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Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2017 November 01.
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Published in final edited form as:

Nat Rev Mol Cell Biol. 2016 November ; 17(11): 679–690. doi:10.1038/nrm.2016.93.

Slowing ageing by design: the rise of NAD+ and sirtuin-activating

compounds
Michael S. Bonkowski1 and David A. Sinclair1,2
1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
2Department of Pharmacology, The University of New South Wales, Sydney 2052, Australia
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Abstract
The sirtuins (SIRT1–7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent
deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice
engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating
compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved
organ function, physical endurance, disease resistance and longevity. Trials in non-human primates
and in humans have indicated that STACs may be safe and effective in treating inflammatory and
metabolic disorders, among others. These advances have demonstrated that it is possible to
rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in
humans.
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The past 100 years have witnessed unprecedented advances in our ability to prevent and treat
disease. Unfortunately, most medicines are designed to only treat one specific condition
while ignoring other comorbidities. Thus, although the inhabitants of most nations are living
longer, healthspan is not increasing. In the United Kingdom, for example, the percentage of
men’s lifespan spent in poor health has risen from 20% to 40% between 1995 and 2006
(REF. 1). Recent progress in longevity research, however, may soon enable doctors to treat
diseases that affect multiple organs with one medicine (or just a few in combination), to
significantly increase healthspan and compress the morbid period of our lives.

“To eat when you are sick is to feed your sickness” wrote Hippocrates, the father of Western
medicine, almost 2,400 years ago. Today, his views seem remarkably prescient. Calorie
restriction without malnutrition is considered the gold standard in biogerontology as the
most robust way to delay ageing and age-related diseases. Since the discovery almost 80
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years ago that calorie restriction can extend the lifespan of rats2, great strides have been
made in understanding why reducing calorie intake results in profound health benefits. Early
theories, which included a delay in development and reduced metabolic rate, were
subsequently ruled out owing to inconsistent experimental observations3.

Correspondence to D.A.S. david_sinclair@hms.harvard.edu.

Competing interests statement
The authors declare competing interests: see Web version for details.
DATABASES:ClinicalTrials.gov: https://clinicaltrials.gov
SUPPLEMENTARY INFORMATION: See online article: S1 (box)
 Bonkowski and Sinclair Page 2

Numerous studies of simple and complex model organisms over the past 20 years have lent
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credence to the idea that a set of evolutionarily conserved longevity pathways are
responsible for the effects of calorie restriction on lifespan4 (FIG. 1). Dozens of genes and
pathways have now been uncovered that compress the period of morbidity and extend the
lifespan of model organisms, from yeast to rodents. Major signalling targets include insulin/
insulin-like growth factor 1 (IGF1) signalling, target of rapamycin (TOR), adenosine
monophosphate-activated protein kinase (AMPK) and the nicotinamide adenine dinucleotide
(NAD+)-dependent sirtuin deacylases5,6. It is believed that these pathways evolved to sense
and respond to the nutritional environment and to promote cellular defence mechanisms in
the face of extrinsic adversity (FIG. 1).

Interestingly, several naturally occurring molecules can activate these survival pathways and
extend lifespan in rodents7,8. Rapamycin, a drug first discovered in a bacterium from Easter
Island, reduces organ transplant rejection by inhibiting mechanistic TOR (mTOR).
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Rapamycin is thought to extend lifespan by mimicking diets that have low levels of essential
amino acids such as methionine or tryptophan9,10. Another potential longevity drug is
metformin, an AMPK-activating molecule from the Hellebore buttercup plant that is a
frontline therapy for type 2 diabetes11. Clinical trials testing the ability of these molecules to
slow aspects of ageing are underway or in the planning stages12. As discussed below, a
promising approach to discovering longevity drugs is to design compounds de novo based
on a molecular understanding of longevity. By designing increasingly potent compounds that
activate sirtuins, it has been possible to pharmacologically mimic some of the physiological
effects of calorie restriction both in animals and humans, with the ultimate goal of creating
medicines that treat multiple age-related diseases and prevent many others.

The yeast silent information regulator 2 (SIR2) gene, which gave sirtuins their name, was
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first shown to extend lifespan in yeast almost 20 years ago, making sirtuins one of the first
families of longevity genes to be discovered13–16. Since then, multiple lines of evidence,
from yeast to humans, have implicated sirtuins in mediating the health benefits of dietary
restriction and exercise. Although sirtuins have attracted considerable attention, they have
also attracted controversy, including finally settled debates about how sirtuins activators
work and even whether sirtuins have a role in ageing17,18. In this Review, we discuss the
trials and tribulations of sirtuin research, which have constructively led to a clearer
understanding of sirtuin structure, enzymatic activity and biological roles. We describe the
ongoing quest to discover and develop safe and potent sirtuin-activating compounds
(STACs) as means to combat multiple age-associated diseases. The use of such compounds
in clinical trials is discussed in the context of the challenges that lie ahead if they are to
reach their full potential in medicine.
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Sirtuins in metabolism and longevity

Sirtuins were initially discovered in the budding yeast Saccharomyces cerevisiae by virtue of
their essential function in gene silencing19 and subsequently found to dictate ‘replicative
lifespan’, which is defined by the number of daughter cells a mother cell can produce15. Sir2
silences transcription at three loci: the HM mating-type loci, telomeres and the ribosomal
DNA (rDNA). As cells age, the Sir2–Sir3–Sir4 complex relocalizes away from telomeres

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and the mating-type locus to accumulate at the rDNA locus, ostensibly to slow the formation
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and toxic accumulation of extrachromosomal rDNA circles (ERCs) that cause replicative
ageing in yeast20,21. Inserting an extra copy of SIR2 (but not SIR3 or SIR4) into the yeast
genome suppresses ERC formation and extends its lifespan22. This finding was corroborated
by an unbiased genome-wide association study that identified the SIR2 locus as the most
significant regulator of yeast replicative lifespan23. In 2000, Sir2 was reported to have
histone deacetylase activity, which results in chromatin compaction and requires NAD+, a
cofactor also required for redox reactions and whose levels change in response to nutrients
and stress24,25. This striking discovery raised the intriguing possibility that yeast Sir2 and its
homologues might act as nutrient and metabolic sensors that relay nuclear changes in the
NAD+/NADH ratio to alter both transcription and genome stability24.

Sirtuins in other species were rapidly identified by virtue of their homology to yeast Sir2,
specifically to the conserved central catalytic core26. In the nematode Caenorhabditis
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elegans27 and the fruitfly Drosophila melanogaster28, sirtuins were shown to control stress
resistance and longevity, although the experimental approaches and magnitude of the effects
have been debated18 (Supplementary information S1 (box)). By now, numerous research
groups have shown that sirtuin overexpression in the nematode29–31 and the fruitfly32,33
results in a reproducible increase in longevity.

On the heels of the work performed in these organisms, there has been considerable effort to
determine the role of the seven mammalian sirtuins (SIRT1–7) and to find small molecules
to modulate their activity for pharmaceutical purposes. There are many points at which to
intervene. Sirtuins are regulated at the level of transcription, translation, protein stability and
oxidation. Sirtuins are also regulated by protein–protein interactions, natural inhibitors such
as nicotinamide, microRNAs, localization within the cell and within organelles, as well as
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by substrate and co-substrate availability34–36. Similar to the yeast sirtuins, the mammalian
sirtuins SIRT1, SIRT6 and SIRT7 act as transcription regulators37,38. These sirtuins also
serve many additional roles, including the control of energy metabolism, cell survival, DNA
repair, tissue regeneration, inflammation, neuronal signalling and even circadian rhythms
(reviewed in REFS 39,40). SIRT1, which is predominately a nuclear protein, deacetylates
histones H3, H4 and H1 (REFS 37,38) but also modifies more than 50 non-histone
proteins41, including transcription factors and DNA repair proteins. Examples of
transcription factors regulated by SIRT1 include p53 (REFS 42,43), nuclear factor-κB (NF-
κB)44, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α)45 and sterol
regulatory element-binding protein (SREBP)46. Examples of SIRT1-regulated DNA repair
proteins include Ku70 (REFS 47,48), poly(ADP-ribose) polymerase 1 (PARP1)49 and
Werner syndrome ATP-dependent helicase (WRN)50,51. Other mammalian sirtuins localize
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to various subcellular compartments where they target non-histone proteins41: SIRT2 is

cytosolic; SIRT3, SIRT4 and SIRT5 are mitochondrial; and SIRT6 and SIRT7 are nuclear
(FIG. 2). Adding to the complexity, there is a growing list of chemical reactions that the
mammalian sirtuins catalyse, including demalonylation, desuccinylation, decrotonylation,
depropionylation, delipoamidation, other long-chain fatty acid deacylations and mono-ADP-
ribosylation52–56, which are generally referred to as deacylation reactions, not to be
confused with deacetylation.

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During sirtuin-mediated deacetylation of target Lys residues, NAD+ is converted to

nicotinamide (NAM) and O-acetyl-ADP-ribose57. NAM then acts as a sirtuin inhibitor by
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binding to the C-pocket of sirtuins, which lies adjacent to the NAD+-binding pocket58,59.
NAM remains one of the most effective and widely used sirtuin inhibitors. Recent studies
have also revealed a link between NAM and its methylated form 1-methylnicotinamide
(MNA) and health. In the nematode, both NAM and MNA increase longevity, and so does
overexpression of the nematode gene required for formation of MNA, anmt-1, which is a
homologue of the mammalian NAM-N-methyltransferase (NNMT), via a pathway
seemingly independent of sirtuin activity30. In mammals, knockdown of NNMT protects
against diet-induced obesity60, although a more recent study found that liver NNMT is
increased by calorie restriction and seemed to improve glucose and cholesterol
metabolism61. Additional studies are needed to understand the potential benefits of sirtuin-
mediated by-products of NAD metabolism in mammals.
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Although the results from experiments in which sirtuins were upregulated genetically or
pharmacologically are generally promising (TABLE 1), the large number of pathways in
which sirtuins are involved is a potential double-edged sword. Sirtuins interact with all the
major conserved longevity pathways, for example AMPK62 and insulin–IGF1
signalling63–68, including targets such as protein kinase A (PKA)63, mTOR64–66, forkhead
box O (FOXO)67 and IGF1 (REF. 68) (FIG. 1). Whether the multiple pathways in which
sirtuins are involved will prove to be advantageous or deleterious for therapy in humans is
unclear, but in the past 10 years, numerous sirtuin-overexpressing mouse strains have been
generated, and at least two of them live longer. Notably, however, a transgenic mouse with
high levels of Sirt1 overexpression recapitulates many of the benefits of calorie restriction,
including increased metabolic activity, reduced blood lipid levels and improved glucose
metabolism, but not a longer lifespan69. Another transgenic mouse with moderate
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overexpression of Sirt1 is protected from inflammation, liver cancer, diabetes and hepatic
steatosis, but it too does not live longer70,71. A brain-specific Sirt1-overexpressing mouse
strain (BRASTO) has a 9–16% longer mean lifespan, depending on the sex of the mice, and
a significant increase in maximal longevity72. Male mice ubiquitously overexpressing Sirt6
live ~15% longer than wild-type mice, although this effect is not observed in females73.
Sirt6-overexpressing mice are also resistant to hypoxia and to the detriments of a high-fat
diet74. In humans, loss of SIRT6 activity has been directly implicated in the progression of
ductal pancreatic adenocarcinoma, a highly fatal form of pancreatic cancer75. These findings
indicate that molecules that activate SIRT1 and/or SIRT6 in humans may provide both broad
health benefits and potent antitumour activities76. Achieving this therapeutic potential,
however, would require a detailed molecular understanding of how sirtuins can be activated.
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Sirtuin-activating compounds
The search for molecules that activate sirtuins began more than a decade ago. In the past 3
years, many of the challenges facing the development of STACs as medicines have been
overcome, including resolving technical issues (see below), increasing the bioavailability of
STACs and determining which diseases to prioritize in clinical trials.

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How do sirtuin activators work?

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The first STACs were discovered for SIRT1 in 2003, the most potent of which was
resveratrol. This initial discovery was important because it proved that allosteric activation
of sirtuins was possible77. High-throughput screening and medicinal chemical efforts have
since identified more than 14,000 STACs from a dozen chemical classes, including stilbenes
(for example, resveratrol), chalcones (for example, butein) and flavones (for example,
quercetin) from plants77. Synthetic STACs include imidazothiazoles (for example,
SRT1720)78, thiazolopyridines (for example, STAC-2), benzimidazoles (for example,
STAC-5) and bridged ureas (for example, STAC-9)79,80 (BOX 1). All of these chemical
classes activate SIRT1 by lowering the Km value of the substrate through a K-type allosteric
activation mechanism. Interestingly, recent work demonstrated that SIRT6 deacetylase
activity can be activated by endogenous fatty acids at the amino terminus of the enzyme56,
hinting that SIRT6 may also be amenable to activation in vivo by synthetic molecules. This
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finding presents an exciting possibility given the ability of SIRT6 to enhance DNA repair, to
modulate the metabolism of cancer cells to prevent malignancy, and to extend mouse
lifespan73,75,81–84.

The original fluorescence-based screens used to discover STACs were criticized because
STACs only seemed to activate SIRT1 when bulky and hydrophobic fluorescent moieties
were attached to the peptide substrates85,86. The most critical paper concluded that
resveratrol and the chemically distinct STACs SRT1720 and SRT2104 were simply binding
to the hydrophobic fluorophores17. This challenge, which spanned a few years, led to the
discovery that SIRT1 has several structural and positional requirements of amino acids that
are adjacent to the acetylated Lys residue for enabling substrate recognition87 and SIRT1
activation79,85,86. A major clue came from the discovery that the activation of SIRT1 is
favoured when a large hydrophobic residue is present on the carboxy- terminal side of the
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acetylated Lys88. A study performed on natural peptide substrates revealed that bulky
hydrophobic amino acids (Trp, Tyr or Phe) at the +1 and +6 positions relative to the acetyl-
Lys could substitute for the fluorescent groups in the original assays89. These findings
indicated that SIRT1 prefers specific hydrophobic amino acids in locations adjacent to the
target Lys for substrate recognition. The same study also identified a mutation in SIRT1
(substitution of Glu230 with Lys) that prevented its activation by resveratrol89 and,
strikingly, also by more than 100 synthetic STACs from multiple chemical scaffolds89,
indicating that both natural and synthetic STACs activate SIRT1 by a common mechanism.

According to Zorn and Wells90, “one of the simplest ways to activate an enzyme with a
small molecule is to bind to an allosteric site within the catalytic domain of that enzyme and
induce a conformational change which changes the affinity of that enzyme for its native
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substrate.” Evidence from crystal structures and enzymological and biophysical studies has
indicated that STACs function not by binding within the catalytic domain but by binding to a
relatively rigid helix-turn-helix N terminus called the STAC-binding domain (SBD)91.
Deletion studies of human SIRT1 have indicated that the N terminus is a key mediator of
allosteric activation. The N terminus is necessary for activation by resveratrol and by
synthetic STACs, which physically bind to this region89, and itself can activate SIRT1 in
trans92. These data indicate that the N-terminal domain may function to activate SIRT1 in

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trans when SIRT1 is a dimer, or in cis to enhance substrate binding to the SIRT1 catalytic
core92.
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Recent crystallographic and amino acid substitution studies of human SIRT1 have identified
the residues with which STACs come in contact and suggest an activation mechanism that is
consistent with a ‘bend-at-the-elbow’ model91. STACs bind to the α-helical SBD in the N
terminus, allowing the domain to flip over the site of interaction between the substrate and
catalytic core91 (FIG. 3a). Next to the SBD are the hinge residues Arg234 and Glu230,
located within a polybasic linker (KRKKRK), which allow the STAC-bound SBD to interact
with the catalytic domain through a salt bridge formed between the guanidinium group of
the SBD and the carboxylate group of Asp475 and hydrogen bonds to His473 and Val459
(FIG. 3b). In this model, the negatively charged Glu230 makes contacts with the positively
charged Arg446, which explains why the SIRT1 substitution of Glu230 to Lys (a negative to
positive charge) blocks SIRT1 activation and why subsequently replacing Arg446 with Glu
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(a positive to negative charge) restores the ability of STACs to activate SIRT1.

Additional clues to the mechanism of the allosteric activation of sirtuins have emerged from
studies of the yeast Sir2 protein, which is the catalytic component of a Sir2–Sir3–Sir4
complex. Molecular dynamic simulations have indicated that the substrate-binding channel
toggles between an open and closed conformation, with Sir4 maintaining the N-terminal
helix in an active conformation89. Interestingly, Glu230 of mammalian SIRT1 structurally
aligns with a similar negatively charged residue in yeast Sir2 (Asp223), which is required for
gene silencing93.

Specificity of SIRT1 activators in vivo

Resveratrol is a nonspecific compound, meaning it interacts with numerous proteins within
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the cell94. In addition to SIRT1, reported targets of resveratrol include AMPK95,96,

phoshodiesterases97, F1-ATPase98, complex III of the mitochondrial electron transport
chain99, PARP1 and Tyr-tRNA synthetase100. Elucidating which effects of resveratrol are
mediated by SIRT1 and which by other effectors has been a considerable task; for example,
whether resveratrol acts on SIRT1 or AMPK. Metformin, a pro-longevity drug that activates
AMPK and produces similar physiological effects to resveratrol, increases NAD+ levels by
activating the NAD salvage pathway enzyme nicotinamide phosphoribosyltransferase
(NAMPT) (FIG. 2) and increases the NAD+/NADH ratio, both of which increase SIRT1
activity96,101. Other studies have shown that resveratrol acts on SIRT1 to activate AMPK by
deacetylating and activating the AMPK kinase LKB1 (also known as STK11)102–105. Thus,
SIRT1 can activate AMPK and AMPK can activate SIRT1. Recent evidence suggests that
resveratrol can activate both AMPK and SIRT1 in vivo; however, the predominant target is
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highly dose-dependent105.

The identification of a SIRT1 mutation that blocked STAC activation in vitro89 has provided
the perfect means to test whether STACs alter physiology by activating SIRT1 directly. In
human HeLa cells and in mouse primary myocytes engineered to express the E230K SIRT1
mutation, resveratrol and synthetic STACs (SRT1720 and SRT2014) failed to induce the
expected changes in mitochondrial mass, gene expression and cellular ATP concentrations.
These results provided strong evidence that these STACs work by directly activating SIRT1

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(REF. 89). To address the question of how STACs function in vivo, it would be interesting to
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repeat these experiments in a mouse carrying a SIRT1 E230K knock-in mutation.

Human and non-human primate studies

Studies using rodents have demonstrated that STACs promote health during ageing or
metabolic stress, including protection from cancer, neurodegeneration, cardiovascular
disease and diabetes, and even lifespan extension. Because regulatory agencies currently do
not consider ageing a disease, the first sirtuin activator is most likely to be used clinically to
treat an age-related disease such as diabetes, non-alcoholic fatty liver disease or an
inflammatory disease (FIG. 4).

The big question is whether sirtuin activators will work in humans106. Resveratrol as a
proof-of-concept molecule is providing valuable clues. Even before it was shown to activate
SIRT1, epidemiological studies in the 1980s suggested that resveratrol may explain aspects
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of the ‘French paradox’, the phenomenon whereby the French have a low incidence of
cardiovascular disease even though they consume high amounts of saturated fats107.
However, the validity of the French paradox has since been challenged108. A conglomerate
of laboratories at the National Institute on Aging in the United States evaluated the benefits
of resveratrol in controlled studies in non-human primates109–111. Following a 2-year high-
fat and high-sugar feeding regimen, they determined that a 2-year resveratrol treatment
prevented high-fat and high-sugar-induced arterial wall inflammation and increased arterial
pulse wave velocity110, which are a major cause of cardiovascular morbidity and mortality
in Western societies112. Additional studies from the National Institute on Aging using the
same experimental approach found that resveratrol led to improvements in insulin sensitivity
of visceral adipose tissue compared with control monkeys109. Moreover, preservation of
pancreatic β-cell morphology and maintenance, and expression of essential β-cell
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transcription factors were observed111.

At least six clinical studies have now evaluated the safety and effects of resveratrol in
humans (TABLE 2). The majority of studies observed improvements in glucose metabolism
and in multiple indicators of protection from cardiovascular disease113–118. In one
randomized double-blind crossover study, a group of healthy obese men were administered
resveratrol (150 mg per day) or placebo for 30 days. The men receiving resveratrol exhibited
a significantly reduced resting metabolic rate, reduced systolic blood pressure and improved
HOMA index118. Muscle samples from those patients showed increased SIRT1 and PGC1α
protein expression, and increased AMPK activity, mitochondrial respiration and fatty acid
oxidation118. Another study in non-obese men, however, found that resveratrol failed to
provide any measurable physiological improvements117. More recently, a phase II study
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evaluating resveratrol in patients with Alzheimer disease showed that resveratrol can delay
cognitive decline in the ability to perform daily tasks119, a finding consistent with studies of
resveratrol and SIRT1 in mouse models of Alzheimer disease120. A comprehensive summary
and meta-analysis of clinical data has been recently published, the conclusion of which is
that the human data are consistent with rodent studies, although more studies are
warranted121.

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The reason for variability in the efficacy of resveratrol in clinical trials is not yet known106.
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One explanation is the choice of human subjects. Studies that have carefully prescreened
patients for advanced age and/or for insulin resistance have seen the greatest benefit of
resveratrol, suggesting that SIRT1 activation restores homeostasis and is most effective in
the context of dysfunctional physiology114. In mice, for example, the strongest effects of
resveratrol are on obese mice. Another confounding issue is that resveratrol and many of the
early STACs are hydrophobic, with low solubility and bioavailability. Thus, the efficacy of
STACs probably depends on the formulation, the size of the chemical particles and whether
the compounds are taken with food106. Nevertheless, for resveratrol to have metabolic
benefits it might not need to be available to all tissues: a recent study using rats found that
resveratrol activates SIRT1 in the gut endothelium to stimulate the secretion of the metabolic
hormone glucagon- like peptide 1, thereby reducing glucose levels without even having to
enter the circulation122.
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More than 14,000 synthetic STACs have been synthesized to date, and dozens of these have
been tested in animal models of type 2 diabetes, colitis, neurodegeneration, fatty liver and
atherosclerosis, among others78,89,123–127. Currently, STACs are in their fifth
generation78–80 and have more than 1,000-fold greater potency in vitro than resveratrol, with
an EC50 in the low nanomolar range. The STAC named SRT2104, which mimics aspects of
calorie restriction and extends male mouse lifespan125, has advanced through multiple phase
I trials with few, if any, side effects128–130 to phase II trials (TABLE 2). Two SRT2104
clinical trials in elderly volunteers and otherwise healthy smokers showed a slight reduction
in body weight, a 15–30% improvement in the cholesterol ratio and a 19% decrease in
triglyceride levels129,130. A separate study of patients with the inflammatory condition
plaque-type psoriasis showed a significant reduction in disease manifestation following 84
days of oral administration of 500 or 1,000 mg per kg SRT2104 (REF. 131). Pharmaceutical
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companies are continuing to develop STACs that have improved pharmaceutical properties
and to conduct additional clinical trials (J. Ellis, personal communication).

NAD+ boosters as sirtuin activators

It has been known since 2003 that upregulation of the NAD salvage pathway, which recycles
NAD+ from NAM, can extend lifespan and mimic calorie restriction in yeast132,133. The
gene responsible for the rate-limiting step in the NAD salvage pathway in yeast, PNC1
(which encodes nicotinamidase), is activated by diverse stresses and calorie restriction,
resulting in greater flux through the pathway and increased Sir2 activity. This early
observation helped to explain how diverse stresses such as calorie restriction, amino acid
restriction, heat stress and osmotic stress extend yeast lifespan.
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In mammals, the homologue of PNC1 is NAMPT, which also catalyses the rate-limiting step
in NAD salvage. NAMPT catalyses the formation of nicotinamide mononucleotide (NMN)
from NAM, which is then converted to NAD by nicotinamide mononucleotide
adenylyltransferase 1 (NMNAT1), NMNAT2 and NMNAT3 (FIG. 4). Nicotinamide riboside,
a naturally occurring precursor of NAD+, enters the salvage pathway after being converted
to NMN by the nicotinamide riboside kinase (NRK) enzymes. CD38 and its homologue
CD157 are glycohydrolases that degrade NAD+ (REF. 134). As humans and mice age, levels

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of NAD+ decline, possibly because the consumption of NAD+ by CD38 outweighs its
synthesis by the kynurenine pathway135.
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NAD-boosting molecules constitute a newer class of STACs gaining attention as a way to

restore NAD+ levels in elderly individuals and potentially activate all seven sirtuins with a
single compound. Examples of NAD-boosting molecules include NMN, nicotinamide
riboside67,136–141 and inhibitors of CD38 such as apigenin142, quercetin142 and GSK
897-78c143. In rodents, these molecules have been administered via various routes, including
intraperitoneal, gavage and in drinking water, at doses ranging from 100 to 1,000 mg per kg
per day67,136–141 for more than 6 months without any apparent adverse side effects140. The
effects of NAD-boosting STACs on physiology are surprisingly broad, with improvements in
glucose metabolism and mitochondrial function, and the recovery from injury of the heart,
ears and eyes67,144–147 (FIG. 4). Nicotinamide riboside supplementation in mice starting at
24 months of age (a very advanced aged for mice), resulted in a modest (~5%) yet
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significant increase in longevity141. Nicotinamide riboside also prevents high-fat diet-

induced glucose dysregulation145 and protects mice from DNA damage148,149, noise-
induced hearing loss150, cardiac injury151 and stem cell-niche depletion141. Treatment with
the related molecule NMN also protected 2-year old mice against a high-fat diet147 and
restored youthful levels of mitochondrial function, ATP production and insulin sensitivity in
muscle, ostensibly through SIRT1 activation137,147. Other indicators of inflammation and
muscle wasting were also reduced137,147. Other NAD+ precursors such as nicotinic acid
adenine dinucleotide or nicotinic acid riboside have yet to be tested but may be equally or
even more efficacious. Non-naturally occurring derivatives of these compounds are also
worth exploring given that the natural compounds have relatively low stability and short
half-lives145.
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Targeting the enzymes that regulate NAD+ levels, such as CD38, CD157 and NAMPT, may
also be worth exploring for their therapeutic potential (FIG. 4). For example, a recent study
indicated that a class of neuroprotective compounds (for example, P7C3) work by
allosterically activating NAMPT152. This finding is consistent with a study showing that
using NMN to increase NAD+ can protect neurons from the degeneration caused by
SARM1, a protein that triggers a precipitous decline in NAD+ levels101. It will be interesting
to test the effects of P7C3 and related molecules and whether they might have benefits
similar to or better than the currently available STACs.

Future perspectives
The sirtuins have generated a considerable amount of excitement and scrutiny over the past
20 years. There is now consensus that sirtuins underlie aspects of calorie restriction and that
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they are key regulators of ageing and age-related diseases. Moreover, STACs can prevent
and treat a variety of diseases in model organisms and in humans by directly binding to and
activating SIRT1.

The path forwards now seems clear. For many years, synthetic STACs have been sufficiently
potent to justify their use in the clinic but proved to be limiting in their bioavailability. Now
with more soluble and specific compounds available89,91,123,125,127, STACs are re-entering

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clinical trials. Currently, given the promising results in patients with psoriasis treated with
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SRT2014 (REF. 131), further clinical trials for this disease seems to be a good path to
follow. Compounds that raise NAD+ levels, such as nicotinamide riboside and NMN, also
show great promise as calorie restriction mimetics to treat numerous age-related conditions,
and possibly extend lifespan. Other possible trials include those to treat rare diseases such as
a the DNA damage syndromes trichothiodystrophy or Cockayne syndrome (which are both
alleviated by increased NAD+ levels in mice), an inflammatory disorder such as psoriasis, or
metabolic diseases such as type 2 diabetes or non-alcoholic fatty liver disease.

Although many questions have been resolved in recent years, many still remain. For
example, the physiological roles of the newly described activities of the sirtuins — namely,
demalonylation, desuccinylation, decrotonylation, depropionylation and delipoamidation —
remain unclear55. The pharmacokinetics and pharmacodynamics of NAD precursors and
how they are affected by the route of delivery need clarification. In addition, identifying the
Author Manuscript

NAD+ pools responsible for eliciting their benefits, both within the body and within
subcellular compartments, is important. Moreover, are other sirtuins in addition to SIRT1
and SIRT6 amenable to allosteric activation? And what are the transport mechanisms for
NAD+ precursors across cell membranes into the bloodstream and into cells153? This last
question is important given new data indicating that there is an intracellular form of NAMPT
(iNAMPT) and an extracellular form (eNAMPT), which is secreted from fat tissue and
controls both systemic and hypothalamic NAD+ bioavailability153. Perhaps the most
practical question is whether STACs will ever be approved as a drug to treat ageing or age-
related diseases in humans. With the elderly population increasing worldwide and health-
care costs threatening the global economy, the answer to that question cannot come soon
enough.
Author Manuscript

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
The authors thank M. B. Schultz for suggestions and edits and are grateful for financial support from the National
Institute on Aging, the National Institutes of Health, the Paul F. Glenn Foundation for Medical Research, Edward
Schulak, and Ovaxon.

Glossary
Replicative ageing
In yeast, the number of daughter cells produced by a mother cell before senescence
Author Manuscript

Redox reactions
Oxidation–reduction (redox) reactions involving the transfer of electrons between two
chemical species

Hepatic steatosis
Also known as fatty liver, is a term used to describe the accumulation of fat in the liver cells

Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2017 November 01.
 Bonkowski and Sinclair Page 11

Allosteric activation
Author Manuscript

Activation of an enzyme by binding of a ligand, which enhances the binding of substrates at

other binding sites

Km
Michaelis constant, which reflects the affinity of an enzyme for its substrate. The Km is
measured as the substrate concentration at which the reaction rate is half of its maximum
rate

K-type allosteric activation

Refers to the major type of allosteric activation, in which the main feature that is altered is
the Michaelis constant (Km)

HOMA index
Author Manuscript

The homeostatic model assessment (HOMA) index is a clinical measure used to predict the
function of pancreatic β-cells and insulin resistance

Bioavailability
The degree and rate at which a substance is absorbed and is made available at the site of
physiological activity

EC50
The concentration of substrate that elicits a half-maximal enzymatic response

Plaque-type psoriasis
The most common form of the disease, which is manifested as raised, red patches covered
with a silvery white build-up of dead skin cells or scale
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Box 1
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The hunt for sirtuin activators

As soon as yeast cells were shown to live longer with an extra copy of the sirtuin-
encoding gene silent information regulator 2 (SIR2), a molecule that can activate sirtuins
was quickly sought after. Generally, although activators are rare and much harder to study
than inhibitors, they have distinct advantages: they are less prone to be rendered
ineffective by residual enzymatic activity, they often have greater target specificity within
an enzyme family and they elicit fewer side effects90. To date, the sirtuin that has
received the most attention as a drug target is SIRT1. The first generation of sirtuin-
activating compounds (STACs) were a group of related plant polyphenols that included,
among others, quercetin, butein and resveratrol, which is a molecule found in red wine77.
These molecules lower the binding affinity of SIRT1 for the substrate to increase
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enzymatic activity by tenfold77. Resveratrol rapidly became the molecule of choice to test
SIRT1 activation because it was potent, nontoxic, naturally available and inexpensive.
Many studies have since shown that resveratrol can extend maximal lifespan in
yeast154–156, worms157,158, flies159–161, fish162–165 and even honeybees166 (TABLE 1).
In mice, resveratrol protects against many of the deleterious effects of a high-calorie diet,
delays age-related diseases, increases exercise endurance, and can mimic many of the
transcriptional changes caused by calorie restriction167–171. These findings have led to
the prevailing model in which SIRT1 imparts many of the benefits of a calorie restriction
diet69–71,136. Additional screens for more potent, soluble and efficacious STACs have
been successful172,173.

In mice, STACs mimic calorie restriction, increase mitochondrial function, protect from
fatty liver and muscle wasting, and have strong antidiabetic, cardioprotective and anti-
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inflammatory effects in disease models78. STACs can also extend a mouse’s lifespan by
up to 15%, even when initially given at 1 year of age123–125. In dozens of studies, the
effects of STACs were shown to be either partially or completely SIRT1-dependent
(reviewed in REF. 174). For example, in mice, the ability of resveratrol to prevent skin
tumours is blunted in the Sirt1 germline knockout175, and mitochondrial function in
skeletal muscles is not induced in a Sirt1-knockout strain105. Furthermore, synthetic
STACs that are much more specific such as SRT1720 and SRT2104 in mice increase
mitochondrial mass in muscle and liver124,125. Mechanistically, STACs trigger the
deacetylation of peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α)
to increase mitochondrial biogenesis; these effects were also shown to be SIRT1-
dependent176.
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Figure 1. Nutrient-responsive signalling pathways that maintain health and extend lifespan
Calorie or dietary restriction increases the concentrations of metabolic effectors such as
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nicotinamide adenine dinucleotide (NAD+) and AMP while reducing the concentrations of
glucose, amino acids and lipids. Exogenous administration of nicotinamide riboside (NR),
nicotinamide mononucleotide (NMN) or the nicotinamide phosphoribosyltransferase
(NAMPT) activator P7C3 can increase NAD+ levels. Calorie restriction also reduces the
concentrations of the hormonal effectors insulin, insulin-like growth factor 1 (IGF1) and
growth hormone (GH). These effectors stimulate or inhibit the activity of metabolic sensors
such as the sirtuins (SIRTs), AMP kinase (AMPK), target of rapamycin (TOR), insulin–
IGF1 signalling (IIS) and forkhead box O (FOXO) transcription factors. Sirtuin-activating
compounds (STACs) such as SRT1720 and SRT2104 can directly activate SIRT1, whereas
rapamycin is a direct inhibitor of TOR. Metformin indirectly activates AMPK. These
metabolic sensors regulate downstream activities such as DNA repair, mitochondrial
biogenesis and function, stress resistance, stem cell and telomere maintenance, autophagy,
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chromatin modifications, reduced inflammation, and translation fidelity. The net effect is to
tip the scale in favour of homeostasis and compressed morbidity, resulting in a disease-free,
more youthful-like state.

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Figure 2. Localization, enzymatic activity and modulation of sirtuins by small molecules

The mammalian sirtuins (SIRT1−7) are a family deacylases that are localized to various
cellular compartments. SIRT1, SIRT6 and SIRT7 are localized primarily to the nucleus,
whereas SIRT3, SIRT4 and SIRT5 are present in the mitochondria. SIRT1 (in some cell
types) and SIRT2 are localized to the cytosol. Their activities are regulated by nicotinamide
dinucleotide (NAD+), which is synthesized de novo from Trp, Asp or niacin (not shown).
NAD+ is also recycled by the NAD salvage pathway from nicotinamide (NAM) by
nicotinamide phosphoribosyltransferase (NAMPT), which converts NAM to nicotinamide
mononucleotide (NMN). NMN is converted to NAD+ by nicotinamide mononucleotide
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adenylyltransferase1 (NMNAT1), NMNAT2 or NMNAT3, which are also capable of

converting nicotinic acid mononucleotide (NaMN) to NAD+. Additional NAD+ precursors
used in the NAD salvage pathway include nicotinamide riboside (NR) and nicotinic acid
riboside, which are converted to NMN and NaMN, respectively (not shown) by nicotinamide
riboside kinase 1 (NRK1) and NRK2. The cell membrane-bound glycohydrolases CD38 and
CD157 degrade NAD+ to NAM135,142. CD73 converts NAD+ to NMN and NMN to NR; it is
a transmembrane protein that may also act as a NR transporter177,178. There is evidence to

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indicate that NAD+ and NMN can be directly transported across the cell membrane in some
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cell types, although the mechanism is not yet known. The inset shows that SIRT1 activation
is stimulated by the presence of a hydrophobic amino acid adjacently to the target Lys (panel
1). When NAM levels are sufficiently high, it binds into the carboxy-terminal pocket of
SIRT1 and inhibits it58 (panel 2). SIRT1 utilizes NAD+ as a co-substrate to promote the
binding and deacetylation of specific protein substrates (panel 3). Sirtuin-activating
compounds (STACs) activate SIRT1 by binding to the STAC-binding domain and primarily
lowering the Km for the peptide substrate, thereby increasing its catalytic activity (panel 4).
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Figure 3. The allosteric activation mechanism of SIRT1

a| The structure of a truncated human sirtuin 1 (SIRT1) in complex with a synthetic sirtuin-
activating compound (STAC) showing the binding site of STAC-1 at the amino-terminal
STAC-binding domain (SBD). STAC binding promotes the interaction of the SBD with the
central catalytic domain to lower the Km for the substrate, thereby increasing SIRT1 activity.
The carboxy-terminal regulatory segment (CTR) stabilizes the deacetylase activity.
Substitution of Glu230 (E230) with a Lys residue impairs enzymatic activation by reducing
the electrostatic interaction between Glu230 on the SBD and Arg446 on the substrate-
binding site. b | Model of how STACs activate SIRT1. Structural analysis and mutagenesis
indicated that the amino acids Glu230 and Arg446 form a salt bridge (red circle) to facilitate
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binding of the amino terminus of SIRT1 with its catalytic core (movement indicated by
arrows). The protein ribbon is rainbow-coloured from blue at the amino terminus to red at
the carboxyl terminus. A STAC1 and an acetylated p53 peptide substrate are shown in cyan
and green, respectively. Adapted from REF. 91, Nature Publishing Group.
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Figure 4. Sirtuin activation and its disease relevance

Sirtuin activity can be stimulated through various mechanisms: allosterically by sirtuin-
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activating compounds (STACs), such as resveratrol, SRT1720 and SRT2104, which lower
the Km for the target proteins; increasing nicotinamide adenine dinucleotide (NAD+) levels
by providing its precursors nicotinamide riboside (NR) or nicotinamide mononucleotide
(NMN); inhibiting the glycohydrolases CD38 or CD157 (with apigenin, quercetin, GSK
897-78c)142,143; inhibiting poly(ADP-ribose) polymerases (PARPs)34; activating
nicotinamide phosphoribosyltransferase (NAMPT) with P7C3 (REF. 152); or by providing
nicotinamide (NAM) analogues, such as methyl-NAM. Nicotinamide riboside kinase 1
(NRK1) and NRK2 convert NR and nicotinic acid riboside (NaR) is to NMN and nicotinic
acid mononucleotide (NaMN), respectively. NaMN and NMN are converted to NAD+ by
nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), NMNAT2 and NMNAT3.
Sirtuins directly or indirectly target more than 100 signalling proteins with relevance to
human disease in a variety of tissues ranging from brain to blood (reviewed in REF. 41).
Although STACs have therapeutic potential, certain diseases, such as cancer in certain
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contexts and Parkinson disease, may benefit from sirtuin inhibition (reviewed in REF. 179).
EF2, elongation factor 2; FOXO, forkhead box O; HNGB1, high-mobility group box 1; IL,
interleukin; LXR, liver X receptor; MMP9, matrix metalloproteinase 9; NF-κB, nuclear
factor-κB; NLRP3, NOD-, LRR- and pyrin domain-containing 3; PGC1α, peroxisome
proliferator-activated receptor-γ co-activator 1α; PPAR, peroxisome proliferator-activated

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receptor; STAT5, signal transducer and activator of transcription 5; TGFβ, transforming

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growth factor-β; UCP2, mitochondrial uncoupling protein 2.

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Table 1

Sirtuins and sirtuin-activating compounds in stress resistance, calorie restriction, exercise and ageing

Species Sirtuin Sirtuin STACs extend Sirtuins mediate Sirtuin Sirtuin Sirtuin
knockout overexpression longevity the effects of CR expression or implicated expression
shortens extends longevity on longevity activity altered in ageing- or activity
longevity by CR or stress altered by
fasting pathways exercise

Yeast (Saccharomyces cerevisiae) >100 citations Not tested

Yes (20–50%)15,22 Yes (30–50%)15,22 • Resveratrol (30–58%)77 • Sir2 is essential132,180 • Yes132,180
• Resveratrol failed86 • Hst2-dependent181 • NAD+ levels unchanged184,185
• Sir2-independent182,183
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Worm (Caenorhabditis elegans) Not tested >40 citations Not tested

Yes (~5–10%)157,186 • * High expression (2.1 line 50%)27 • Resveratrol (~10–65%)157,159,189,190 • Essential31,186

• Low gene copy number (10–25%)29,30,157,187,188 • SRT1720 failed158 • Non-essential191,192

• Eleven independent low copy overexpressing lines (~5–15%)188

Fruit fly (Drosophila melanogaster) >24 citations Not tested

• Yes (~50%)193 • *Yes (~18–29%)28,161 • Resveratrol (8–29%)157,197 •* Essential28 Yes195,197
• No change194,195 • Fat-body specific dSir2 (REFS 32,196) • Resveratrol failed190 • Fat-body specific dSir2 (REFS 32,196)
• Dose-dependent197

Honey bee (Apis mellifera) Not tested Not tested Not tested Not tested Not tested Not tested
Resveratrol (33–38%)166

Fish (Nothobranchius furzeri and Nothobranchius guentheri) Not tested Not tested Not tested Not tested Not tested Not tested
Resveratrol (27–59%)163–165

Mouse (Mus musculus) >100 citations >800 citations >100 citations

• Elevated embryonic and postnatal lethality198 • Brain Sirt1 (9–16%)72 • Resveratrol in animals fed with high-fat diets167 Essential199,200
• Yes (~20–50%)68,199,200 • Sirt6 in males (9–17%)73 • SRT1720 (~8–22%)124
• SRT2104 in males (~10%)125

Multiple studies from the same laboratory were removed to limit citations. CR, calorie restriction; Hst2, homologous to Sir2; NAD+, nicotinamide adenine dinucleotide; Sir2, silent information regulator 2;
SIRTs, sirtuins; STACs, sirtuin-activating compounds.
*
Disputed by REF. 18.

Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2017 November 01.
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Table 2

Clinical trials of sirtuin-activating compounds (2000–2016)

Sirtuin-activating compound Phase I (safety) Phase II (safety and efficacy) Phase III–IV (pivotal and post-marketing)

Resveratrol or trans-resveratrol • Cardiovascular: • Cardiovascular: • Cardiovascular:

NCT01564381, NCT02415114, NCT01449110, NCT01564381, NCT01914081

NCT02616822, NCT01842399, NCT01842399, NCT02409537,
NCT01185067 NCT01615445, NCT02062190 • Pulmonary:
Bonkowski and Sinclair

• Cancer: • Diabetes: NCT02245932

NCT00256334 NCT02114892, NCT01158417, • Diabetes:

NCT02549924, NCT02247596,
• Diabetes: NCT01451918, NCT01714102, NCT01997762,
NCT02216552 NCT02244879,
NCT01677611, NCT02502253 NCT02219906,
• Neuropathy: NCT02216552,
• Neuropathy: NCT02766803
NCT01339884, NCT02621554,
NCT01339884, NCT01126229, NCT01504854, NCT02062190 • Inflammation:
NCT01321151, NCT01640197
NCT01492114,
NCT02244879,
NCT02475564,
NCT02433925,
NCT02130440

• Neuropathy:

NCT02621554,
NCT02336633,
NCT00678431,
NCT01219244

SRT2104 • Cardiovascular: • Inflammation: None

NCT01031108, NCT01702493 NCT01154101

• Diabetes: • Diabetes:

NCT01031108 NCT01018017, NCT00937326

• Inflammation:

NCT01453491, NCT01014117

Nicotinamide riboside • Phamacokinetics: • Diabetes: None

Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2017 November 01.
NCT02689882, NCT02300740, NCT02303483
NCT02721537, NCT02712593,
NCT02191462

• Cardiovascular:

NCT02678611

Clinical trials of sirtuin-activating compounds are listed in the table with their unique ClinicalTrials.gov identifier number. At the time of this publication, we excluded studies with unknown status.
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