of the organisms that meets these represents the number of cell divisions a mother cell can un- needs is the budding yeast (Saccharomyces cerevisiae), a unicellu- dergo before death and is a model for proliferating cells (e.g. larfungusthatiswidelyusedasamodelfor human agingand undifferentiated stem cells) (Sinclair 2013). As budded daugh- age-related diseases. Its unparalleled genetic tractability com- ter cells have to be separated from the mother cells, RLS meth- bined with the availability of whole genome homozygous and ods are usually time-consuming and difficult to adapt to high- heterozygous gene deletion collections as well as overexpres- throughput approaches. sion libraries, make S. cerevisiae a versatile toolbox for chemoge- CLS models aging in post-mitotic/differentiated cells and is nomic screens. Due to its short generation time (∼90 min) and assessed by monitoring cell survival in stationary batch cul- modest culturing requirements, yeast can also be grown rapidly tures after the diauxic shift. CLS is compatible with several in high-throughput experimental setups. high-throughput techniques such as flow cytometry (Carmona- In this minireview, we summarize strategies for yeast-based Gutierrez et al. 2010), outgrowth (Murakami et al. 2008) or spot- drug discovery and give examples for successfully employed ting assays (Teng and Hardwick 2013). It should be noted, pharmacological screens in yeast with a focus on aging and age- though, that the batch culture aspect makes this model sensi- related diseases. tive to growth behavior and the accumulation of degradation products (especially acetic acid) in the medium, which might confound the interpretation of aging phenotypes. Thus, growth rates and media pH should always be controlled, at least when YEAST AS A MODEL FOR HUMAN AGING validating results from high-throughput experiments. Recently, When selecting an experimental model for the identification of continuous flow cultures such as retentostat cultures (Boender anti-aging compounds, a high degree of evolutionary conserva- et al. 2011), where the cellular biomass is retained in the growth tion is crucial. In yeast, many pathways that are relevant for chamber by filtration and cells stop dividing, have gained at- aging and disease in humans are well conserved, including nu- tention as CLS models since they combine post-mitotic aging trient signaling, cell cycle regulation, DNA repair mechanisms, with constant nutrient supply, thus eliminating the disadvan- mitochondrial homeostasis, lipostasis, protein folding and se- tages of batch cultures. Although these culturing methods must cretion, proteostasis, stress response, and regulated cell death still be trimmed to fit high-throughput demands, they are attrac- tive tools in the validation chain of batch culture-based screens. (Longo and Fabrizio 2002; Tenreiro and Outeiro 2010; Eisenberg and Buttner ¨ 2014; Lasserre et al. 2015; Janssens and Veenhoff During yeast aging, typical age-associated phenotypical 2016; Knorre et al. 2016; Bilinski, Bylak and Zadrag-Tecza 2017; markers emerge, such as the accumulation of reactive oxygen Postnikoff, Johnson and Tyler 2017; Carmona-Gutierrez et al. species (ROS), the buildup of damaged organelles and proteins, 2018). About 90% of the ∼6000 yeast genes have already been DNA fragmentation, loss of membrane integrity and the increase characterized and approximately 30% of the yeast genome is of apoptotic/necrotic cell populations (Carmona-Gutierrez et al. conserved to humans, based on sequence similarity (Stefanini, 2010; Janssens and Veenhoff 2016; Carmona-Gutierrez et al. 2018) De Filippo and Cavalieri 2013). Transgenetic studies have re- which bears many similarities to hallmarks of human aging vealed, however, that sequence similarity is a poor predictor (Lopez-Ot ´ ´ın et al. 2013). To some extent, yeast might even be of orthology (i.e. functional homology). In fact, about half (in better suited to monitor aging on a cellular level other than some pathways over 90%) of the essential yeast proteins can human cell cultures because (i) due to the smaller size and growth in suspension of yeast cells, a larger number of cells be replaced with their human orthologs, even though the re- spective sequence similarity ranges from over 90% to as little can be monitored in a given culturing platform, especially com- pared to adherent human cell lines, which usually grow in as 9% (Kachroo et al. 2015). The growing awareness that path- ways related to human senescence display a high degree of con- monolayers (Montague et al. 2014); (ii) in contrast to mam- servation in S. cerevisiae and other unicellular fungi has increas- malian cell culture, where cells are extracted from their phys- ingly promoted the use of yeast to understand signaling path- iological environment within a heterogeneous tissue and thus ways and identify molecular players involved in aging, as well only represent an isolated entity from a multicellular organism, as to unveil and/or test potential anti-aging interventions. In- yeast cell cultures represent an in vivo situation. There is even deed, yeast-aging phenotypes are surprisingly similar to human evidence for differentiation-like behavior of yeast cells when post-mitotic cellular aging (Longo and Fabrizio 2012). In the pres- forming colonies on solid media (Cap ´ et al. 2012), and some ence of sufficient nutrients, yeast cultures grow exponentially studies suggest a degree of crosstalk between individual cells ´ ´ ´ by asymmetric budding of daughter cells from mother cells. (Herker et al. 2004;Palkova, Wilkinson and Vachova 2014). In During cell division, mother cells retain damaged cellular ma- terms of pharmacological screens, this may offer the possibil- ity of using yeast as a rudimentary archetype for tissue mi- terial that accumulates over time, ensuring maximal health of their offspring. Eventually (after 20–25 cell divisions), the mother croenvironments, although studying tissue-specific responses to drugs, including drug metabolization or prodrug activation, cells die and release their cellular material into the environment (Steinkraus, Kaeberlein and Kennedy 2008;Longo et al. 2012). might exceed the power of the model (Resnick and Cox 2000). Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 Zimmermann et al. 3 survival during aging may yield bioactive compounds that act on different targets but have the same phenotypical outcome. FCG screens allow assessment of all substances that might al- ter a desired phenotype, while potentially effective candidates with poor pharmacokinetic properties or side-effects masking the desired effect are sieved out automatically. Nevertheless, narrowing down a specific target of a bioactive drug remains challenging (Schenone et al. 2013). RCG screens, on the other hand, circumvent this problem as the drug target is defined a priori, and chemical ligands are selected in vitro before testing their activity in vivo. Although there is still no comprehensive molecular characterization of the aging process, numerous po- tential targets for anti-aging drugs have been identified in ge- nomic screens. For example, a large-scale screen with a collec- tion of yeast deletion strains has indicated a lifespan-shortening role of purine biosynthesis (Matecic et al. 2010). Given a feasi- Figure 1. Advantages and limitations of yeast for anti-aging drug discovery. ble readout (e.g. activity measurements of the enzymes involved The budding yeast, S. cerevisiae, offers a genetically well-defined cellular envi- in the pathway), an RCG screen could help to identify purine ronment paired with a fast and economic experimental workflow, including a biosynthesis modulators that, in turn, affect lifespan. However, variety of mutant libraries for drug target identification. Due to its unicellular a common disadvantage of RCG screens is that results from in nature, studying tissue-specific responses might be limited. Other constraints vitro experiments, which usually use highly purified targets of are limited drug metabolization and suboptimal permeability of the fungal cell interest isolated from their physiological environment, are of- wall to xenobiotics. See the main text for further details. ten not transferable to the situation in a cellular context. More- over, substances that act highly specifically in vitro might still Consequently, while many intracellular mechanisms of aging exert significant side-effects in vivo that interfere with the de- regulation are well-conserved in yeast, extrinsic, intercellular sired phenotypical outcome. Therefore, hybrid setups, or in situ factors, such as insulin-like growth factor (IGF-1) levels or in- RCG screens, where the activity or downstream effects (e.g. in flammation, which influence aging in multicellular organisms the case of a toxic transgene) of the target of interest is tested (Franceschi et al. 2007; Fontana, Vinciguerra and Longo 2012; in genetically engineered yeast strains rather than in vitro, allow Bartke 2017), cannot be investigated adequately in yeast. In ad- the advantages of combining both approaches. dition, the fungal cell wall represents a barrier to some com- pounds, which, however, can be weakened genetically (see the chapter on ‘Pharmacokinetics and off-target effects in yeast’). YEAST AS A CHEMOGENOMIC SCREENING Nevertheless, the easy handling and well-defined, highly con- PLATFORM served cellular environment have predestined yeast as a tool for drug discovery (Figure 1). Limiting factors in the direct applicability of RCG and FCG Besides S. cerevisae, other yeast species are used as aging screens in animals and human cell culture are the correspond- models as well, in particular the fission yeast Schizosaccharomyces ing time, resource and experimental constrictions. In addition, pombe. In fact, some cellular components that are poorly con- animal models are less suited for FCG screens, because the com- served between S. cerevisiae and mammals, such as the mRNA plexity of genetic engineering impedes high-throughput testing splicing machinery and nuclear structural proteins, are well- of genetic mutants once a lead substance has been identified. conserved in S. pombe (Roux et al. 2010). Fission yeast is also Yeast, as an established model for a plethora of physiological preferably used to study mitochondrial inheritance, since this and disease-relevant settings, offers a technical addendum to process is similarly regulated in human cells (Lin and Austri- extensively exploit the quantitative and qualitative potential of aco 2014). In addition, S. pombe has been used in pharmacolog- such screens. One of the main advantages of yeast is the avail- ical screens for anti-cancer agents (Satoh et al. 2017)aswellas ability of a variety of strain libraries with loss-of-function or genome wide screens to unravel the genetic network of drug- gain-of-function mutations of almost all genomic open reading induced ROS accumulation (Hagihara et al. 2017). frames (ORFs). Upon identification of an anti-aging compound candidate, such genetic libraries can help to identify drug targets without having to fundamentally change experimental setups. PHARMACOLOGICAL SCREENING STRATEGIES The yeast knock-out collection (available via the Euro- In the search for novel bioactive compounds, there are two ex- pean Saccharomyces cerevisiae Archive for Functional Anal- perimental approaches: (i) forward chemical genetics (FCG) or ysis, www.euroscarf.de; or GE Dharmacon, dharmacon. phenotypic screens, and (ii) reverse chemical genetics (RCG) or gelifesciences.com) comprises ∼5100 strains in haploid genome target-based screens (Figure 2). In FCG screens, cells are treated background with deletions of non-essential genes (∼80% of all with substance libraries and hits are selected based on a pheno- yeast genes). Essential genes can be studied in heterozygous type (e.g. cell survival). When combined with libraries of differ- diploid deletion strain libraries, or temperature-sensitive condi- ent yeast mutants (e.g. gene deletion or overexpression strains), tional mutants. The deletion strains are barcoded (unique DNA this strategy facilitates rapid identification of potential targets of identifier for each strain), which enables enrichment analyses a lead compound. Untargeted anti-aging screens typically follow from a pooled mixture of strains via DNA barcode microarrays or this paradigm since aging is generally regarded as a multifac- barcode sequencing. Under the premise that in such a mixture torial phenomenon which can be influenced by numerous (and of strains long-lived mutants will be enriched and short-lived often uncharacterized) cellular pathways in parallel (Lopez-Ot ´ ´ın mutants depleted over time, the knockout collection has suc- et al. 2013). Thus, an unbiased, multifactorial readout such as cessfully been employed in screens for genetic determinants Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 4 FEMS Yeast Research, 2018, Vol. 18, No. 6 Figure 2. Screening strategies for chemogenomic drug discovery. (A) Forward chemical genetics (FCG) start with a compound library, which is administered to wild type cells or engineered yeast cells expressing a protein of interest. Hits are selected based on a phenotypic readout, ending up with bioactive compounds. Targets can be identified by treating genetic libraries (loss-of-function or gain-of-function mutants) with the identified hits and reversal of the phenotype as a readout. (B) Reverse chemical genetics (RCG) start with the selection of a target of interest, which is treated with a compound library in vitro, using ligand binding or enzymatic activity as readouts. Wild type cells or genetic libraries are treated with the selected hits to identify a phenotype and/or target. of aging (Powers et al. 2006; Murakami et al. 2008; Matecic et al. this technique only works when the compound causes some 2010). In a chemical screening context, drug-resistant mutants degree of growth inhibition. Nevertheless, when working with (when the compound inhibits growth) are enriched, while compounds that prolong lifespan but do not inhibit growth, drug-sensitive strains show impaired growth compared to the modified applications of such arrays may still be useful (see untreated control and become underrepresented over time. This the chapter on ‘High-throughput methods to monitor yeast approach is even suitable for parallel identification of bioactive aging’). It should be noted that the yeast knockout collection compounds and their respective targets. It is also termed drug- does harbor a substantial amount of secondary mutations induced haploinsufficiency profiling (HIP) when working with which might interfere with the chosen readout (Teng et al. 2013). heterozygous diploid strains, or homozygous profiling (HOP) Thus, it is imperative to validate hits from such libraries by (i) when working with homozygous/haploid deletion strains. In complementation assays (e.g. by plasmid-based expression of contrast to HOP assays, where the potential target is deleted, HIP the deleted gene) and/or (ii) recapitulation of the phenotype is based on a direct interaction of a chemical with its target. Both in de novo created deletion strains, ideally in different strain strategies may reveal mechanistic insights into disease-relevant backgrounds. drugs (Sun and Zhou 2016); however, they are highly sensitive to The yeast ORF library (GE Dharmacon) offers over 4900 growth behavior per se and might lead to a high number of false strains with galactose-inducible overexpression of single yeast hits. Indeed, in a HOP study which aimed at characterizing the genes which can be used for multicopy suppression profiling influence of single amino acid substitutions in histones H3 and (MSP). The rationale of MSP is that a larger number of target H4 on replicative aging, only 36% of the mutants identified in proteins can increase the efficient dose of a drug that impairs the initial DNA barcode enrichment screen could be validated growth. Again, this assay relies on differences in growth, which in the subsequent low-throughput experiments (Sen et al. 2015). might be difficult to screen for in aging setups, especially when As an alternative to enrichment assays, the knockout collection a drug does not cause an apparent growth phenotype. Never- strains can also be tested independently in parallel using cell theless, such approaches confirmed a number of drug-target in- arrays and automated micorarraying devices. By pinning small teractions, including rapamycin-Tor1p, tunicamycin-Alg7p and amounts of liquid yeast cultures onto agar plates containing fluconazole-Erg11p (Giaever et al. 2004; Hoon et al. 2008). the TORC1 inhibitor rapamycin, and scanning for mutants Random mutation suppression (RMS) relies on spontaneous that were able to form colonies, this procedure has been used induced mutations of the genome, which suppress the effect to gain new insight into the rapamycin-responsive cellular of drug treatment. Random mutation libraries derived from network (Xie et al. 2005). A considerable restriction is that the transposon mutagenesis can easily be generated and used to substance of interest has to be introduced into the agar plates; identify loci linked to drug-resistance (Kumar 2016). Given the hence, the required amount of compound is several orders of decreasing costs for whole genome sequencing, screens for magnitude higher than for setups in liquid culture. Moreover, spontaneous resistance by single nucleotide polymorphisms Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 Zimmermann et al. 5 (SNPs) have become economically justifiable, simply by treating A typical (FCG) yeast compound screening procedure has a large number of wild type cells and selecting for clones that three phases: (i) an initial high-throughput compound screen are still able to form colonies in the presence of the compound. using wild type yeast or a strain, specifically designed for the de- The genome of the resistant clone(s) can be sequenced to un- sired phenotype, for example, activation of a pathway of interest ravel the mutated locus. In fact, this rationale was followed to (using reporter- or growth-based assays) or amelioration of the link a gain-of-function mutation in the peroxiredoxin Tsa1p to toxicity of a transgene; (ii) immediate validation in other exper- oxidative stress resistance after chemical mutagenesis (Timmer- imental (animal) models before proceeding with target identifi- mann et al. 2010), and has also been used to identify genetic loci cation. This ensures that compounds only active in yeast can be associated with drug treatment (Tardiff et al. 2013). filtered out as they are less relevant for applications in humans In order to investigate specific pathways for in situ RCG (although the mechanism of action might still shed light on con- screens, yeast can be genetically manipulated to facilitate high- served aging processes). When the initial screen was performed throughput readouts. For example, target of rapamycin (TOR)- in a yeast disease model, the validation should be carried out in pathway activity can be monitored in strains carrying TOR- a multicellular model for the respective pathology (if available); responsive promoters fused to fluorescent proteins and in and (iii) treatment of selected genetic mutant libraries (or any quantification of the promoter activities by flow cytometry. In- combination thereof) with the validated compound(s) using HIP, deed, this method was used to identify a novel TORC1 in- HOP, MSP or mutagenesis to identify genetic loci, which mod- hibitor among a library of ∼320 000 compounds (Chen et al. ify the effects of the drug (Figure 3). Tardiff et al. followed ex- 2012). Of note, even though several pathway activities correlate actly this strategy to identify chemical compounds which ame- with aging (e.g. mTOR signaling in neurons (Yang et al. 2012), liorate proteinopathies (Tardiff et al., 2012, 2013). Notably, the or the DNA damage response signaling routes p16INK4a/Rb readout for the third phase of these screens was acute growth (Ressler et al. 2006) and p19ARF/p53 (Krishnamurthy et al. 2004: impairment upon exposure to the drug, which is usually eas- 4)), there is no single signaling cascade that governs the ag- ier to assess than aging. Therefore, it is worth testing a range of ing process, or is specifically activated/deactivated during ag- concentrations of the candidate compound: if high substance ing. There are several transcriptional pathways that have been concentrations cause growth impairment then subsequent tar- linked with longevity, including several epigenetic modifications get identification can be performed under more stringent con- (Fahrenkrog 2015) such as silencing by the histone demethylase ditions, potentially yielding more unambiguous results. Rph1p (Schroeder, Raimundo and Shadel 2013), or the activation of stress-resistance transcription factors Msn2p/Msn4p (Fabrizio 2001) and Yap1p (Yiu et al. 2008). However, at present, it is diffi- HIGH-THROUGHPUT METHODS TO MONITOR cult to apply a reporter-based technique to unbiased screens for YEAST AGING anti-aging compounds, simply because a comprehensive, strin- gent transcriptional response to aging that could be harnessed One of the challenges when monitoring aging in any model or- to identify compounds, which alter the response globally, has ganism is the lack of dichotomous (e.g. positive or negative) yet to be unraveled. readouts. Most yeast-based screens rely on differences in growth As an alternative to reporter-based screens, simple genetic in liquid or solid media, which requires stringent experimental ‘tricks’ may be used to amplify the phenotype after a given setups such as a clear growth defect of the control condition. treatment, e.g. by linking the activity of a pathway to the ex- However, a compound with anti-aging properties does not nec- pression of an essential gene. The difference in growth upon essarily improve cell growth per se, but may only compensate pathway modulation can be harnessed to embed pharmacolog- the loss of regrowth ability during prolonged aging. There are ical screens in pathway-specific approaches. For example, his- (at least) three possible strategies to meet the requirements of tone deacetylase (HDAC) Sir2p-mediated silencing of an engi- anti-aging screens: (i) following population survival over time to neered URA3 gene (coding for a protein essential for growth in identify substances which decelerate loss of viability; (ii) linking uracil-free media) at a telomeric region (strongly deacetylated the effects of drug treatment to more unambiguous phenotypes by Sir2p) was used to identify novel Sir2p inhibitors by treat- (e.g. by linking pathway activities to essential genes); or (iii) in ing the cells with different compounds and to monitor growth the case of target identification procedures, increasing the con- in media lacking uracil (Bedalov et al. 2001). A similar strat- centration of the substance of interest until a more distinct phe- egy served to search for Sir2p inhibitors, but this time scor- notype can be observed. ing decreased growth in media containing 5-fluoroortic acid, Classic, low-throughput clonogenicity assays, where the abil- which is converted to toxic 5-fluorouracil (5-FOA) by the prod- ity to form cultures from single cells on solid media is mon- uct of the URA3 gene (Grozinger et al. 2001). In theory, varia- itored, can be performed in a high-throughput fashion by tions of this approach can be used to screen for substances assessing outgrowth or microcolony formation. The outgrowth that activate Sir2p, e.g. by altering the genomic locus of the capacity (i.e. the ability to divide in fresh liquid media) of an ag- URA3 insertion to a region which is silenced upon Sir2p acti- ing culture declines with age and can be monitored by measur- vation (Li et al. 2013) and monitoring growth in 5-FOA medium ing, over several hours, the optical density of a culture aliquot (thus, higher HDAC activity would reduce the levels of Ura3p that has been diluted in fresh media. There are commercially and allow growth). Since Sir2p and its human homolog SIRT1 are available growth curve measuring devices which combine pho- widely implicated in the regulation of aging (see below), such ap- tometric measurements with integrated shaking incubators, fa- proaches could yield attractive molecules for pharmacological cilitating automated readouts. This technique has been used to anti-aging interventions. Alternatively, URA3-expression could study the effects of media composition and osmolarity on yeast also be put under control of a transcription factor of interest aging (Murakami et al. 2008), and also in a large-scale screen to identify activators/inhibitors of specific transcriptional sig- for genes associated with longevity among the deletion mu- naling. However, this workflow is only compatible with semi- tants of the yeast knockout collection (Powers et al. 2006). The biased screens, as it limits the number of targets to the pathway setup can easily be adapted to compound screens by replacing of interest. different yeast strains with different treatments. Microcolony Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 6 FEMS Yeast Research, 2018, Vol. 18, No. 6 Figure 3. Yeast as a pharmacological screening tool in forward chemical genetics. Compound libraries can be tested in wild type yeast in an unbiased manner, or in semi-biased approaches in engineered/humanized yeast strains. To investigate specific pathways which do not result in an apparent phenotype, y east can be manipulated to translate pathway activities into growth behavior. For instance, a promoter that is regulated by the pathway of interest can be linked to an essential gene (e.g. URA3). The growth on uracil-free or 5-fluoroorotic acid (5-FOA) containing media is an indirect measure for increased or decreased pathway activity, resp ectively. Humanized yeast, expressing human disease-relevant genes can be used to find compounds that specifically interfere with the function of the transgene . Compounds derived from unbiased screens should be validated in multicellular models before screening for potential targets in different yeast mutant libraries. Among others these libraries include heterozygous or haploid deletion as well as overexpression collections for different genes (Gene XY). In addition, de novo derived mutants by spontaneous ( ) or transposon (Tn7) mutagenesis can be used to identify loci associated with the compound’s mechanism of action. formation is based on the same principle as clonogenicity, but pound screens, yeast can be treated with compound libraries in with a faster readout and a high-throughput workflow. Aliquots 96-, 384-, or even 1536-well plates, and aged in a shaking in- of an aging yeast culture are spotted onto agar plates and the cubator. At multiple time points during aging, culture aliquots growth of colonies can be tightly monitored with a BioSpot R An- can be taken manually or in an automated fashion using alyzer, which is able to distinguish colonies with a diameter as liquid-handling workstations (e.g. QIAGEN BioRobot), then spot- smallas25 μm (Teng and Hardwick 2013). For anti-aging com- ted, and microcolonies counted after approximately 18 hours. Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 Zimmermann et al. 7 Compounds that prolong lifespan result in improved formation erally, exponentially growing cells appear to take up externally of microcolonies over time compared to untreated controls. It is supplied molecules better than non-dividing cells (De Nobel and of note that both methods measure cell growth and the ability Barnett 1991), which should be kept in mind when performing to divide, which does not necessarily correspond to cell viability. pharmacological screens. Cells may be unable to divide for several reasons, e.g. mutation Yeast possesses pleiotropic drug resistance (PDR) efflux of growth-relevant genes, but still maintain their viability. More- pumps which might alter the effective dose of a bioactive com- over, chemicals that prolong lifespan but also inhibit growth pound. To overcome drug efflux, PDR deletion mutants can be might not be detectable with this approach. For example, cells employed in pharmacological yeast screens (Griffioen et al. 2006). show reduced growth rates under rapamycin treatment, and In some cases, this may increase the sensitivity to drugs by up to when transferred to rapamycin-free media only recover slowly 200-fold compared to wild type cells (Rogers et al. 2001). Conse- by passive dilution of the drug (Evans, Burgess and Gray 2014). quently, a combination of deletion mutants of the paralogs PDR1 Here, cell vitality as a measure for the physiological capabilities and PDR3, which are transcription factors for the pleiotropic of a cell (Carmona-Gutierrez et al. 2018) can be measured in situ, drug response, with disruption of the ergosterol biosynthesis e.g. by flow cytometry-based cell stainings with vital cell dyes gene ERG6, which results in increased membrane permeability, (e.g. FUN-1 or alamarBlue R ). It should be noted, though, that greatly enhanced the efficacy of growth-inhibiting compounds vital dyes usually detect metabolic activity, for instance, metab- in a screen performed at the National Cancer Institute (Simon olized FUN-1 shifts from green to red fluorescence (Millard et al. and Bedalov 2004a). However, the presence of a drug efflux sys- 1997). Thus, compounds that attenuate metabolic activity may tem can also be considered as an advantage of the yeast system, also reduce FUN-1 conversion and cause a similar phenotype as it models the situation in human cells, which usually com- as cell death, although the cell is still viable. Another option is prise functional drug export systems (Chen et al. 2016). There- the flow cytometric/photometric quantification of cell death by fore, finding substances which can withstand immediate ex- membrane integrity dyes (e.g. propidium iodide) or cellular ROS port is most likely to more efficient in cells with intact drug levels (e.g. by measuring the ROS-dependent conversion of di- efflux pumps. In fact yeast has been suggested as a tool for hydroethidium to hydroxyethidium and ethidium, respectively), identifying inhibitors of multi drug resistance in cancer cells which are good estimators for yeast aging (Pan 2011;Kainz et al. (Mart´ın-Cordero et al. 2013). In addition, interference with the 2017). Importantly, all fluorescence/absorbance-based readouts fungal drug response and cell membrane composition might en- have to be controlled for background signals that might be tail undesired side-effects which could mask normally occurring caused by the tested compounds. For unbiased anti-aging FCG responses to pharmacological treatment. Nevertheless, when screens, a combination of these methods will give the most ro- dealing with low amounts of substances, drug efflux mutants bust results and minimize the occurrence of both false-positive are worth considering as models. and false-negative hits. Yeast is highly compatible with multi- Enzymatic modification of xenobiotics, which plays a major assay approaches, as it can be aged in multi-well formats, role in drug detoxification/degradation in mammals, might only permitting maximal interoperability with automated pipetting occur to a limited extent in yeast. While yeast does not har- robots and high-throughput screen (HTS) readouts. bor mixed function type 1–3 cytochrome P450 oxidases (Cresnar For high-throughput RLS screens, several single-cell mi- and Petricˇ 2011), which mainly mediate drug metabolization in crofluidics methods have been established to overcome the mammals (Zanger and Schwab 2013), it has a conserved detox- time-demanding manual separation of budded cells (Jo et al. ification system by covalent conjugation of xenobiotics to glu- 2015). Large-scale genomic screens have also made use of the tathione (Penninckx 2002; Townsend and Tew 2003;Ubiyvovk so-called ‘old mother cell sorting’, where the surface of young et al. 2006;Prev ´ er ´ al et al. 2009). However, if specific modifi- mother cells are labeled with biotin, and after a couple of genera- cation of compounds or activation of prodrugs is required, tions the aged mother cells can be extracted from the culture us- yeast-expressing human drug metabolization enzymes can be ing streptavidin-coated magnetic beads (Park, Mcvey and Guar- employed. For instance, transgenic yeast-expressing human cy- ente 2002;Sen et al. 2015). In addition, microfluidic approaches to tochrome P450 1A2 (CYP1A2) has been used to study the bioacti- monitor replicative age-dependent intracellular modifications, vation and subsequent toxicity of the hepatotoxin Aflatoxin B1 such as protein abundance and localization changes, have also (Guo et al. 2005). been developed (Cabrera et al. 2017). However, the adaptability Cell-based screens offer the possibility of estimating toxic of these methods to pharmacological screens has yet to be es- off-target effects of the tested compounds. Many substances tablished. that are toxic to mammalian cells also kill yeast cells, e.g. the HDAC-inhibitor valproate, the chemotherapy medication paclitaxel, or the anti-cancer compound bleomycin (Almeida et al. 2008). Moreover, yeast-based portable instruments have PHARMACOKINETICS AND OFF-TARGET been developed to estimate genotoxic hazard in environmen- EFFECTS IN YEAST tal monitoring (Knight et al. 2004). Yeast can undergo regu- Yeast has a cell wall that is mostly composed of glucans, manno- lated cell death when exposed to apoptotic stimuli (Madeo, proteins and low amounts of chitin, and it has long been be- Frohlic ¨ h and Frohlic ¨ h 1997; Madeo et al. 1999, 2002). Interest- lieved that the cell wall acts as a potent barrier for molecules ingly, inter- and intracellular triggers of mammalian apoptosis with an M > 700. However, some studies have demonstrated such as hypochlorite (produced during the oxidative burst in that molecules with significantly higher M (of up to 400 000) can immune cells) or ceramide (the trigger of caspase-independent traverse the cell wall (De Nobel and Barnett 1991). There is little cell death) also induce apoptosis in yeast in a regulated manner data regarding the influence of the yeast cell wall on the bioavail- (Carmona-Gutierrez et al., 2011, 2013, 2018). Humanized yeast ability of small molecules. As a benchmark, the TORC1 inhibitor has been used to unravel the adverse side-effects of phenoth- iazines which are prescribed as antipsychotics (Li et al. 2008): rapamycin has an M of ∼914 and an effective concentration (EC) of 1–10 nM (Powers et al. 2006;Alvers et al. 2009), which is com- a yeast model expressing the human fatty acid transport pro- tein 2 (FATP2) was treated with a compound library using a parable to the EC in human cells (Foster and Toschi 2009). Gen- Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 8 FEMS Yeast Research, 2018, Vol. 18, No. 6 Figure 4. Versatile transferability of yeast phenotypical screens. Yeast acts as a cellular sieve for substances which cannot pass the plasma membrane, are exported by pleiotropic drug efflux pumps (PDRs), or generally exhibit no biological activity. Chemicals toxic to yeast can be tested in human cell culture for cyt otoxicity. Substances which specifically kill cancer cells are potential chemotherapeutics, while substances with no toxicity might be further tested in pathogenic fungi as potential anti- fungal drugs. Lifespan-extending compounds should be validated in multicellular model organisms (nematodes, flies, mice). Substances which only e xtend yeast lifespan may be considered for biotechnological applications (e.g. yeast bioreactors) where yeast survival is desired. Chemicals which inhibit growth can be further tested in both directions (e.g. when they inhibit nutrient signaling), but are less suitable for biotechnological use. fluorescent fatty acid analog as a readout. Phenothiazines naling pathways. Deletion of genes that promote nutrient sig- efficiently blocked fatty acid uptake, consistent with their naling, such as RAS2 or SCH9 (Fabrizio et al. 2003), results in lifes- metabolic side-effects such as hypertriglyceridemia. pan extension in yeast, which can also be observed when reduc- In summary, yeast provides a cellular environment that is ing the activity of the corresponding pathways in mice (Yan et al. suited for estimating basic pharmacokinetic properties as well 2007; Selman et al. 2009). Similarly, treatment with the TORC1 as the off-target effects of a compound of interest (Figure 4). Nev- inhibitor rapamycin prolongs yeast lifespan (Powers et al. 2006) ertheless, ECs have to be reconfirmed when transferring the re- and delays aging in mice (Harrison et al. 2009). Low-dose applica- sults to other organisms, and a substance that does not exhibit tion of rapamycin in healthy adults might decrease senescence any toxicity in yeast cannot be considered safe for use in other markers, but does not improve overall frailty (ClinicalTrial Iden- organisms without further testing. tifier: NCT01649960; Singh et al. 2016). It is of note that rapamycin treatment leads to immunosuppression (hence its main use against organ rejection), which complicates its use in (healthy) CONSERVED MECHANISMS OF ANTI-AGING humans (Li, Kim and Blenis 2014). Nevertheless, the mechanistic COMPOUNDS IN YEAST parallels of rapamycin’s mode-of-action across species are stun- ning: it forms a complex with FK506-binding protein 12 (FKBP12) The relevance of a yeast in anti-aging drug discovery is best in mammalian cells, which is necessary for the inhibitory judged by its successful application both in the mechanistic in- effects on the TOR complex. This ultimately results in the vestigation of aging processes and the identification of strategies inhibition of translation and the activation of autophagy, an in- to promote longevity. In fact many pharmacological anti-aging tracellular degradation that recycles macromolecules and or- interventions follow a conserved mode of action in eukaryotes ganelles (Yin, Pascual and Klionsky 2016). In yeast, rapamycin from yeast to mammals (Table 1). A common target of anti-aging binds to Fpr1p, an FKBP12 homolog, and then this complex in- molecules is the regulation of cell growth/proliferation (de Cabo hibits yeast TORC1, also leading to translational arrest and ac- et al. 2014; Jimenez, Ribeiro and Clotet 2015). This is not surpris- tivation of autophagy (Loewith and Hall 2011). Interestingly, au- ing, since growth signaling has been implicated in aging progres- tophagy activation is essential for rapamycin-mediated lifespan sion by promoting ROS generation (Weinberger et al. 2010), and extensionbothinyeast(Alvers et al. 2009) and in fly models genetic interference with cellular growth regulation (e.g. by in- (Bjedov et al. 2010). This observation agrees with other stud- hibiting ribosomal function) has repeatedly been shown to pro- ies that indicate the general anti-aging properties of autophagy mote RLS in yeast (Chiocchetti et al. 2007; Steffen et al. 2008; (Madeo et al. 2015). The AMP-sensing kinase (AMPK) can also in- McCormick et al. 2015). Along similar lines, reducing nutrient duce autophagy by inhibition of TOR and activation of the Atg1 signaling, which is most efficiently achieved by caloric restric- complex upon nutrient scarcity. Activation of AMPK in mam- tion (CR), represents an evolutionary conserved means of ex- mals, for example by supplementation of metformin, which el- tending lifespan (Fontana, Partridge and Longo 2010;deCabo evates cytosolic AMP levels by inhibiting respiratory complex et al. 2014). Therefore, a promising strategy for the identification I, extends lifespan (Martin-Montalvo et al. 2013). In yeast, met- of anti-aging compounds is to screen for their ability to mimic formin supplemented in the millimolar range has been shown to the effects of CR. counteract aging, which is accompanied by a transcriptional re- Nutrient availability in yeast is mainly sensed by the target sponse similar to glucose deprivation (Borklu-Yucel, Eraslan and of rapamycin/S6 kinase (TOR/Sch9p), G-protein/protein kinase A Ulgen 2015). However, the molecular target in yeast remains to (Ras/PKA) and AMP-activated kinase (AMPK, Snf1p in yeast) sig- Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 Zimmermann et al. 9 Table 1. Anti-aging compounds from yeast to mammals. Selected compounds that extend lifespan in yeast and at least two higher eukaryotes. Molecular target Molecular target in Anti-aging Aging-related clinical Substance in yeast mammals mechanism trials Reference Spermidine Histone acetyl- Histone Protein Observational study (Eisenberg et al. 2009, transferases acetyl-transferase deacetylation, (NCT01649960) 2016) EP300 autophagy induction completed; Phase II clinical trial (NCT03094546) ongoing Ethanolamine Phosphatidyl- Phosphatidyl- Increase of Atg8-PE none (Rockenfeller et al. 2015) ethanolamine ethanolamine levels; autophagy induction Rapamycin Tor1 mTORC1 TOR inhibition; Phase I trial (Alvers et al. 2009; autophagy (NCT01649960) Harrison et al. 2009; induction; inhibition completed; Phase II trial Prev ´ er ´ al et al. 2009) of translation (NCT02874924) ongoing Resveratrol Sir2 SIRT1, TyrRS Sirtuin activation; Several beneficial (Howitz et al. 2003;Baur protein effects in Phase II et al. 2006;Berman et al. de-acetylation, clinical trials against 2017) autophagy induction age-related diseases Aminoguanidine unknown 3-deoxyglucosone Decreased advanced none (Oudes et al. 1998;Wang glycation et al. 2007;Kazi et al. end-products 2017) Metformin unknown Respiratory AMPK activation, Phase II clinical trial (Martin-Montalvo et al. complex I autophagy induction (NCT03309007) ongoing 2013; Borklu-Yucel, Eraslan and Ulgen 2015) a b trial-identifier at clinicaltrials.gov in parentheses; direct interaction controversial. be determined. In humans, metformin is primarily used against cellular material for degradation in the vacuole (the yeast type 2 diabetes, since its action on AMPK suppresses gluco- equivalent of mammalian lysosomes) (Yin, Pascual and Klion- neogenesis (Rojas and Gomes 2013). Trials in healthy individu- sky 2016). Supplementation of the PE precursor ethanolamine als are ongoing (NCT03309007) or have recently been completed induces autophagy and prolongs lifespan in yeast, while block- (NCT02432287). age of PE biosynthesis has opposite effects (Rockenfeller et al. The decisive role in elucidating the regulation and mechan- 2015). Ethanolamine induces autophagy in human cells culture ics of the autophagic machinery make yeast an excellent model and slows down senescence in mammalian cells and flies, to screen for autophagy-activating compounds, which may hold representing yet another molecule that promotes autophagy great promise for anti-aging interventions. Along these lines, and lifespan across species. While a phase II clinical trial using yeast served to identify the anti-aging properties of the nat- orally administered PE in patients with solid tumors is ongoing ural polyamine spermidine (Eisenberg et al. 2009). Spermidine (NCT02950103), no clinical trials in healthy adults are currently treatment reduces global histone acetylation and leads to an being, or have been, conducted. upregulation of autophagy-related genes. Once again, disrup- The polyphenol resveratrol was identified as a sirtuin- tion of autophagy inhibits lifespan extension by spermidine in activating compound in a screen using yeast RLS as a readout yeast as well as fly and nematode models. Genetic screens in (Howitz et al. 2003). Sirtuins are NAD -dependent protein dea- yeast led to the identification of histone acetyltransferases as cylases, with a wide variety of substrates and functions (Bheda possible targets of spermidine, a notion that has been corrob- et al. 2016). Interestingly, resveratrol (promoting protein deacety- orated by experiments in human cell culture (Pietrocola et al. lation) and spermidine (reducing protein acetylation) have simi- 2015). Oral spermidine supplementation also prolongs the lifes- lar effects, including the induction of autophagy in human cells pan of mice, improves cardiovascular health in an autophagy- (Morselli et al. 2011). Although the direct interaction of resver- dependent fashion, and consumption of spermidine-rich food atrol with the yeast sirtuin Sir2p has been challenged in later inversely correlates with cardiovascular morbidity in humans studies (Kaeberlein et al. 2005), the groundwork performed in (NCT03378843; Eisenberg et al. 2016). Other benefits of spermi- yeast fostered experiments in higher eukaryotes. In fact there dine treatment, such as improved T-cell response (Puleston et al. have been other targets identified for resveratrol in human cells, 2014), anti-cancer immunosurveillance (Pietrocola et al. 2016) including the tyrosyl transfer-RNA synthetase (TyrRS), which and protection against age-related memory loss (Gupta et al. binds resveratrol’s tyrosine-like phenolic ring and then translo- 2013, 2016), make it a potential all-round anti-aging interven- cates to the nucleus to activate stress response pathways (Sajish tion, with autophagy induction as the general mechanism of and Schimmel 2015). It remains to be determined whether this action (Madeo et al. 2018). A clinical trial built on spermidine’s interaction contributes to resveratrol’s health-promoting effects capability to promote cognitive abilities is currently ongoing and if a similar molecular mimicry occurs in yeast. Resveratrol- (NCT03094546). mediated health benefits include prolonged lifespan of mice Autophagy involves the formation of phos- when fed a high-calorie diet (Baur et al. 2006), and improved phatidylethanolamine (PE)-conjugated Atg8p (the yeast ho- health parameters in nonhuman primates under an adverse molog of the autophagy related protein LC3), which is essential diet (Jimenez-Gomez et al. 2013; Mattison et al. 2014). The ther- for the buildup of double-membrane vesicles that sequester apeutic potential in humans has been corroborated in a series Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 10 FEMS Yeast Research, 2018, Vol. 18, No. 6 of clinical trials in patients with diverse morbidities (Berman lators. Such genes frequently exert cytotoxic effects when ex- et al. 2017). However, some trials in healthy adults reported pressed in yeast, which enables screening for cell viability or improved biomarkers for atherosclerosis (NCT01244360; Agar- cell growth phenotypes (Figure 3). By using different genetic iso- wal et al. 2013) and inflammation (NCT01492114; Bo et al. 2013), forms of a heterologous gene, yeast can be engineered to fa- but mixed effects on cerebral blood flow and no effects on cilitate the screening of isoform-/organism-specific therapeutic cognitive abilities (NCT01640197, NCT01794351; Kennedy et al. compounds. Such an approach has been performed, for in- 2010). Several compounds that have been discovered as poten- stance, to identify acetyl-CoA carboxylase 2 (AAC2)-specific in- tial anti-aging drugs in yeast screens still await validation in hibitors (Marjanovic et al. 2010). multicellular organisms, including the bile acid lithocholic acid To some extent such concepts follow the rationale of (Goldberg et al. 2010) and the fungal secondary metabolite beau- RCG, as the target or pathway of interest is usually se- veriolide I (Nakaya et al. 2012). Chemical genetic screens in lected a priori, but compound treatment is performed in an fission yeast have revealed several pharmacologically acces- in vivo cell-based fashion. Heterologous expression of disease- sible cellular processes against aging (Stephan, Franke and related genes, such as neurotoxic proteins, often results in + + Ehrenhofer-Murray 2013). The K /H exchanger nigericin and yeast cell death (Shrestha and Megeney 2015; Heinisch and + + Na /H exchanger monensin prolong fission yeast lifespan by Brandt 2016; Ruetenik et al. 2016; Speldewinde and Grant 2017). improving vacuolar acidification and reducing vacuolar frag- Importantly, a common molecular etiology of neurotoxicity mentation in a vacuolar-ATPase-dependent manner. Interest- is the disturbance of protein quality control. Similar to hu- ingly, studies with these compounds in human neuroblastoma man cells, yeast is equipped with multiple levels of proteosta- cell lines have linked cytosolic acidification to mitochondrial sis control, including chaperone-mediated protein (re-)folding quality control by autophagy (Berezhnov et al. 2016). Here, high (Winkler et al. 2012; Mathew and Stirling 2017), endoplas- concentrations of nigericin led to decreased cytosolic pH and ac- mic reticulum-associated degradation (ERAD) of misfolded pro- tivated mitochondrial-specific autophagy (termed ‘mitophagy’) teins (Brodsky and Skach 2011), unfolded protein response independent of the canonical PINK1/Parkin signaling cascade. (Wu, Ng and Thibault 2014) and proper protein localization It remains to be determined if this effect can be harnessed to through vesicular trafficking (Bonifacino and Glick 2004). For extend lifespan in higher eukaryotes. Vacuolar acidification has example, expression of the Parkinson’s disease (PD)-related been linked to lifespan extension upon methionine restriction protein α-synuclein in yeast leads to disturbance of Rab1p- in yeast (Ruckenstuhl et al. 2014). It is noteworthy that lysosomal mediated ER-to-Golgi trafficking and ER stress (Cooper et al. pH in mammalian cells is regulated by mTOR via the lysosomal 2006), as well as reduced vacuolar proteolytic function (Auf- ATP-sensitive Na channel lysoNaATP in response to nutrient schnaiter et al. 2017) and results in cytotoxicity, which in- 2+ availability (Cang et al. 2013). Lysosomal acidification/function volves the Golgi-localized Ca transporter, Pmr1p, and the mi- is an appealing target for pharmacological lifespan extension tochondrial nuclease, Nuc1p (yeast homolog of EndoG). Impor- (Carmona-Gutierrez et al. 2016), and the evolutionary conserva- tantly, these mechanisms are conserved in multicellular organ- tion allows screening for compounds in yeast. isms and neuroblastoma cell lines, respectively, consolidating The accumulation of advanced glycation end-products the use of yeast in elucidating disease mechanisms (Buttner ¨ (AGEs), which are products of non-enzymatic addition of car- et al. 2013a, 2013b). Such yeast strains can be used to screen bohydrates to other macromolecules, has been linked to the for substances which ameliorate the phenotype. Indeed, a small progression of age-related vascular decline, especially in pa- molecule screen in yeast using optical density as well as vi- tients with diabetes (Goldin 2006). Glycation inhibitors such as tality staining with alamarBlue R has been effectively used to aminoguanidine have been shown to delay replicative senes- discover compounds that ameliorate α-synuclein toxicity by cence in human fibroblasts (Wang et al. 2007)aswellasextend- restoring vesicular transport (Fleming et al. 2008;Su et al. 2010). ing lifespan in Drosophila (Oudes et al. 1998). Recently, a study A similar screen performed in a yeast model of Alzheimer’s has suggested that aminoguanidine acts in a conserved manner disease (AD) identified a set of small molecules with the in yeast and prolongs lifespan by reducing AGEs (Kazi et al. 2017). capacity to modulate aggregation of amyloid beta, the ma- In summary, yeast is a suitable cellular environment for the jor pathological hallmark of AD (Amen and Kaganovich 2016; identification or mechanistic characterization of anti-aging sub- Park et al. 2016). Different yeast-expressing proteotoxic pro- stances (Figure 4), provided that the results can be reproduced in teins, namely α-synuclein, transactive response DNA-binding higher eukaryotes. protein 43 (TDP-43, involved in frontotemporal dementia) and htt-72Q (the mutant form of huntingtin causing Huntington’s disease) revealed 8-hydroxyquinolines (8-OHQs) as potential lead substances against neurodegenerative diseases. The pri- YEAST CHEMOGENOMIC SCREENS AGAINST mary readout in this screen was the reconstitution of the AGE-RELATED DISEASES transgene-mediated growth defect by treatment with a sub- In addition to its application as a model for the aging process, stance library of ∼190 000 compounds. Remarkably, mechanis- yeast has been increasingly used to study specific human age- tic investigations in yeast confirmed that 8-OHQs ameliorated related diseases. On the one hand, specific molecular defects proteotoxicity mainly by intracellular metal chelation, in line might be modeled by genetic manipulation to obtain mecha- with their known ability to form complexes with metal ions nistic insights into corresponding disorders, e.g. congenital dis- (Tardiff et al. 2012). Another hit from this screen that showed orders due to defective N-glycosylation or lysosomal storage promising effects against α-synuclein-mediated toxicity was in- diseases arising from deficiencies in lysosomal proteins and vestigated in detail using a combinatory approach with three pathways (Hauptmann et al. 2006; Rajakumar, Munkacsi and different yeast mutant libraries. After a primary screen testing Sturley 2017). On the other hand, humanized yeast models het- synthetic analogs of the lead substance for their ability to inhibit cell growth at high concentrations, the most promising candi- erologously express human (disease-relevant) genes (Menezes et al. 2015; Heinisch and Brandt 2016; Laurent et al. 2016)and date, N-aryl benzimidazole (NAB), was administered to an over- expression library, a collection of random transposon insertions can be used to screen compound libraries for specific modu- Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 Zimmermann et al. 11 as well as cells with spontaneous genomic point mutations. The ing cellular respiration, it should be kept in mind that yeast does three libraries revealed a network surrounding the ubiquitin lig- not possess a typical respiratory chain complex I, but instead ase, Rsp5p, which is involved in endosomal transport, as the regenerates NAD by a set of NADH dehydrogenases located main target for NAB. Importantly, the results could be confirmed on both sides of the inner mitochondrial membrane (Lasserre in neuronal cell cultures (Tardiff et al. 2013) and neurons derived et al. 2015). from humans with PD (Chung et al. 2013). Interestingly, Rsp5p has a C2 calcium-binding domain and, together with the known THE ADDED VALUE OF YEAST-BASED 2+ involvement of Ca in α-synuclein-mediated toxicity, this fos- SCREENS ters the view of a causal calcium signaling network governing PD, which can be studied in a unicellular organism. The ex- In the realm of model organisms, yeast has unique properties tensive screening strategy preceding the identification of NAB that allow transferring the results of a single screen to different represents the blueprint for the optimal use of yeast as a drug applications. First, yeast is a valid model for human aging, and discovery tool. In fact, a comparable approach in neuronal cell compounds that prolong yeast lifespan are good candidates for culture would not have been feasible, both technically and eco- anti-aging interventions in humans. Important selection crite- nomically. ria for anti-aging compounds include improved lifespan (CLS or Given the conservation of cancer-relevant pathways in yeast, RLS or both) or modulation of aging-related pathways (e.g. nu- proteins involved in cancer etiology can be expressed in yeast trient/growth signaling, autophagy, proteostasis or ROS genera- both to study their impact on growth and to identify potential tion), drug bioavailability (in yeast mainly influenced by mem- inhibitors (Simon and Bedalov 2004b). In an effort to find novel brane permeability or drug export), no or low toxicity (tested by inhibitors of human sphingosine kinase 1 (SphK1), which phos- assessing cell viability/vitality). Lifespan-extending compounds phorylates sphingosine and shows elevated expression in var- should be validated in established multicellular model organ- ious cancers, Kashem et al. employed a yeast model heterolo- isms such as nematodes, flies or mice (Buffenstein, Edrey and gously expressing SphK1 (Kashem et al. 2016). Expression of this Larsen 2008). Thereby, appropriate control experiments for each transgene resulted in a lethal phenotype, consistent with pre- model (e.g. food consumption, drug bioavailability) should be vious findings that reported toxicity upon elevation of sphin- included, especially when the compound is administered ad li- golipid long-chain base phosphates in yeast (Zhang et al. 2001). bitum via food or drinking water. To facilitate the translational Importantly, some of the bioactive compounds found in the use of compounds, pharmacokinetic properties should be de- yeast model had not been identified in a parallel in vitro screen, termined for each compound in vivo or pre-estimated based on underlining the relevance of a living cellular screening environ- the chemical properties using online tools such as SwissADME ment (Kashem et al. 2016). Due to the high grade of conserva- (http://www.swissadme.ch). Lifespan-extending hits from yeast tion in cell cycle genes, pharmacological screens for inhibitors screens that cannot be validated in other organisms may have of cyclin-dependent kinases (CDKs), which are essential for cell biotechnological applications in cases where yeast aging has ad- division and therefore attractive targets for chemotherapy, can verse effects, such as beer fermentation (Bran ´ yik et al. 2008). be performed in yeast. The yeast CDC28 gene can be functionally Here, the underlying mechanism and ways to reproduce the ef- complemented by its human homologs, CDK1 and CDK2, allow- fects by genetic means rather than the substance itself are of ing expression of the human proteins in a cdc28 strain. By us- interest, as the addition of uncharacterized compounds to biore- ing growth inhibition upon compound treatment as a readout, actors is usually not preferred. Second, yeast can be used as this model has already revealed selective inhibitors of the hu- a model for fast-growing mitotic cells to identify anti-cancer man CDKs (Mayi et al. 2015). agents (Simon and Bedalov 2004b). Compounds toxic to yeast Mitochondrial function is intimately connected to healthy can be tested in human cell culture (both in malignant and non- aging, while mitochondrial disorders are involved in a variety of malignant cell lines) for cytotoxicity. Substances that specifically age-related diseases (Bratic and Larsson 2013). Yeast has been kill cancer cells are potential anti-cancer agents. Notably, sub- successfully used to identify novel substances that could in- stances that are cytotoxic in yeast, or inhibit yeast growth but do crease mitochondrial membrane potential and total ATP con- not show any activity in human cancer cell lines, might be con- tent, both of which negatively correlate with aging (Montague sidered as potential anti-fungal compounds, provided that they et al. 2014). A screen performed in cells lacking a subunit of the do not exhibit any toxic effects in non-malignant cell lines and mitochondrial succinate dehydrogenase (SDH) has been used to mice. Indeed, due to its close relatedness to fungal pathogens screen 200 000 compounds for potential therapeutics against like Candida (Berman and Sudbery 2002), S. cerevisiae is a widely- disorders linked to succinate accumulation, such as familial used model for the mechanistic examination of antifungal drugs paraganglioma. Interestingly, two of the screen hits were in- (Tebbets et al. 2012; Roberts, Miller and Atkinson 2017;Serratore hibitors of the fungal alcohol dehydrogenase, which in cancer et al. 2017). Thus, given an appropriate readout (e.g. lifespan or cells plays a similar role in regenerating NAD as lactate dehy- cell stress), results from a single screen can be used for a variety drogenase (LDH). Accordingly, human HEK293 cells treated with of translational studies (Figure 4). siRNA against the SDH component SDHB showed increased sus- ceptibility to the LDH inhibitor oxamate, suggesting LDH as a CONCLUDING REMARKS potential target in paraganglioma therapy (Bancos et al. 2013). In addition to single mitochondrial gene deletions, yeast cells de- Yeast has emerged as one of the most versatile organisms both void of mitochondrial DNA (ρ strains) can be used to investigate in basic research and applied science. The small and compara- bioactive compounds in the absence of mitochondria-encoded tively well-characterized genome makes yeast a prime model to genes, which distinguishes yeast from other model organisms. study complex cellular processes in a simple environment. The Indeed, screens with inhibitors of sphingolipid biosynthesis in fruitful translation of findings from yeast to higher eukaryotes the background of ρ strains have revealed distinct suscepti- has helped to understand aging processes in humans, including bilities to different compound classes depending on the pres- the mode of action of anti-aging molecules like rapamycin, sper- ence of mitochondrial DNA (Kemmer et al. 2009). When target- midine or resveratrol. Multiple pharmacological screens have Downloaded from https://academic.oup.com/femsyr/article/18/6/foy020/4919731 by DeepDyve user on 15 July 2022 12 FEMS Yeast Research, 2018, Vol. 18, No. 6 used yeast for initial drug discovery, and often the screening pro- Berman AY, Motechin RA, Wiesenfeld MY et al. The therapeutic cedure would have been unfeasible in other experimental mod- potential of resveratrol: a review of clinical trials. Npj Precis els. Yet yeast has long been overlooked as a tool to screen for Oncol 2017;1, DOI: 10.1038/s41698-017-0038-6. anti-aging compounds. The growing number of available tools Berman J, Sudbery PE. Candida albicans: A molecular revolu- and methods to assess lifespan in a high-throughput fashion tion built on lessons from budding yeast. Nat Rev Genet further paves the way for yeast-based anti-aging screens. In ad- 2002;3:918–32. dition, yeast viability is a powerful readout that allows trans- Bheda P, Jing H, Wolberger C et al. 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Published: Sep 1, 2018