Low‐Molecular Weight Compounds that Extend the Chronological Lifespan of Yeasts, Saccharomyces cerevisiae, and Schizosaccharomyces pombe

Yeast is an excellent model organism for research for regulating aging and lifespan, and the studies have made many contributions to date, including identifying various factors and signaling pathways related to aging and lifespan. More than 20 years have passed since molecular biological perspectives are adopted in this research field, and intracellular factors and signal pathways that control aging and lifespan have evolutionarily conserved from yeast to mammals. Furthermore, these findings have been applied to control the aging and lifespan of various model organisms by adjustment of the nutritional environment, genetic manipulation, and drug treatment using low‐molecular weight compounds. Among these, drug treatment is easier than the other methods, and research into drugs that regulate aging and lifespan is consequently expected to become more active. Chronological lifespan, a definition of yeast lifespan, refers to the survival period of a cell population under nondividing conditions. Herein, low‐molecular weight compounds are summarized that extend the chronological lifespan of Saccharomyces cerevisiae and Schizosaccharomyces pombe, along with their intracellular functions. The low‐molecular weight compounds are also discussed that extend the lifespan of other model organisms. Compounds that have so far only been studied in yeast may soon extend lifespan in other organisms.


Introduction
[22][23] Such nutritional restriction can easily control CLS of yeast, and nutritional restriction that does not cause malnutrition can also extend the lifespan of higher organisms. [5,24]Third, drugs can extend the lifespan of various organisms.Rapamycin, an inhibitor of TOR complex 1 (TORC1), extends CLS of yeast, worms, flies, and mice. [25]Lifespan control using drugs is easier to perform and apply than genetic manipulation or nutritional restriction.Therefore, identifying drugs and compounds that extend lifespan in model organisms is a growing research area. [26]This review focuses on two yeasts, S. cerevisiae and S. pombe, unicellular eukaryotes that diverged ∼600 million years ago. [27,28]Various low-molecular weight compounds have been reported to extend the CLS of these yeasts, and by summarizing this knowledge, we hope to provide new options and possibilities for drugs that mediate aging and lifespan in model organisms.

Low-Molecular Weight Compounds that Extend the Chronological Lifespan of S. cerevisiae
The budding yeast S. cerevisiae is a unicellular eukaryotic model organism that has been used to considerably progress lifespan research, and >30 low-molecular weight compounds have been reported as drugs that extend CLS (Figure 1).For several compounds, the intracellular factors, pathways, and processes involved in extending CLS have been identified.Additionally, several compounds can extend CLS in fission yeast as well as in multicellular organisms such as nematodes, flies, and mice. [29]Figures 2 and 3).
The TORC1 pathway is an evolutionarily conserved signaling pathway that regulates aging and lifespan.Inhibition of TORC1 by various compounds, including rapamycin, myriocin, and caffeine, has been reported to extend CLS in S. cerevisiae.

Rapamycin
The macrolide compound rapamycin is one of the most commonly used drugs to extend lifespan in model organisms.[32][33] In S. pombe, rapamycin does not affect cell growth but does extends CLS. [34,35]38][39][40][41][42][43][44][45] The mechanism of lifespan extension by rapamycin has been intensively studied in organisms other than yeast.[48] Furthermore, rapamycin-induced lifespan extension of C. elegans is abolished by the suppression of heat shock factor 1 (HSF-1), indicating that this is required for lifespan extension. [49]n studies using flies, rapamycin induced extended lifespans by suppressing the TORC1 pathway through ribosomal S6 protein kinase (S6K), eIF4E-binding protein (4E-BP), and autophagy. [50]However, rapamycin was reported to not extend the lifespan of males. [51]Sex-based differences in the effects of rapamycin on lifespan have also been reported in mice.Similar to flies, female mice have a greater lifespan extension effect than male mice; furthermore, a lack of significant rapamycin-induced lifespan extension has been reported in male mice. [52,53]apamycin is one of the most studied compounds that successfully extends lifespan in model organisms.Several other compounds have also been discovered that induce CLS extension by inhibiting TORC1.

Myriocin
Myriocin (ISP-1) inhibits serine palmitoyl transferase, the first enzyme in sphingolipid synthesis in all eukaryotes. [54]Myriocin treatment at low concentrations extends CLS in S. cerevisiae by lowering amino acid pools and inducing autophagy by inhibiting sphingolipid-controlled Phk1/2-Sch9 signaling and the PKA and TORC1 pathways and activating the Snf1/AMPK pathway. [55,54]In addition, CLS extension by myriocin reportedly requires stress-induced ubiquitin Ubi4 and ubiquitination of the methionine transporter Mup1. [55]Myriocin treatment induces Ubi4 transcription to promote Mup1 ubiquitination and endocytic clearance and reduce the amino acid pools, resulting in cellular amino acid restriction. [55]Therefore, myriocin-induced changes in nutrient-sensing signaling pathways may be due to amino acid restriction.

Caffeine
Caffeine, a methylxanthine analog of the purine bases adenine and guanine, exerts a pleiotropic effect on cells, causing activation or inhibition of various cell integrity pathways. [62]Caffeine suppresses the TORC1 pathway and extends CLS in a manner dependent on Rim15, a protein kinase downstream of the TORC1 pathway, suggesting that caffeine-induced CLS extension is achieved by TORC1 pathway suppression. [62,63]n fission yeast, caffeine also extends CLS. [35]This does not occur in strains lacking Tco89, a nonessential core component of TORC1, suggesting that caffeine-induced CLS extension is involved in TORC1 suppression. [35]  Conversely, studies using C. elegans have shown that caffeine contributes to lifespan extension through the insulin/IGF-1like pathway. [64,65][67][68] In addition, RNA interference (RNAi) of daf-16 and of cbp-1, which encodes an acetyltransferase that acetylates DAF-16 and inhibits nuclear translocation. [69]prevents lifespan extension by caffeine. [65,70,67]Caffeine contributes to CLS extension in both yeasts and to lifespan extension in nematodes and is mediated by nutritional signaling pathways, but the detailed pathways appear to differ.However, caffeine-induced lifespan extension is also observed in daf-16 mutant strains. [64,71]Furthermore, caffeine-induced lifespan extension in nematodes can be suppressed in a concentration-dependent manner by exposure to adenosine. [64]Since caffeine blocks adenosine receptors in mammals, lifespan extension by caffeine may be partially dependent on adenosine signaling. [64]tudies using Drosophila melanogaster reported that caffeine can shorten rather than extend lifespan and is involved in remodeling the gut microbiota. [72,73]Thus, although caffeine seems to have a positive effect on lifespan extension in relatively simple organisms, further research is needed to determine its effect in higher organisms.Simple oral administration of candidate compounds that extend lifespan may not produce beneficial effects in higher organisms that coexist with intestinal bacteria.This possibility is discussed not only for caffeine but also for metformin, which is described later. [74]In addition, caffeine also shortens the lifespan of mosquito. [75]

Methionine Sulfoximine
Methionine sulfoximine, an inhibitor of glutamine synthetase, extends CLS in S. cerevisiae by reducing intracellular glutamine levels and suppressing TOR signaling. [76]Restriction of some amino acids can extend CLS in both S. cerevisiae and S. pombe. [77,78,76]

Morusin, Mulberrin, and Cudraflavone B
Morusin is extracted from the bark of Morus alba, a species of mulberry tree, and has a wide range of biological activities. [79]ulberrin (Kuwanon C) is a Morusin analog and a key component of the Chinese medicine Romulus Mori with various biological functions. [80]Morusin and mulberrin extend CLS in S. cerevisiae and the lifespan of C. elegans. [68]CLS extension by morusin and mulberrin in budding yeast was significantly reduced in calorie-restricted medium (glucose concentration was reduced from 2% to 0.5%), suggesting that these CLS extensions are involved in inhibiting nutrient-sensing pathways. [68]In addition, mulberrin-induced CLS extension was not observed in strains with deletions of Tor1 kinase of TORC1 or several factors downstream of TORC1, including Sch9, suggesting that the mulberrin-induced CLS extension is dependent on the TORC1 pathway. [68]udraflavone B, an analog of morusin and mulberrin, reportedly extends CLS of S. cerevisiae in a TORC1 pathwaydependent manner. [68]However, because morusin extends CLS even in Δsch9 cells, not all analogs of morusin, mulberrin, or cudraflavon B may cause CLS extension through the same mechanism.
The effects of these compounds on lifespan have been observed in C. elegans.Morusin and mulberrin extend lifespan in nematodes, and this is dependent on AKT-1 and AKT-2 that function downstream of DAF-2 and share high similarity with the C-terminal region of yeast Sch9. [68]Morusin inhibits the phosphorylation of T389 of p70S6K1, a target of TORC1, and induces autophagy in human HeLa cells. [68]The regulation of p70S6K1 phosphorylation and the induction of autophagy are involved in the regulation of aging. [68]6.Gentirigeoside B Gentirigeoside B from Gentiana rigescens Franch increases RLS and CLS in S. cerevisiae.[81] In addition, gentirigeoside B inhibits Sch9 downstream of TORC1, increases Rim15 and Msn2 activities downstream of TORC1, and induces autophagy.[81] Therefore, gentirigeoside B may extend RLS and CLS by suppressing the TORC1 pathway via the downstream factors Sch9, Rim15, and Msn2.[81] Autophagy plays an important role in regulating the lifespan of various organisms, including the CLS of yeast.Suppressing the TORC1 pathway also contributes to autophagy induction.[82] Compounds such as spermidine and 4,4′-dimethoxychalcone extend CLS through the induction of autophagy.

Spermidine
[85][86] Spermidine also induces autophagy and extends the lifespan of mice. [87]Spermidine stimulates autophagy in mammals via posttranslational hypusination of eukaryotic initiation factor 5A (eIF5A), which is essential for synthesizing the autophagy transcription factor TFEB. [88,89] Spermidine reportedly extends the lifespan of mice in a manner dependent on the microtubule-associated protein MPA1S, which positively regulates autophagy. [90]Research on Drosophila reported that spermidine-induced lifespan extension does not occur in strains with reduced insulin/IGF signaling because of the deletion of three insulin-like peptides and thus may be involved in insulin/IGF signaling. [91]Similarly, methionine metabolism is also important for lifespan extension because spermidine-induced lifespan extension is not observed in strains lacking the enzyme, glycine N-methyltransferase, which regulates methionine metabolism. [91]Intriguingly, spermidine increases the survival of the bdelloid rotifers Philodina acuticornis and Adineta vaga. [92]However, in a study using middle-aged male Sprague-Dawley rats, no extension of lifespan was observed with spermidine treatment. [93]8.4,4ʹ-Dimethoxychalcone 4,4ʹ-dimethoxychalcone (DMC) is a natural flavonoid found in the traditional Chinese herbal remedy Angelica sinensis.[94,95] DMC induces autophagy and extends CLS and lifespan in budding yeast, nematodes, and flies, and slows aging in cultured human cells.[96,97] In S. cerevisiae, screening using mutant strains of the autophagy-signaling pathways revealed that DMC-induced CLS extension decreased in deletion strains of GATA transcription factor Gln3, catalytic subunits of Pph21/22 of type 2A phosphatase (PP2A), which regulate Gln3 activity, and a regulatory subunit Tpd3 of PP2A.[98] suggesting that Gln3 plays an important role in CLS extension by DMC.[96] Gln3, a target of PP2A, acts downstream of TORC1.Under favorable growth conditions, TORC1 phosphorylates Tap42, which acts as another regulatory subunit of PP2A and subsequently interacts with and inhibits Pph21/22 to maintain Gln3 phosphorylation and inactivation.[99,100] Conversely, under unfavorable growth conditions, TORC1 is suppressed, Tap42 releases the catalytic subunits of PP2A, and Gln3 is dephosphorylated and activated. [99] DM treatment produces a similar intracellular amino acid profile and metabolic imprint to Δgln3 cells and reduces gene expression induced by Gln3, suggesting that DMC suppresses Gln3.[96] Rapamycin can activate Gln3 by inhibiting TORC1, and inhibition of Gln3 by DMC has the opposite effect to rapamycin.[96] Rapamycin increases the viability after 3 days of culture and autophagy induction regardless of the presence or absence of Gln3, and the effects of rapamycin and Gln3 are additive.[96] Therefore, DMC appears to operate Gln3 independently from TORC1.
DMC-induced lifespan extension via Gln3-like GATA transcription factors was also observed in C. elegans.Silencing of the GATA transcription factor ELT-1, a homolog of Gln3, abolished the DMC-induced lifespan extension. [96]9.Ethanolamine Ethanolamine, a precursor of phosphatidylethanolamine, increases the level of phosphatidylethanolamine and induces autophagy in budding yeast and cultured mammalian cells to extend the CLS of yeast and the lifespan of flies and mammalian cells.[101]

Inokosterone and Cucurbitacin B
Inokosterone from Gentiana rigescens Franch extends RLS and CLS in budding yeast and the lifespan of mammalian cells. [102]LS extension by inokosterone does not occur in strains deficient in antioxidant genes, including SOD1 and SOD2, which are involved in CLS regulation. [103]ATG2, which encodes a core autophagy-related protein of macroautophagy. [82]and ATG32, which encodes a mitochondrial outer membrane receptor recognized by the autophagy machinery. [102,104]Therefore, inokosterone induces antioxidant stress tolerance and mitophagy to extend RLS. [102]Although RLS and CLS do not necessarily match. [15]a similar mechanism may be involved in inokosteroneinduced CLS extension.
Cucurbitacin B (CuB) induces autophagy, extends RLS and CLS, and increases resistance to oxidative stress in S. cerevisiae. [105]CuB-induced CLS extension is not observed in Δatg2 or Δatg32 cells. [105]and this is therefore dependent on autophagy and mitophagy.The intracellular functions of CuB and inokosterone may be similar because both compounds have similar structures of steroid skeletons, increase resistance to oxidative stress, induce mitophagy, and extend CLS.

Selenomethionine
Supplementation of a selenium (0.00045% selenomethionine in media) increases RLS and CLS in S. cerevisiae. [106]Full CLS extension by selenium supplementation does not occur in strains lacking ATG32, which encodes a core mitophagy factor, or ALT1, which encodes a transaminase, indicating that mitophagy and Alt1 are required for CLS extension by selenium. [106] factor widely studied in aging and lifespan research is Sir2. [107]Sir2 proteins are highly conserved and act as either nicotinamide adenine dinucleotide (NAD.+ -dependent protein deacetylases or mono-ADP-ribosyltransferases and are involved in lifespan regulation in various organisms.In budding yeast, Sir2 contributes to the extension of RLS but not to CLS. [15] Therefore, although many studies have been conducted on drugs that target Sir2 and related factors, including resveratrol and nicotinamide (NAM), it seems difficult to find a clear consensus regarding their effects on CLS regulation.For example, CLS is extended by resveratrol, which activates Sir2, and NAM, which inhibits Sir2.

Resveratrol
Results of lifespan research with resveratrol, which activates Sir2, have been mixed.][112][113][114][115][116] In S. cerevisiae, resveratrol is reported to reduce CLS. [110,117]Resveratrol is structurally similar to tyrosine and acts as a tyrosine antagonist and binds to human tyrosyl-tRNA synthetase (TyrRS). [110]Resveratrol has cis-trans isomers that have opposite effects on the activation of TyrRS-regulated poly-ADP-ribose polymerase 1 (PARP1) and concomitant production of NAM, which inhibits Sir2/SIRT1. [110]hus, the different results of studies on the effects of resveratrol may be due to differences in the cis-trans isomers of resveratrol.

Nicotinamide
Sir2, which is activated by NAD.+ and suppressed by NAD.+ precursor, NAM, promotes RLS extension but has a negative effect on CLS in S. cerevisiae; Δsir2 cells have a long CLS. [15,118,119]Although NAM extends CLS in S. cerevisiae and the lifespan in C. elegans, it does not extend the lifespan in mice. [25,120,118,121]In budding yeast, CLS extension by NAM is dependent on Sir2, and NAM extends CLS by suppressing Sir2 activity. [118,117]Phosphoenolpyruvate carboxykinase Pck1 is a key enzyme in the gluconeogenesis pathway, and Sir2 inactivates Pck1 by deacetylation of Lys514. [122]NAM-induced CLS extension does not occur in the absence of Pck1, and NAM may therefore suppress Sir2-induced deacetylation of Pck1 and be involved in activating the gluconeogenesis pathway. [118,117]NAM also extended the lifespan of nematodes lacking SIR-2.1, the homolog of Sir2. [121]indicating that SIR-2.1 is not necessarily required for lifespan extension by NAM.

Ehretiquinone
Ehretiquinone increases antioxidant activity, induces autophagy, and extends RLS and CLS in budding yeast and CLS in mammalian cells. [123]Analysis using the YOM36 strain, which does not show CLS extension by deletion of SIR2, shows that ehretiquinone increases SIR2 expression. [123]Ehretiquinone-induced CLS extension is not observed in the SIR2-deficient strain, and SIR2 may therefore be involved in CLS extension by ehretiquinone. [123]any low-molecular weight compounds can extend CLS, although no clear intracellular targets are known in S. cerevisiae.These compounds, including hydrogen sulfide (H 2 S) and metformin, extend CLS of budding yeast and the lifespan of other model organisms.

Hydrogen Sulfide
H 2 S is a potent endogenous gasotransmitter that converts protein cysteine thiols and protein sulfenic acids to persulfides during persulfidation. [124][127][128] In budding yeast, the AGC-type protein kinase Ypk2, which regulates cell wall integrity, may be involved in H 2 S-release-induced CLS extension by using the H 2 S donor, NaHS. [128]The budding yeast Ypk2, an ortholog of mammalian SGK1 and fission yeast Gad8, is recognized along with Ypk1 as a substrate that is directly phosphorylated by TOR complex 2 (TORC2), which is a sensor and master regulator of plasma membrane-and cell wall-related events. [129]Thus, Ypk1/2 acts as a central regulator of the pathways and processes necessary for homeostasis of plasma membrane lipids and protein.H 2 S treatment increases YPK2 expression, and CLS extension by H 2 S does not occur in a YPK2-knockout mutant. [128]Therefore, H 2 S seems to extend CLS by inducing YPK2.However, the deletion of YPK2 also extends CLS. [128]n C. elegans, lifespan extension occurs with the use of AP39, a mitochondrion-targeted sulfide-delivery molecule, but the lifespan extension effect disappears in animals where daf-16 is suppressed. [130]Therefore, daf-16 may be required for lifespan extension by H 2 S. Furthermore, H 2 S is reportedly important for extending the lifespan of nematodes by inhibiting TORC1 or protein translation. [124,131]34]

Metformin, Aminoguanidine, and Hydralazine
The glycation inhibitor aminoguanidine (AMG), its analog metformin, and the mild glycation inhibitor hydralazine reduce modification of protein advanced glycation end product (AGE) and extend CLS in S. cerevisiae. [135]Metformin is a biguanide commonly used for treating type 2 diabetes mellitus, and its effects on lifespan have been studied in various species. [136,137]Metformin is thought to activate AMPK, which is partially mediated by inhibition of complex I in the mitochondrial electron transport chain. [138,137]Metformin-induced CLS extension has also been reported in fission yeasts. [139]he effects of metformin on lifespan are highly dependent on species, genetic background, sex, and dose used.In mice, metformin treatment that started at 5 weeks of age extended the lifespan of males but not of females. [25]In nematodes, the effects of metformin on lifespan also vary depending on genetic variants and drug concentration. [140,141]In Drosophila, metformin has been reported to extend lifespan but also reported to be insufficient to do this. [74,142,137,143]Studies using nematodes have reported that lifespan extension by biguanides such as metformin requires AMPK, transcription factor SKN-1, and ether lipid biogenesis. [144,145]

Curcumin
][149] but does not extend the lifespan in mice. [113]In S. cerevisiae, curcumin increases intracellular reactive oxygen species (ROS), and curcumininduced CLS extension is not observed in strains deficient in antioxidant genes, including SOD1 and SOD2. [147]Therefore, curcumin may act as a hormetin, exerting mild stress on cells, triggering hormesis, and slowing the aging process. [147]urcumin extends the lifespan of nematodes, and several factors are necessary for this extension.Curcumin-lifespan extension was not observed in mutants of AGE-1, MEK-1, OSR-1, SEK-1, SIR-2.1,SKN-1, or UNC-43, suggesting that these factors regulate this lifespan extension. [150]AGE-1 is a catalytic subunit of a phosphoinositide 3-kinase in the insulin/IGF-1-like pathway, MEK-1 is a MAPK kinase (MAPKK) in the JNK pathway, OSR-1 is an osmosis regulatory factor related to SEK-1 and UNC-43, SEK-1 is a MAPKK of the p38 pathway, and UNC-43 is a calmodulin kinase II.
Interestingly, the structure of curcumin may be similar to that of DMC, which induces CLS extension in an autophagydependent manner through inhibition of Gln3 in budding yeast (Figure 1).Thus, the function of curcumin may also be similar to that of DMC, and like DMC, curcumin induces autophagy in mammals. [151]18.Epigallocatechin Gallate (-)-Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea.[152] A high-throughput rapid CLS assay showed that EGCG, which extends the lifespan of worms, flies, and rats.[153,151,[154][155][156] also extends CLS in S. cerevisiae.[138] The longevity of C. elegans induced by EGCG depends on the AMPK, SIR-2.1, and p38 MAPK pathways.[157,156] These factors and pathways may also be involved in yeast CLS regulation by EGCG. Howevr, EGCG has also been reported not to extend the lifespan of nematodes under normal culture conditions.[152] In addition, a study using flies reported that the lifespan-extending effect of EGCG occurs in male flies, but not in female flies.[155] In experiments using mammalian cells, EGCG acts on 67-kDa laminin receptor and activates the PKA pathway.[158] However, studies on various organisms have shown that suppression of the PKA pathway generally contributes to longevity.

L -Carnitine, Propionyl-L-Carnitine, and Acetyl-L-Carnitine
L-Carnitine, propionyl-L-carnitine, and acetyl-L-carnitine extend CLS in S. cerevisiae. [164]CLS extensions by these carnitines were reduced in caspase Yca1-deficient or rho 0 strains, which have lost mitochondrial DNA, suggesting that CLS extension by carnitines is related to apoptosis and mitochondrial function. [164]Although L-carnitine also extends lifespan in C. elegans, the lifespan extension disappears with RNAi suppression of daf-16 and skn-1 or a mutation of daf-2, suggesting that lifespan extension by Lcarnitine depends on these factors. [165]

Lithocholic Acid
Lithocholic acid (LCA), a bile acid with a steroid skeleton, extends the CLS of S. cerevisiae under both normal and calorie-restricted (CR) conditions. [166]LCA-induced CLS extension under CR conditions was significantly reduced in deletion strains of carnitine acetyl-CoA transferase Cat2, carnitine-dependent acetyl-CoA transporter Crc1, or carnitine acetyltransferases Yat1/2, suggesting that LCA-induced CLS extension under CR conditions is involved in transporting acetyl-CoA from peroxisomes to mitochondria via the carnitine shuttle. [167]LCA also reportedly extends the lifespan of flies. [168]

Quercetin, Morin, and Steppogenin
[171] Quercetin significantly extends CLS even under CR conditions, and the CLS extension mechanism may therefore be independent on the nutrient-sensing pathway. [169]Further analysis is required to elucidate the detailed CLS extension mechanism.Quercetin also extends the lifespan of nematode, mosquitoes, and bees. [172,173,75]

Protocatechuic Acid
Protocatechuic acid, a bioactive compound found in Hom Dang rice bran pericarp, reduces ROS and extends CLS in S. cerevisiae. [170]Protocatechuic acid also extends the lifespan of nematode. [174]lthough many low-molecular weight compounds have been reported to extend CLS in S. cerevisiae, the underling mechanisms are often unknown.In addition, many of these compounds that extend CLS in budding yeast have not yet been confirmed to extend the lifespan of other organisms, although several of them may be good candidates for this.

Gentiopicroside
Gentiopicroside (GPS), a secoiridoid glycoside from Gentiana rigescens Franch, extends RLS and CLS in S. cerevisiae. [177]The GPS-induced extension of RLS was abolished in a strain lacking ATG32, a gene required for mitophagy, and RLS extension by GPS may therefore depend on mitophagy. [178]However, CLS extension also occurs in Δatg32 cells, so mitophagy may be not involved in CLS extension by GPS. [165]

Menadione
Menadione, which participates in redox cycling and generates superoxide, extends CLS at low concentrations. [160]Because CLS extension by menadione was not observed in Δtor1 cells, menadione-induced CLS extension might be related to CLS extension by TORC1 inhibition. [160]However, although the effect of menadione on lifespan has been investigated in nematodes, no effect of menadione on lifespan extension has been observed. [179]

Neohesperidin
Neohesperidin, a citrus flavonoid, reduces ROS and extends CLS in S. cerevisiae, but the detailed mechanism is unknown. [180]Neohesperidin has a similar molecular structure to that of gentirigeoside B, which may extend CLS by suppressing the TORC1 pathway (Figure 1), and then CLS extension by neohesperidin may also be involved in TORC1 suppression.Neohesperidin dihydrochalcone and neohesperidin dihydrochalcone-O-glycoside, which are extracted and processed from neohesperidin, have been reported to downregulate the mechanistic TOR pathway in mammalian cells. [181].29.Glyceollin I Glyceollin I, an induced phytoalexin isolated from soybean, has various bioactivities including antifungal and anticancer activities and insulin sensitivity enhancement and extends CLS at low (nM) concentration but decreases this at high (μM) concentration.[182] As mentioned above, many low-molecular weight compounds have been discovered that extend CLS in S. cerevisiae.However, a lack of extension of CLS was reported with caffeine, curcumin, metformin, resveratrol, and spermidine, although these may be due to differences in the background of strains or culture conditions.[183,117] Currently, only a limited number of lowmolecular weight compounds are understood via the mechanism by which they extend CLS.Further studies of these drugs will reveal how they contribute to CLS regulation.

Low-Molecular Weight Compounds that Extend the Chronological Lifespan of S. pombe
Research into aging and lifespan using the fission yeast S. pombe began later than for S. cerevisiae.However, because the two organisms are evolutionarily very different (diverged million years ago). [184]comparing research using these two yeasts will likely contribute to understanding lifespan regulation.In addition, aging and lifespan research is currently being conducted in various model organisms; and among these, S. pombe has the sixth largest number of aging-related genes registered in the database, after S. cerevisiae, C. elegans, Homo sapiens, D. melanogaster, and Mus musculus. [185]Although fewer studies on fission yeast have been reported than for budding yeast, many low-molecular weight compounds that extend CLS in fission yeast have been reported (Figure 2).Among these, several compounds have been studied for their CLS extension mechanism and have been observed to extend lifespan in other organisms (Figure 3).Note, because caffeine, metformin, myriocin, and rapamycin extend CLS in budding and fission yeasts and have already been described in the section on budding yeast, their description is omitted here.
The evolutionarily conserved lifespan-regulating signaling pathway TORC1 is also involved in CLS regulation in S. pombe.In addition to caffeine, myriocin, and rapamycin, several compounds that target TORC1, including torin-1, are thought to extend CLS in fission yeast through inhibition of TORC1.

Wortmannin
Wortmannin, an inhibitor of phosphoinositide 3-kinases and TOR kinases, extends CLS in S. pombe. [188]D. melanogaster-based studies have investigated the effects of wortmannin at concentrations of 0.005 to 5 μM on lifespan. [189,190]Wortmannin has been reported to increase the lifespan of males at concentrations of 0.5 and 5 μM, but decrease that of females at a concentration of 5 μM. [189,190]In addition, ly294002, an inhibitor of phosphoinositide 3-kinases such as wortmannin, extends the lifespan of C. elegans and D. melanogaster. [47,189,190]

Ethylenediaminetetraacetic Acid
Supplementation with ethylenediaminetetraacetic acid (EDTA), a metal chelating agent, extends CLS in S. pombe. [191]The longevity effect of EDTA may be due to metal starvation.In fission yeast, starvation of zinc, a trace metal necessary for organisms, extends CLS in an ecl gene-dependent manner. [191,192][199][200] In addition, Ecl protein inhibits the activity of TORC1 under starvation. [201,202]Thus, EDTA-induced CLS extension may mimic zinc starvation and may occur via activation of ecl genes and suppression of their downstream TORC1 pathway.
The metal starvation-induced lifespan extension has been observed not only in CLS of fission yeast but also in the lifespan of nematodes.Worms treated with the chelating agent N,N,N′,N′tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) during early development have an extended lifespan. [203]Furthermore, the longevity of C. elegans caused by the chelating agent is partially attenuated in daf-16, hsf-1, and skn-1 mutant strains, suggesting that chelating agent-induced lifespan extension is mediated by these factors. [203][206][207] Pmk1 is a MAPK that is a core effector of the cell integrity pathway and an extracellular signal-regulated kinase ERK ortholog. [208,209]The Pmk1 pathway is expected to have a negative effect on CLS extension because deletion of MAPK, MAPKK, and MAPKK kinase (MAPKKK) leads to extended CLS. [210]CLS extension caused by a defect in the cell integrity pathway is consistent with deletion of YPK2, a homolog of Gad8 and an upstream factor in the Pmk1 pathway, is long-lived in S. cerevisiae. [208,128]eanwhile, activation of the Sty1 pathway extends CLS while inhibition of the PKA pathway also extends CLS in S. pombe. [59,211]or example, deletions of a catalytic subunit of PKA, Pka1, or the G-protein coupled receptor Git3, which detects extracellular glucose. [208,212]extend CLS. [213,214]ntriguingly, these three pathways that regulate CLS are closely interrelated.Sty1 MAPK activates its downstream transcription factor Atf1 and then induces transcription of the phosphodiesterase Cgs2, leading to suppression of the PKA pathway. [22,215]n addition, Atf1 is activated by both the Sty1 pathway and the Pmk1 pathway. [208]ow-molecular weight compounds, including micafungin, tschimganine (TMN), and prostaglandin J 2 (PGJ 2 ), extend CLS in fission yeast via these pathways.

Micafungin and Calcofluor White
Micafungin and calcofluor white, which cause cell wall stress, extend CLS in S. pombe when used at low concentrations. [216]icafungin is a nonreversible inhibitor of 1,3--glucan synthase and inhibits fungal cell wall synthesis, whereas calcofluor white binds to the liner structure of 1,3--glucan polysaccharides and causes cell wall stress. [217,216,218]Micafungin activates Sty1 and Pmk1 pathways. [219]and Sty1 and Pmk1 are required for full CLS extension by micafungin. [216]therefore, micafungin extends CLS via these pathways.However, because deletion of Pmk1 extends CLS, it remains unclear how Pmk1 activation is involved in CLS extension.CLS extension by calcofluor white may also be mediated by Sty1 and Pmk1, although this has not been verified.
Meanwhile, because calcofluor white reportedly activates Rho1, a member of the Rho family GTPase, and then inhibits TORC1 in S. cerevisiae. [62,100]CLS extension by calcofluor white may also be involved in the TORC1 pathway in S. pombe.

Tschimganine, Mochimganine, 𝜶-Hibitakanine, and 𝜷-Hibitakanine
TMN is extracted from Ferula tschimganica roots and exhibits estrogenic, insecticidal, and fungicidal activities. [220,221]TMN extends CLS in S. pombe, and subsequent analysis revealed that activation of Sty1 is important for extending CLS by TMN. [222,188]The analogs of TMN, -hibitakanine, -hibitakanine, and mochimganine, also extend CLS in S. pombe, and Sty1 is required for full CLS extension by these drugs. [222,223]Although TMN and hibitakanine are not effective in extending CLS in S. cerevisiae, hibitakanine extends CLS through an unknown mechanism. [222]

Prostaglandin J 2
Prostaglandin belongs to a subclass of eicosanoids and is an important lipid mediator involved in the inflammatory response. [224]PGJ 2 extends CLS in S. pombe. [188]and this may be mediated by suppressing the PKA pathway because longevity was not observed in Δgit3 and Δpka1 cells. [188]In addition, because this extension requires Dnm1, an outer mitochondrial membrane GTPase required for mitochondrial fission. [225]GJ 2 -induced CLS extension may be related to mitochondrial fission. [188]However, although other prostaglandins (PGA 1 , PGB 1 , PGE 1 , PGE 2 , and PGF 2 ) were also investigated, none of them extended CLS. [188]tudies using various model organisms have shown that decreased protein synthesis, decreased translation, and increased fidelity of protein synthesis slow aging and extend lifespan. [226,124,227,34,131]Signaling pathways involved in CLS extension also influence translational regulation.For example, the TOR and PKA pathways regulate ribosomal gene transcription in S. cerevisiae. [228]and the TOR inhibitor Torin-1 reduces global protein translation in S. pombe. [186]In S. pombe, deletion strains of several ribosomal proteins or ribosomal RNAs extend CLS. [229,59]imilarly, CLS extension by drugs involved in ribosome biosynthesis has been confirmed.

Diazaborine and Ribozinoindole-1
Diazaborine and ribozinoindole-1 (Rbin-1) suppress ribosomal biogenesis and extend CLS in S. pombe. [59,230]Diazaborine prevents ribosomal biogenesis by inhibiting AAA-ATPase Drg1, which is necessary for the maturation of the 60S ribosomal subunit. [231]Rbin-1 reduces ribosomal biogenesis by inhibiting AAA-ATPase midasin (Mdn1), which is involved in 60S subunit assembly. [232,233]Although both diazaborine and Rbin-1 can significantly extend CLS, Rbin-1 has minimal effect on cell growth, whereas diazaborine affects cell growth in a concentrationdependent manner. [230]However, antibiotic G418, which inhibits protein synthesis, did not cause markedly extend CLS as did diazaborine or Rbin-1. [230]lthough no clear intracellular targets are known in fission yeast, several compounds can extend CLS in fission yeast and the lifespan in other model organisms.Conversely, although extension of CLS in fission yeast has been induced by several lowmolecular weight compounds, this has not been demonstrated in other organisms.

Plumbagin
The botanical naphthoquinone pesticide plumbagin extends CLS in S. pombe. [188]Low concentrations of plumbagin extend the lifespan of C. elegans, and SKN-1 activity is required for this. [179]n addition, menadione, a naphthoquinone similar to plumbagin, extends CLS in S. cerevisiae. [160]but does not extend the lifespan of C. elegans. [179]

Mycophenolic Acid and Acivicin
Mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH), which controls the rate of GMP synthesis in the de novo pathway of purine synthesis, extends CLS in S. pombe. [188]MPA reportedly extends RLS in S. cerevisiae by inhibiting GMP synthesis. [165]Similar to MPA, acivicin, a glutamine analog that selectively and irreversibly abolishes the glutaminase activity of GMP synthetase by covalent modification, extends CLS in S. pombe. [188]Therefore, similar to RLS in budding yeast, CLS extension by MPA or acivicin may be related to the inhibition of GMP synthesis, but no additional verification results have been reported. [188]

Rotenone
Although Rotenone, a mitochondrial complex I inhibitor, moderately increases CLS in S. pombe. [188]this reduces the lifespan due to toxicity in C. elegans. [234]n fission yeast, CLS is influenced by membrane proteins involved in the intracellular pH maintenance system. [235,188]Several drugs that affect cellular pH regulation have been reported to extend CLS.

Monensin and Nigericin
Monensin and nigericin have similar structures and properties. [236]and extend CLS in S. pombe by maintaining the acidic pH of the vacuole and suppressing vacuolar fragmentation. [188]Monensin transports metal cations through lipid membranes and acts as an antiporter for Na.+ H. + . [237,238]he CLS extension effects of monensin and nigericin are lost in strains lacking a V-ATPase, such as Vma1 and Vma3. [188]12.Vanadate Vanadate, an inhibitor of the P-type ATPase family.[239] extends CLS, probably because of the inhibition of the action of P-type ATPase Pma1.[235] Different Pma1 mutations can extend CLS in S. pombe.[235,17] Although a few low-molecular weight components have been reported that extend CLS in fission yeast, other information is limited. Thereore, future studies are expected to elucidate the mechanism of CLS regulation in fission yeast and the lifespan regulation in other organisms.

Actinomycin D
Actinomycin D, a chromopeptide anticancer drug, extends CLS in S. pombe. [195,59]Actinomycin D exerts anticancer activity through tight binding with DNA and subsequent inhibition of transcription elongation by blocking RNA polymerase. [240]

3,3′-Diindolylmethane
A compound derived from the digestion of indole-3-carbinol, found in the broccoli family, 3,3′-diindolylmethane (DIM) induces autophagy and extends CLS in S. pombe. [241,188]Autophagy is an important cellular process for maintaining a sufficient CLS in yeast and lifespan of higher organisms, but whether autophagy is involved in DIM-induced CLS extension has not been verified.

Conclusion
Currently, over 60 drugs extend CLS in either budding yeast or fission yeast (Figures 1 and 2).Among them, multiple lowmolecular weight compounds have been analyzed for lifespan not only in yeast but also in multicellular model organisms, and several drugs that extend lifespan across different species have been identified (Figure 3).Many drugs that mediate lifespan in multiple organism do so by regulating the TORC1 pathway, autophagy, and protein translation.These pathways and processes can be adjusted through genetic mutations and nutritional environment management, and through such modulation, it is possible to regulate lifespan.However, drug treatment remains the simplest and most effective way to control the lifespan of living organisms.Currently, among these lifespan-controlling drugs, rapamycin appears to be the most well-analyzed, reliable, and effective lifespan-extending drug for a variety of organisms.However, TORC1 kinase, which rapamycin directly affects, is located upstream of the intracellular signal transduction pathway and mediates many cellular processes in addition to lifespan regulation.Therefore, as a lifespan control drug, rapamycin is limited by acting on many other cellular processes.Future drug-based lifespan analysis may provide an alternative to rapamycin with fewer side effects and more reliable lifespan-extending agents.CLS research using both S. cerevisiae and S. pombe, which take advantage of their ease of handling and speed of research, is expected to contribute to the search for drugs more effective than rapamycin and to the elucidation of the mechanisms of effect of various lifespan-extending drugs.Furthermore, although several CLS studies have been conducted to date, complete control of CLS remains achieved.Interventions in CLS using drugs will bring about various knowledge about CLS, thereby contributing to understanding of its essence.

Figure 1 .
Figure 1.Low-molecular weight compounds that extend the chronological lifespan of S. cerevisiae.Many compounds extend CLS in S. cerevisiae.The action of each drug is described in the text.The effects of drugs and intracellular factors are indicated by red and green arrows, respectively.The colors of atoms in the molecular model of each low-molecular weight compound are: hydrogen, silver; carbon, black; nitrogen, blue; oxygen, red; sulfur, yellow; and selenium, purple.Compounds that have been confirmed to extend lifespan in other model organisms are written in bold.

Figure 2 .
Figure 2. Low-molecular weight compounds that extend the chronological lifespan of S. pombe.Many compounds extend CLS in S. pombe.The action of each drug is described in the text.The effects of drugs and intracellular factors are indicated by red and green arrows, respectively.The colors of atoms in the molecular model of each low-molecular weight compound are: hydrogen, silver; boron, magenta; carbon, black; nitrogen, blue; oxygen, red; sulfur, yellow; fluorine, lime; vanadium, cyan; and chlorine, olive.Compounds that have been confirmed to extend lifespan in other model organisms are written in bold.

Figure 3 .
Figure 3. Various compounds that extend CLS in yeast cause lifespan extension in multicellular organisms.References in black indicate those where lifespan (for yeasts, CLS) extension was observed.Conversely, references that include results where lifespan extension was not observed are shown in red.