Finding Ponce de Leon’s Pill: Challenges in Screening for Anti-Aging Molecules

The mTOR inhibitor rapamycin

mTOR is a conserved serine/threonine kinase that senses and responds to nutrient availability, growth factors, and environmental stress and plays a key role in triggering growth^6,29. In multicellular eukaryotes, mTOR exists in two distinct multi-protein complexes, mTORC1 and mTORC2, distinguished by their association with regulatory-associated protein of mTOR (RAPTOR) and rapamycin-insensitive companion of mTOR (RICTOR), respectively^30,31. Rapamycin forms a complex with the FKBP12 protein, which binds to mTORC1 and inhibits its activity³². Importantly, chronic treatment with rapamycin also inhibits mTORC2³³. mTORC1 activity is regulated by nutrients (glucose and amino acids), cytokines, hormones (insulin or IGF1), energy (ATP levels), and oxidative stress via PI3K, AKT, and AMPK signaling⁶. Key downstream mediators of mTORC1 signaling are pathways that control cell growth, proliferation, stress response, and autophagy^29,34. mTORC1, therefore, critically integrates cellular growth and maintenance with nutrient availability, hormonal cues, and other environmental stimuli.

A number of studies have established a link between mTOR signaling pathways and longevity in organisms ranging from yeast to mammals. Inhibition of mTOR signaling by genetic or pharmacologic means extends lifespan in yeast^35–37, nematodes^38,39, fruit flies⁴⁰, and mice^33,41–47. Likewise, genetic deletion in mice of the downstream mTORC1 effector, S6 kinase 1, increases oxidative metabolism, protects against age- and diet-induced obesity, and increases female lifespan^47,48. Consistently, enhanced activity of the mTORC1 target 4E-BP1 in skeletal muscle results in increased oxidative metabolism and protects mice from diet- and age-induced metabolic dysfunction⁴⁹.

In a landmark study, NIA’s Interventions Testing Program (ITP) showed that treatment of a genetically heterogeneous mouse stock with the mTOR inhibitor rapamycin (administered at 14 mg/kg food; 2.24 mg/kg body weight/day) initiated at either 9 months or 20 months of age extended lifespan in both sexes^43,50. A follow-up study demonstrated that the increase in mouse lifespan induced by rapamycin is dose and sex dependent. At a given chow concentration of rapamycin, female mice showed a greater increase in lifespan than did males, which correlated with higher blood levels of rapamycin achieved in females relative to males⁵¹. Rapamycin treatment induced entirely distinct gene expression changes in males and females, implying the existence of sex-specific responses to mTOR inhibition⁵¹. Furthermore, the expression patterns of xenobiotic-metabolizing enzymes in the livers of rapamycin-treated (14 mg/kg food) mice differed strikingly from those in DR-exposed animals at 12 months of age⁵¹. Indeed, DR is less effective in lifespan extension when initiated later in life^52–54, while rapamycin treatment extends the lifespan of mice, even when started in middle age^43,55. Crucially, rapamycin-induced lifespan extension in mice has also been observed in diverse genetic backgrounds^41,42,44,56.

Mechanisms of longevity extension by rapamycin remain a hotly debated topic in aging biology^56,57. Rapamycin has anti-neoplastic properties^58–60, and cancer is the major cause of death in most mouse strains that show rapamycin-mediated lifespan extension^43,61. In this context, one plausible explanation for the extension of mouse lifespan by rapamycin is that this drug suppresses the onset and/or aggressiveness of lethal cancers. However, some investigators have reported that rapamycin also inhibits age-associated phenotypes besides neoplasia^62,63, strongly suggesting that this drug has broader anti-aging effects. In contrast, a recent exhaustive study by Neff et al. claimed that the effects of rapamycin on aging phenotypes per se were quite limited⁵⁶. In this regard, conflicting observations have been made concerning the effects of rapamycin treatment in mouse models of AD⁶⁴. Long-term rapamycin treatment led to behavioral improvements in mouse AD models and induced an autophagy-mediated decrease in Aβ and hyperphosphorylated tau levels^65,66. Conversely, rapamycin has been shown to promote Aβ production^67,68 and led to an increase in Aβ-induced cell death⁶⁹.

Rapamycin has significant side effects – metabolic dysfunction, cataract, and testicular atrophy in particular – that may limit its long-term utility as an anti-aging treatment in humans^70,71. Most importantly, due to the immunomodulatory effects of mTOR inhibitors, treatment of human patients with the rapamycin-like drug everolimus/RAD001 is associated with a higher incidence of infection in individuals with diseases such as cancer^72,73 and tuberous sclerosis complex (TSC)⁷⁴. Conversely, a recent study showed that short-term administration of everolimus/RAD001 to healthy older individuals enhanced the immunological response to influenza vaccination, with modest side effects⁷⁵. Decreased influenza vaccine response is a major clinical challenge in older populations⁷⁶. These findings suggest that intermittent or short-term administration of rapamycin or other mTOR inhibitors might suppress certain functionally important effects of aging, such as poor immunization response, while avoiding the negative consequences associated with chronic use of these agents. A recent study in mice is consistent with this view, identifying an intermittent rapamycin administration regimen in mice that minimizes metabolic dysfunction, while maintaining chronic mTORC1 suppression in adipose tissue, though not in other tissues⁷⁷. It will be of great interest to evaluate the effects of such intermittent dosing regimens on a wide range of age-associated phenotypes and on lifespan.

Metformin and other biguanides

Metformin, an oral biguanide antiglycemic agent, is the most widely used drug in the treatment of metabolic syndrome and T2D. Metformin’s mechanism of action is not completely understood and is likely to be multi-factorial. It was reported to decrease serum glucose levels by inhibiting respiratory chain Complex I in hepatocytes⁷⁸, resulting in reduced ATP production, leading to activation of the LKB1 and AMPK kinases, suppressing hepatic gluconeogenesis^79,80. Metformin has been reported to activate AMPK in many other tissues, including adipose, skeletal muscle, heart, pancreatic β-cells, and hypothalamus with potential beneficial physiological effects in patients with T2D^81,82. However, metformin also exerts important effects independent of AMPK and LKB1⁸³, e.g. by antagonizing the action of glucagon⁸⁴. Recently, another AMPK-independent mechanism has been revealed for metformin. A study by Madiraju et al. showed that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, increasing the cytosolic redox state and decreasing the mitochondrial redox state⁸⁵. This suppresses hepatic gluconeogenesis by reducing the conversion of lactate and glycerol to glucose⁸⁵. Although metformin is currently approved for treatment of T2D, a large literature suggests efficacy of metformin against other conditions, particularly cardiovascular diseases and cancer⁷⁸. In this regard, a recent study demonstrated that metformin reduces tumorigenesis by inhibiting mitochondrial Complex I in cancer cells⁸⁶.

AMPK activation provokes longevity in flies and worms^87,88. A number of studies suggest that metformin treatment can recapitulate some effects of DR. In this context, several studies have examined the effects of metformin and other biguanides on lifespan and reported a variety of outcomes. Metformin and other biguanides extend C. elegans lifespan in a dose-dependent manner^89–91. The increase in C. elegans lifespan by metformin is mediated through inhibition of bacterial folate and methionine metabolism, which in turn alters methionine metabolism in the worm, resulting in reduced S-adenosylmethionine and increased S-adenosylhomocysteine levels⁸⁹. However, metformin apparently does not extend longevity in D. melanogaster^92,93. Indeed, despite robust activation of AMPK, high doses of metformin actually decrease lifespan of both male and female flies⁹³, perhaps due to disruption of intestinal fluid homeostasis⁹³. However, metformin treatment suppressed age-related phenotypes in intestinal midgut stem cells⁹⁴ and also exerted beneficial effects in a fly obesity model⁹⁵. A recent study showed that metformin treatment causes a significant extension in mean and maximal lifespan in both sexes of the cricket Acheta domesticus⁹⁶.

Several studies have been performed in rodents to test the effects of metformin and other biguanides on lifespan; the outcomes have varied with genotype, sex, and dose and duration of treatment⁹⁷. Chronic treatment with metformin (100 mg/kg in the drinking water) enhanced the mean lifespan of cancer-prone HER-2/neu transgenic, outbred SHR, and inbred 129/Sv female mice by 8% (p<0.05), 37.8% (p<0.01), and 4.4% (p<0.05), respectively^98–100. Metformin treatment also extended the maximum lifespan of HER-2/neu transgenic and outbred SHR female mice by 9% and 10.3%, respectively, while no effect was observed on maximal lifespan in inbred 129/Sv female mice^98–100. Conversely, treatment of inbred 129/Sv male mice with a similar dose of metformin actually reduced mean lifespan by 13.4%¹⁰⁰. However, metformin treatment (2 mg/mL in drinking water) in a transgenic mouse model of Huntington disease (HD) prolonged male mean lifespan by 20.1% (p=0.017), but did not affect female survival¹⁰¹. It has been reported that metformin treatment (100 mg/kg in the drinking water) of female outbred SHR mice initiated at 3 months of age induced a trend towards increased mean lifespan¹⁰². Metformin treatment also postponed the onset of detectable tumors when started at young or middle ages, but not at old age¹⁰². Neonatal metformin treatment of 129/Sv mice (100 mg/kg via subcutaneous injection) led to a 20% (p<0.001) increase in male mean lifespan and also slightly increased maximum lifespan by 3.5%¹⁰³. However, in females, the mean and maximum lifespan in metformin-treated groups were decreased by 9.1% and 3.8%, respectively¹⁰³. In a recent study by Martin-Montalvo et al., male C57BL/6 mice supplemented with 0.1% metformin in the diet showed a 5.8% increase in mean lifespan (p=0.02, Gehan–Breslow survival test), whereas supplementation with 1% metformin was toxic and reduced mean lifespan by 14.4%¹⁰⁴. However, supplementation of B6C3F1 male mice with 0.1% metformin resulted in extension of mean lifespan only by 4.2% (p=0.064, Gehan–Breslow)¹⁰⁴. Treatment with another biguanide, phenformin (2 mg/mouse in 0.2 mL of drinking water), significantly reduced spontaneous tumor development in female C3H/Sn mice and prolonged mean lifespan by 21% or more (p<0.05)^105,106 and maximum lifespan by 26%¹⁰⁵. Evaluation of the lifespan effects of metformin in mice by the ITP consortium is ongoing, and the results should be available soon.

In rats, buformin treatment (5 mg/rat in 1 mL of drinking water) led to a non-significant 7.3% increase in mean lifespan of female LIO animals, while phenformin (5 mg/rat in 1 mL of drinking water) had no effect¹⁰⁵. However, administration of both buformin and phenformin increased the maximum lifespan of female LIO rats by 5.5% and 9.8%, respectively¹⁰⁵. Treatment with metformin (300 mg/kg/day) did not increase either mean or maximum lifespan of male F344 rats¹⁰⁷. However, in the same report, a parallel group of male F344 rats exposed to DR also failed to exhibit lifespan extension¹⁰⁷, leaving the metformin results in this study somewhat inconclusive. Mechanistically, treatment with metformin has been proposed to mimic some effects of DR, in particular by increasing AMPK activity and also activating antioxidant responses, leading to a reduction in both oxidative damage accumulation and chronic inflammation¹⁰⁴.

Although no study has formally analyzed the effects of long-term metformin treatment on lifespan in healthy humans, randomized clinical trials of metformin showed beneficial effects on health and survival in overweight/obese patients with T2D, as shown by decreased incidences of cardiovascular disease and cancer and reduced overall mortality^108–110. However, when combined with sulfonylurea, metformin increased the risk of diabetes-related death and all-cause mortality in a mixed group of non-overweight and overweight/obese individuals with T2D^78,108. Consistent with these observations, a recent study by Bannister et al. reported that patients with T2D treated with metformin displayed improved survival compared to matched, non-diabetic controls, whereas those treated with sulfonylureas showed reduced survival¹¹¹.

Given the relatively promising rodent data, the hints that metformin might suppress cancer and other age-associated conditions in humans, and metformin’s relatively benign safety profile, there is great current interest in formally testing the ability of this drug to delay age-associated disease in humans¹¹². Indeed, the US Food and Drug Administration (FDA) recently approved a study termed Targeting Aging With Metformin (TAME) for the evaluation of metformin as an anti-aging drug. The TAME project will involve approximately 3000 participants between the ages of 70 years and 80 years who either already have one, two, or all three of the conditions: cancer, heart disease, or cognitive impairment or are at risk of developing them. The trial will take place at roughly 15 centers around the United States over 5–7 years, costing approximately $50 million¹¹³. The goal of the study is to determine whether metformin can prevent the onset of age-associated disease. This landmark trial will represent the first testing of a candidate anti-aging compound in humans.

Resveratrol and other sirtuin-activating compounds

The sirtuins are a family of NAD⁺-dependent deacetylases/ADP-ribosyltransferases/deacylases implicated in regulating nutrient responses and numerous other aspects of cell biology⁸. Overexpression of Sir2, the founding member of the sirtuin family, extends replicative lifespan in the budding yeast Saccharomyces cerevisiae by repressing the accumulation of extrachromosomal rDNA plasmids, promoting segregation of an undamaged proteome to the daughter cell, enforcing subtelomeric silencing, and perhaps other mechanisms^114,115. Several, though not all, investigators have found that overexpression of sirtuins in worms and flies modestly increases lifespan in these organisms^116–123. Interestingly, the Sir2 homolog Sir-2.1 can extend C. elegans lifespan in a manner independent of its deacetylase activity¹¹⁶. Indeed, nicotinamide (NAM), a product of sirtuin activity, and its metabolite, 1-methylnicotinamide (MNA), are capable of extending worm lifespan, potentially by inducing transient ROS signaling¹¹⁶. In mammals, SIRT1 is the closest Sir2 homolog; overexpression of this protein in the brain (but not the whole organism) extends lifespan¹²⁴, probably by enhancing hypothalamic function during aging¹²⁵. Global overexpression of another sirtuin, SIRT6, extends mouse lifespan in males specifically, at least in part via suppression of lung cancer, a major cause of death in males of the mouse stock used^126,127. SIRT2 overexpression stabilizes levels of the mitotic checkpoint protein BubR1 in progeroid BubR1^H/H mice and extends both median and maximum lifespan in male mice of this strain¹²⁸. No information is available concerning the potential effects of chronic SIRT2 overexpression in WT animals. Accumulating evidence suggests that NAD⁺ levels may decline during aging, impairing sirtuin activity, and that the ability of sirtuin overexpression to increase lifespan partially counters this effect by maintaining sirtuin function in the face of a diminished NAD⁺ pool in older organisms¹²⁹.

Resveratrol and certain other polyphenols were originally identified as Sir2/SIRT1 activators that extended the average and maximal lifespan of yeast¹³⁰. It is important to note that resveratrol is a highly promiscuous drug and exerts functionally important effects on many cellular targets¹³¹. Treatment of worms and flies with resveratrol (dosed at 100 μM in worms and 10–100 μM in flies) has also been reported to extend lifespan, dependent on the presence of functional Sir-2.1 and dSir2, respectively¹³². However, a study by Bass et al. claimed that resveratrol treatment (1–1000 μM) had no significant effects on Drosophila lifespan¹³³. The same study also reported that resveratrol treatment at 100 μM induced only a slight and sporadic increase in C. elegans lifespan in both WT and sir-2.1 mutant animals, suggesting that these small increases in C. elegans lifespan induced by resveratrol may be Sir-2.1 independent¹³³. Resveratrol protects worms from oxidative stress, radiation-induced damage, and amyloid toxicity^134–136 and also induces radioprotection in flies¹³⁷. Resveratrol treatment increases mean and maximum lifespan in the honeybee¹³⁸ and the short-lived fishes Nothobranchius furzeri and Nothobranchius guentheri^139–141.

It was reported that resveratrol and other sirtuin-activating compounds (STACs) activate Sir2/SIRT1 allosterically¹³⁰. However, other groups have found that these compounds were unable to enhance SIRT1 activity towards native peptides in vitro^142,143. In this context, it has been suggested that increased SIRT1 activity induced by resveratrol depends on the presence of a non-native fluorophore conjugated to the peptide sequence originally used in screening for SIRT1 activators^142,143. Recent reports, however, have shown that resveratrol and other STACs directly bind to SIRT1 and allosterically enhance its deacetylase activity towards non-tagged peptide substrates^144,145. Resveratrol has also been reported to inhibit the catalytic activity of human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS), resulting in its nuclear translocation and stimulation of NAD⁺-dependent activation of poly (ADP-ribose) polymerase 1 (PARP1)¹⁴⁶. PARP1 plays important roles in both DNA repair and transcription¹⁴⁷.

In mice, resveratrol is protective against some damaging effects of high-fat/high-calorie diets^148–151, substantially reduces the growth and development of multiple types of cancers^152–154, and delays or prevents the onset of AD^155,156. Moreover, in rodents and humans, resveratrol is protective against both type 1 diabetes and T2D^157,158 and cardiovascular disease¹⁵⁹ and possesses anti-inflammatory¹⁶⁰ and anti-viral activities¹⁶¹. Resveratrol supplementation (either at 0.016–0.1% of diet or 25 mg/kg/day) has been reported to increase lifespan in mouse models of obesity¹⁴⁸, AD¹⁶², HD¹⁶³, and amyotrophic lateral sclerosis^164,165. Resveratrol treatment (2–8 mg/kg/day) increases the lifespan of LPS-treated mice¹⁶⁶ and attenuates catecholamine-induced mortality in obese rats (20 mg/kg/day)¹⁶⁷. Furthermore, resveratrol (10 mg/mL, intraperitoneal injection) prolongs survival in a mouse model of sepsis-induced acute kidney injury and restores renal microcirculation¹⁶⁸. Resveratrol administration (18 mg/kg/day in the diet) also improves survival in a rat hypertension model¹⁶⁹. Importantly, however, resveratrol treatment (100–1200 mg/kg food) does not increase lifespan in normal chow-fed mice^50,170,171. Resveratrol supplementation induces gene expression changes in several tissues that resemble those associated with calorie restriction in mice^171,172.

In humans, 30-day resveratrol supplementation (150 mg/day) in obese men induced metabolic changes, including reductions in sleeping and resting metabolic rate, intrahepatic lipid content, circulating glucose levels, inflammatory markers, and systolic blood pressure¹⁷³. Skeletal muscle from resveratrol-treated objects displayed increased AMPK activity, increased SIRT1 and PGC-1α protein levels, and improved mitochondrial respiration of fatty acids¹⁷³. In contrast, 12 weeks’ supplementation with resveratrol (75 mg/day) in non-obese, postmenopausal women with normal glucose tolerance induced no apparent change in body composition, insulin sensitivity, resting metabolic rate, plasma lipids, or inflammatory markers¹⁷⁴. Moreover, resveratrol supplementation had no effect on its putative molecular targets, including AMPK, SIRT1, NAMPT, and PPARGC1A, in either skeletal muscle or adipose tissue¹⁷⁴.

An important recent study by Cai et al. demonstrated a non-linear dose response for the protective effects of resveratrol in humans and mice¹⁷⁵. When co-administered with high-fat diet (HFD), low-dose resveratrol (~0.07 mg/kg/day) appeared to be more efficacious than high-dose (14 mg/kg/day) in reducing adenoma number and decreasing overall tumor burden in Apc^min mice, a model of intestinal carcinogenesis. Interestingly, female mice on the lower dose of resveratrol exhibited significantly higher expression and activation of AMPK in intestinal mucosa than those in the high-dose group¹⁷⁵. Consistently, human colorectal tissues exposed to low dietary concentrations (0.01 to 0.1 μM) of resveratrol ex vivo displayed rapid AMPK activation and increased autophagy at low concentrations and a less pronounced or even no effect at higher doses (1 to 10 μM)¹⁷⁵. This unusual effect may help rationalize the conflicting reports of resveratrol’s efficacies in humans, and future human studies using resveratrol must be designed with careful attention paid to dosage and serum levels and to a thorough assessment of effects on resveratrol’s putative molecular targets.

Other STACs have been synthesized and are reported to enhance healthspan and extend lifespan in mice. The STAC SRT1720 (100 mg/kg/day) has been reported to extend mean lifespan of adult male C57BL/6J mice fed a standard diet by 8.8% (p=0.096), and up to 21.7% (p=0.0193) on a HFD, without increasing maximal lifespan in either context^176,177. SRT1720 treatment improved physiological parameters in HFD-fed animals, reducing liver steatosis, increasing insulin sensitivity, enhancing locomotor activity, and also inducing a gene expression profile similar to that associated with a standard diet¹⁷⁶. SRT1720 supplementation inhibited pro-inflammatory gene expression in liver and muscle of mice fed a standard chow diet and delayed the onset of age-related metabolic disease¹⁷⁷. Similarly, dietary supplementation (100 mg/kg) with SRT2104, another synthetic STAC, increased both mean and maximal lifespan of male C57BL/6J mice fed a chow diet by 9.7% (p<0.05) and 4.9% (p<0.001), respectively, and increased insulin sensitivity and motor coordination while reducing inflammation¹⁷⁸. Short-term treatment with SRT2104 preserves bone and muscle mass in an experimental atrophy model¹⁷⁸. These findings indicate that resveratrol and other STACs can exert beneficial effects on health, particularly in the context of HFD, and that some STACs can modestly extend lifespan under normal feeding conditions; however, additional studies are warranted to better evaluate their effects on longevity in females and other strains of mice. In this regard, there is great current interest in evaluating the effects of NAD⁺ precursors as therapies for metabolic disease and candidate anti-aging drugs¹²⁹.

Other potential candidate anti-aging drugs

In recent decades, numerous compounds with pro-healthspan and -longevity effects have been identified. Due to space limitations, we restrict our discussion to a few key small molecules that have shown beneficial effects, from invertebrate models to mice (Figure 2).

Figure 2. Pharmacological interventions targeting aging-related pathways and processes.

Representative compounds (yellow boxes) target various processes or pathways that contribute to aging and either promote or suppress their activities/progression, resulting in improved health and enhanced lifespan.

Spermidine is a member of the polyamine family, involved in numerous critical cellular processes including DNA stability, transcription, translation, apoptosis, cell proliferation, and cell growth¹⁷⁹. In multiple organs, levels of polyamines have been reported to decline with age^180,181. Indeed, a study by Pucciarelli et al. suggested that maintaining high levels of spermidine during aging might promote longevity¹⁸². Administration of exogenous spermidine extended the lifespan of yeast, flies, worms, and cultured human peripheral blood mononuclear cells¹⁸³. Spermidine also reduces the age-related decline of locomotor performance in flies¹⁸⁴. Furthermore, it has been reported that a polyamine-rich diet reduced age-related pathology and increased lifespan in Jcl:ICR male mice¹⁸⁵. Conversely, depletion of endogenous spermidine by genetic manipulation of the polyamine pathway shortens lifespan in yeast¹⁸³ and mice¹⁸⁶. Spermidine supplementation reduces levels of age-related oxidative damage in mice¹⁸³ and also increases stress resistance in yeast¹⁸³ and flies¹⁸⁷. The beneficial effects of spermidine are mediated mainly via induction of autophagy^183,187, allowing the regulated degradation and recycling of dysfunctional cellular components¹⁸⁸. Defective autophagy prevented the onset of spermidine supplementation-associated benefits^183,187.

Aspirin, a derivative of salicylic acid, is the prototypical cyclooxygenase inhibitor and non-steroidal anti-inflammatory agent¹⁸⁹. Aspirin is a versatile drug, with antithrombotic and antioxidant properties^190,191. Indeed, chronic aspirin use in humans reduces the risk of mortality from a variety of age-associated diseases, including atherosclerosis, diabetes, and a variety of cancers^192–196. Aspirin use has been reported to be associated with increased survival in extreme old age in humans¹⁹⁷. In a recent study by Ayyadevara et al., aspirin was shown to upregulate the expression of antioxidant genes (superoxide dismutase, catalases, and glutathione-S-transferases), resulting in attenuation of endogenous ROS levels and extension of C. elegans lifespan¹⁹⁸. Another study showed that aspirin treatment leads to lifespan extension in the cricket A. domesticus⁹⁶. In studies by the ITP, aspirin treatment (21 mg/kg diet) led to an increase in the mean lifespan of male mice, but there was no effect in females¹⁹⁹.

Nordihydroguaiaretic acid (NDGA), also known as masoprocol, is a naturally occurring dicatechol, with antioxidant, antiviral, antineoplastic, and anti-inflammatory activities²⁰⁰. It has been reported to be a potent antagonist of the inflammatory cytokine TNFα. Dietary administration with NDGA delayed motor deterioration in a mouse model of amyotrophic lateral sclerosis and significantly extended lifespan²⁰¹. Consistently, the ITP reported that NDGA (2500 mg/kg diet) increased the lifespan of UM-HET3 male mice^199,202. Lifespan extension by NDGA was not observed in female mice, even at a dose that produced blood levels equivalent to those in males²⁰². One possible explanation for this sex discrepancy could be that male controls in this study showed a somewhat short lifespan at two of the three ITP testing sites²⁰². Additional studies will be required to fully address this issue.

Acarbose is an inhibitor of α-glucosidases, intestinal enzymes that convert complex carbohydrates into simple sugars to facilitate their absorption²⁰³. Acarbose treatment thus impairs carbohydrate digestion and inhibits the normal postprandial glucose rise²⁰³. The ITP found that acarbose administration (1000 mg/kg diet) induced a significant increase in median and maximal lifespan in both sexes, although the impact was much more pronounced in males²⁰². Acarbose treatment increased male median lifespan by 22% (p<0.0001), but female median lifespan by only 5% (p=0.01). Similarly, maximum lifespan extension in males and females was 11% (p<0.001) and 9% (p=0.001), respectively²⁰². Acarbose-treated mice had a significant increase in levels of serum fibroblast growth factor 21 (FGF21) and also a mild reduction in IGF1 levels²⁰². FGF21 plays important roles in the regulation of glucose, lipid, and energy homeostasis²⁰⁴. Transgenic mice with constitutive FGF21 secretion displayed an increase in both mean and maximal lifespan, probably occurring via reduced IIS^205,206.

17-α-estradiol is a non-feminizing estrogen, with reduced binding affinity for estrogen receptors²⁰². It inhibits the activity of the enzyme 5α-reductase, responsible for the reduction of testosterone to the more potent androgen dihydrotestosterone²⁰⁷, which has higher affinity for the androgen receptor than does testosterone²⁰⁸. 17-α-estradiol has been reported to be neuroprotective against cerebral ischemia, Parkinson’s disease, and cerebrovascular disease^209–211. Recently, it has been shown to diminish metabolic and inflammatory impairment in old male mice by reducing calorie intake and altering nutrient sensing and inflammatory pathways in visceral white adipose tissues, without inducing feminization²¹². In ITP studies, administration of 17-α-estradiol (4.8 mg/kg diet) from 10 months of age increased male median lifespan by 12%, without significant effect on maximum lifespan or effects on female lifespan²⁰². Similar to NDGA, the relatively short lifespan of male controls might contribute to this apparent sex discrepancy²⁰² and further longevity studies are warranted using this drug.

β-adrenergic receptor (β-AR) antagonists bind to β-ARs (β1, 2, and 3-AR) and block the action of the endogenous catecholamines epinephrine and norepinephrine. Increased activity of β-ARs may hasten the development of age-related pathologies and increase mortality in genetically modified mice^213–218. Consistently, chronic administration of β-AR agonists leads to increased mortality and morbidity²¹⁹. In humans, increased production of β2-AR due to specific genetic variants is associated with reduced lifespan²²⁰. Conversely, dietary administration of β-AR blockers metoprolol (1.1 g/kg in the diet) and nebivolol (0.27 g/kg in the diet) increased the median lifespan of C3B6F1 male mice by 10% (p=0.016) and 6.4% (p=0.023), respectively, without affecting food intake or utilization²²¹. However, no effect was observed on maximal lifespan. Consistently, treatment with metoprolol (5 mg/mL diet) and nebivolol (100 μg/mL diet) extended the median lifespan of Drosophila by 23% (p≤0.0001) and 15% (p≤0.001), respectively, without impact on food intake or locomotion²²¹. Similar to β-AR blockers, an α1-AR antagonist, doxazosin mesylate, which inhibits the binding of norepinephrine to α1-AR on the membrane of vascular smooth muscle cells, extends C. elegans lifespan by 15%²²². Given that some of these agents are routinely administered clinically as antihypertensives and their safety profiles are well characterized, they may warrant further evaluation in humans specifically for their potential anti-aging effects.

Antioxidants, compounds conferring resistance to oxidative stress, have in some cases also proven successful in increasing lifespan, particularly in lower organisms. Dietary supplementation with the glutathione precursor N-acetylcysteine (NAC) increased resistance to oxidative stress, heat stress, and UV irradiation and significantly extended both the mean and the maximum lifespan of C. elegans²²³ and D. melanogaster²²⁴. Furthermore, treatment with EUK-134 and EUK-8, small molecule synthetic catalytic mimetics of superoxide dismutase (SOD) and catalase, was reported to extend C. elegans lifespan²²⁵; however, as discussed by Gems and Doonan, other groups have not observed this effect²²⁶. Treatment of a mixed group of male and female C57BL/6 mice with another SOD mimetic, carboxyfullerene (C3, at 10 mg/kg/day), reduced age-associated oxidative stress and mitochondrial superoxide production and modestly extended mean lifespan²²⁷. Consistently, oral administration of carboxyfullerene (C60; 4 mg/kg/day) dissolved in olive oil to male Wistar rats leads to a 90% increase in median lifespan as compared to water-treated controls²²⁸. Similarly, some other studies have shown an ability of antioxidants to extend lifespan in multiple organisms^229,230.

Conversely, there are many reports that do not support the idea that dietary supplementation with antioxidants can increase the lifespan of healthy animals or humans as a general rule. Dietary supplementation with either vitamin E (α-tocopherol) or vitamin C (ascorbic acid) significantly shortened the lifespan of short-tailed field voles²³¹. Similarly, treatment of male mice with a nutraceutical mixture enriched in antioxidants was ineffective in extending lifespan²³². Moreover, as described in a recent review by Bjelakovic et al., systematic review and meta-analyses of a large number of randomized clinical trials evaluating the effects of dietary supplementation with various anti-oxidants (β-carotene, vitamin A, vitamin C, vitamin E, and selenium) in humans did not reveal any overall benefit; indeed, in some cases, there was evidence for increased mortality occurring in response to these agents²³³. Deleterious effects of antioxidant supplementation may result from inappropriate suppression of the normal signaling functions ROS play in cells, including in crucial cell populations such as stem cells²³⁴.

Selective deletion of senescent cells by senolytic drugs

Cellular senescence refers to permanent cellular growth arrest, which can be induced by multiple stressors, including serial passage, telomere attrition, inappropriate mitotic stimuli, and genotoxic insult²³⁵. Senescence is thought to play an important role in tumor suppression in mammals^236,237. However, senescent cells develop an altered secretory phenotype (termed the SASP) characterized by the release of factors such as proteases, growth factors, interleukins, chemokines, and extracellular remodeling proteins²³⁸. With advancing age, senescent cells accumulate in various tissues^239–241 and potentially contribute to pathological states, as factors they secrete induce chronic inflammation, loss of function in progenitor cells, and extracellular matrix dysfunction^236,242. The functional impact of senescent cells in vivo has been a hotly debated topic in aging biology for many years. Recently, genetic approaches to delete senescent cells in mice have been described, via activation of a drug-inducible “suicide gene”²⁴³. Depleting senescent cells in a progeroid mouse model substantially delayed the onset of multiple age-related phenotypes, including lordokyphosis (a measure of sarcopenia in this model), cataract, loss of adipose tissue, and impaired muscle function²⁴³. However, the overall survival of these mice was not extended substantially by deletion of senescent cells, perhaps because the suicide gene was not expressed in the heart or aorta; cardiac failure is thought to represent a major cause of mortality in this strain²⁴³. A recent landmark study by Baker et al. showed that clearance of naturally occurring senescence cells in non-progeroid mice maintained the functionality of several organs with age, delayed lethal tumorigenesis, and extended median lifespan in mixed and pure C57BL/6 genetic backgrounds by 27% (p<0.001) and 24% (p<0.001), respectively²⁴⁴. This study provides very strong evidence that age-associated accumulation of senescent cells contributes to age-associated pathologies and shortens lifespan in WT animals.

Pharmacologic, as opposed to genetic, approaches to deplete senescent cells have posed a major technical and conceptual challenge. A recent study showed that senescent cells display increased expression of pro-survival factors, responsible for their well-known resistance to apoptosis²⁴⁵. Interestingly, small interfering RNA (siRNA)-mediated silencing of many of these factors (ephrins, PI3Kδ, p21, BCL-xL, and others) selectively killed senescent cells but left dividing and quiescent cells unaffected. These siRNAs were termed “senolytic” siRNAs²⁴⁵. Small molecules (senolytic drugs) targeting the same factors also selectively killed senescent cells. Out of 46 agents tested, dasatinib and quercetin were particularly effective in eliminating senescent cells. Dasatinib, used in cancer treatment, is an inhibitor of multiple tyrosine kinases²⁴⁶. Quercetin is a natural flavonol that inhibits PI3K, other kinases, and serpins^247,248. Dasatinib preferentially eliminated senescent human preadipocytes, while quercetin was more effective against senescent human endothelial cells and senescent bone marrow-derived murine mesenchymal stem cells (BM-MSCs). The combination of dasatinib and quercetin was effective in selective killing of senescent BM-MSCs, human preadipocytes, and endothelial cells²⁴⁵. The combination was more effective in killing senescent mouse embryonic fibroblasts compared to either drug alone. Treatment of chronologically aged WT mice, radiation-exposed WT mice, and progeroid Ercc1 hypomorphic mice with the combination of dasatinib and quercetin reduced the burden of senescent cells. Following drug treatment, old WT mice showed improved cardiac function and carotid vascular reactivity, irradiated mice displayed improved exercise capacity, and progeroid Ercc1^-/Δ mutants demonstrated delay of age-related symptoms and pathologies²⁴⁵. Similarly, a recent study by Chang et al. identified ABT263 (Navitoclax, a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as another potent senolytic agent²⁴⁹. ABT263, which is used for the treatment of multiple cancers^250–252, induced apoptosis and selectively killed senescent cells in a manner independent of cell type or species²⁴⁹. In culture, senescent human lung fibroblasts (IMR90), human renal epithelial cells, and mouse embryo fibroblasts (MEFs) were more sensitive to ABT263 treatment than their non-senescent counterparts²⁴⁹. In contrast, another study found that ABT263 is not a broad-spectrum senolytic; instead it acts in a cell type-specific manner²⁵³. In this study, ABT263 was found to be senolytic in human umbilical vein cells (HUVECs), IMR90 cells, and MEFs, but not in human primary preadipocytes²⁵³.

Treatment of either irradiated or naturally aged mice with ABT263 not only reduced the burden of senescent cells, including those among bone marrow hematopoietic stem cell (HSC) and muscle stem cell (MuSC) populations, but also suppressed the expression of several SASP factors and rejuvenated the function of aged HSCs and MuSCs²⁴⁹. These results, together with the impressive results obtained in genetic models described previously, indicate that senolytic drugs may have a role in improving tissue function during aging. However, some senolytic drugs are associated with toxic side effects, like thrombocytopenia and neutropenia in the case of ABT263, which are major potential hurdles in their use as anti-aging therapies. These toxicities may be mitigated somewhat if these drugs can be administered intermittently, rather than chronically, to achieve their senolytic effects.

Major results concerning the small molecules discussed in this review are summarized in Figure 2.