The examples of chemical genetics studies using
The budding yeast Saccharomyces cerevisiae is a useful eukaryote model organism for application to chemical biology studies, for example, drug screening, drug evaluation, and target identification. To use yeast for chemical biology research, however, it has been necessary to construct yeast strains suitable for various compounds because of their high drug resistance. Hence, the deletion of all multidrug resistance genes except for those that are important for viability and for genetic experiments/manipulation could increase the drug sensitivity without influencing the transformation, mating, or sporulation efficiency. There are two major factors conferring multidrug resistance in S. cerevisiae: one is the drug efflux system and the other is the permeability barrier. We therefore constructed a strain which shows high sensitivity to multiple drugs by disrupting the drug efflux system using ATP-binding cassette transporters and suppressing the membrane barrier system by introducing an ERG6-inducible system. In this review, we discuss the construction of our multidrug-sensitive yeast strains and their application in chemical biology.
- multidrug-sensitive yeast
- drug efflux system
- permeability barrier system
- drug target identification
- drug screening
1.1. Screening and target identification of bioactive small molecules: important processes in chemical genetics
The screening of bioactive small molecule compounds is the most important process in drug development. Natural products which have structural diversity isolated from microorganisms, plants, and animals are useful sources in the field of drug development . Structurally, new natural products might show novel activities such as antimicrobial, antiviral, and antitumor activities. These natural products also provide useful information for medicinal chemistry, and allow the development of new synthetic compounds as novel medicines. For example, eribulin, a semi-synthetic derivative of halichondrin B, has been approved as an anti-cancer drug [2, 3, 4]. Therefore, the screening and identification of new small molecules open new avenues for drug development. There are two major ways to identify bioactive small molecules: phenotypic screening and target-based screening. Phenotypic screening is based on cytotoxicity [5, 6, 7], cell cycle arrest , immune-suppression , and morphological changes  of drug-treated cells, fungi, and bacteria. Target-based screening is performed based on measurable readouts such as enzymatic activity inhibition  or drug-protein interaction . These approaches have identified useful small molecules and medicines.
Target identification (Target ID) of small molecules is also quite important in order to develop safe and useful drugs . Thalidomide, a cautionary example, was used as a sedative a half-century ago before it was found to be teratogenic and to cause multiple birth defects . However, thalidomide is also used in the treatment of Hansen’s disease, myeloma , and so on. In addition, immunomodulatory drugs derived from thalidomide have been developed as a new class of anti-cancer drugs and novel medicines for treating ribosomopathies such as 5q-syndrome . Recently, cereblon, a substrate receptor of the CRL4 E3 ubiquitin ligase, has been identified as a primary target of thalidomide teratogenic  and anti-cancer  activity. These lines of research provide useful information that cereblon may pose a risk of teratogenic activity and simultaneously serve as an attractive molecular target for immunomodulatory drug development. To identify the relevant target molecules and target pathways, indirect and direct approaches have been used . The indirect approaches include phenotypic analysis and large-scale analysis such as proteomic and genome-wide analyses. Some specific changes in cell morphology, cell cycle arrest, and other phenotypes provide us useful information for predicting targets of the drugs. Based on this property, Morphobase, an encyclopedic database of the morphological changes that occur in drug-treated cells, has been constructed and applied to drug target discovery . Large-scale analyses such as proteomics, metabolomics, and transcriptome analysis of drug-treated cells have been performed to predict the target pathways of bioactive small molecules . Genome-wide genetic studies are also frequently used for drug target ID. For example, synthetic lethal/sick genetic interaction analyses [19, 20], genome-wide overexpression screening , and haploinsufficiency-chemical sensitive assays  have been used to analyze the mode of action of various drugs. On the other hand, direct approaches, such as affinity probe approaches and genetic analyses, are quite useful to identify the direct target molecules of drugs. By using affinity probe approaches, the targets of thalidomide  and FK506  have been identified. Genetic analysis is another powerful method of identifying not only drug targets [24, 25, 26, 27, 28, 29] but also the signaling pathway affected by a drug. Genetic studies using model organisms such as yeast have contributed to identification of the target molecules of bioactive compounds.
The identification of new bioactive small molecules and elucidation of their target molecules/signaling pathways are important not only for developing medicines but also for basic science. Such compounds are a useful tool for understanding the fundamental protein functions in cells. Well-known examples are famous immunosuppressants such as FK506, cyclosporine, and rapamycin. These compounds inhibit immunophilin and T-cell activation through different mechanisms . Studies of these compounds have revealed their detailed immunoreaction mechanisms . Mitotic inhibitors are another example. Mitotic spindle formation and chromosome segregation are fast processes that are completed within approximately 1 hour. Therefore, by taking advantage of rapid pharmacological intervention, studies using microtubule inhibitors (αβ-tubulin inhibitors [31, 32, 33] or γ-tubulin inhibitor ), mitotic kinesins (Eg5 [34, 35]), and mitotic kinase inhibitors (aurora kinases [36, 37], Cdk1 , Plk1 [39, 40], Mps1 [41, 42]) highlighted useful information regarding the temporal regulation of mitotic spindle architecture and faithful chromosome segregation. These findings could in turn contribute to further drug development. Therefore, target ID of newly found useful bioactive compounds is quite an important process in both basic science and medicine development.
Saccharomyces cerevisiae, a useful model organism for chemical genetics
|Benomyl||Pathway analysis||Identification of Mad1, Mad2, Mad3 as mitotic spindle checkpoint proteins by using benomyl sensitive mutants|||
|Benomyl||Pathway analysis||Identification of Bub1, Bub2, Bub3 as mitotic spindle checkpoint proteins by using benomyl sensitive mutants|||
|Reveromycin A||Target ID||Identification of |||
|Curvularol||Target ID||Identification of |||
|Rapamycin||Target ID||Identification of |||
|Eudistomin C||Target ID||Identification of |||
|Splitomicin||Screening||Identification of splitomicin as a NAD+-dependent histone deacetylase inhibitor|||
|Mammalian cell line (HeLa)||Budding yeast (BY4741)|
|Latrunculin A (nM)||0.2||>240|
|4-Nitroquinoline 1-oxide (μM)||0.1||7.1|
In this review, we discuss the construction of two multidrug-sensitive yeast strains, 12geneΔHSR  and 12geneΔHSR-iERG , which are available for genetic analysis. We also discuss the application of these strains in drug screening and target ID .
2. Construction and application of multidrug-sensitive yeast strains
2.1. Construction of multidrug-sensitive yeast strains
We constructed a multidrug-sensitive yeast strain by disrupting 12 ABC transporter-related genes and suppressing the
|Transformation efficiency (Cfu/μg)||Mating efficiency (%)||Sporulation efficiency (%)|
|BY4741||9.6 × 105 ± 2.2 × 105||17.7 ± 7.5||21.9 ± 6.8|
|55.0 ± 51.3||4.8 ± 1.7||9.4 ± 4.7|
|12geneΔ0||1.2 × 105 ± 2.0 × 104||15.7 ± 5.3||5.0 ± 2.9|
|12geneΔ0HSR||N.D.||N.D.||28.8 ± 4.6|
|12geneΔ0HSR-iERG6 (under glucose condition)||7.0 ± 8.2||6.4 ± 2.2||0.0 ± 0.0|
|12geneΔ0HSR-iERG6 (under galactose condition)||3.0 × 104 ± 2.4 × 104||N.D.||10.7 ± 3.0|
2.2. Application 1: drug screening
2.2.1. Availability of 12geneΔ0HSR-iERG6 in drug screening
|Number of broth||Number of hit broth||Hit ratio (%)|
|Number of broth||Number of hit broth||Hit ratio (%)|
To identify the mitochondrial inhibitors, we used the difference in cell growth between the glucose medium and the glycerol medium. Yeast can use glycerol as a respiratory substance after the conversion to dihydroxyacetone phosphate via glycerol-3-phosphate by cytosolic and mitochondrial enzymes, GUT1p and GUT2p, respectively. Therefore, yeast could grow even in the presence of a mitochondrial inhibitor in glucose medium because of anaerobic respiration, but not in glycerol medium in which one of the metabolites in glycolysis, dihydroxyacetone phosphate, could not be produced. Therefore, we compared the growth inhibition induced by microbial broth samples on glucose medium (1% yeast extract, 2% polypeptone, 2% glucose, 1.5% agar) with that on glycerol medium (1% yeast extract, 2% polypeptone, 3% glycerol, 1.5% agar), and chose the broth which inhibited yeast growth on glycerol medium but not on glucose medium . Growth inhibition activities of microbial broth samples were evaluated using the paper disc method on agar plates inoculated with recombinant
To determine whether it is possible to isolate the novel compounds or not, we selected the microbial broths which were detected using 12geneΔ0HSR-iERG6 but not using the quadruplex mutant. We found a total of 46 broths (fungus origin: 16 broths; actinomycetes origin: 30 broths) which inhibited the growth of 12geneΔ0HSR-iERG6 specifically. Among these broths, we selected two fungus broths for further purification of active metabolites, and isolated 4,6′-anhydrooxysporidinone (
2.2.2. Screening of readthrough compounds
Because the usefulness of our strains was confirmed, we next performed the preliminary screening of compounds that show readthrough activities. Readthrough compounds allow the translational machinery to skip nonsense mutations encoding premature termination codons (PTCs) and could become medicines for hereditary diseases caused by PTCs (Figure 4). To date, many small molecules have been developed as readthrough drug candidates. Several forms of aminoglycoside antibiotics, such as gentamicin (
To discover novel readthrough compounds, we constructed yeast strains for the screening of readthrough compounds using 12geneΔ0HSR.
Next, we initiated a high-throughput screening of the readthrough compounds based on the halo assay using chemical library. This screening is underway, but already several hit compounds have been found, including rapamycin (
2.3. Application 2: target ID
Since our strains show multidrug sensitivity without a decrease in genetic availability, they should also be useful for performing target ID for drugs and the mechanism evaluation of compounds, especially those which are only available in limited amounts, such as natural products. Here we show an example of target ID . Eudistomin C (EudiC, Figure 8), a natural product isolated from the Caribbean tunicate
Collectively, our target ID studies of EudiC suggested the mode of action of EudiC cytotoxicity and indicated that our sensitive strains would be quite useful for performing drug target IDs in a relatively short period.
3. Conclusions and perspective
In the field of chemical biology, several model organisms, including yeast, worms, flies, and mice, have been used. Yeast is one of the most-used model organisms due to its ease of handling and its genetic availability, but its drug resistance is sometimes an obstacle to investigation. To overcome this problem, we constructed two multidrug-sensitive yeast strains, 12geneΔ0HSR and 12geneΔ0HSR-iERG6. These strains not only show a broad spectrum of drug sensitivities against compounds for which resistance is shown by both ABC transporters and ergosterol without influencing transformation, mating, or sporulation efficiency, but they are also useful for drug screening. Indeed, we performed a screening of antifungal compounds and protein translation regulators which skip stop codons and found some promising candidates. Using 12geneΔ0HSR-iERG6, we succeeded in improving the hit rate of drug screening from microbial broth. The screening of microbial broth which inhibits the growth of 12geneΔ0HSR-iERG6 but not of the quadruplex mutant identified novel compounds suggested that our multidrug-sensitive strain-based screening using previously tested chemical sources in yeast screening could identify new bioactive compounds. Furthermore, as our screening system for readthrough compounds, genetically modified multidrug-sensitive strains can be applied for several types of screening such as a yeast 2-hybrid system-based protein-protein interaction modulators screening. Recently, a yeast 3-hybrid system has been applied for drug-protein interaction analysis . In this study, the
Recently, it has been reported that RNAseq combined with Crisper/Cas9-based genome-editing technologies is useful for target ID in mammalian cells . Identification of the drug target using our multidrug-sensitive strains and confirmation of the identified mutation in mammalian cells by Crisper/Cas9-based genome editing will reveal the mechanisms of drugs in more detail. Our multidrug-sensitive strains have the potential to facilitate chemical genetic studies and contribute to the development of medicines in the future.