David SINCLAIR, et al
Nicotinamide ( NAM ) vs Ageing
University of NSW research finds
compound that can reverse ageing
AUSTRALIAN researchers have found what could become the elixir of
life - a chemical compound that can reverse ageing.
The discovery of nicotinamide mononucleotide (NMN) by University
of NSW researchers could lead to new treatments for ageing,
cancer, type 2 diabetes and muscle wasting and inflammatory
diseases within five to ten years.
Human trials of the compound that turns back ageing by improving
communication between parts of a cell could start as early as next
The only hiccup is the cure is unaffordable for most people
because it costs $1,000 per gram.
The research used the equivalent of 500mg of NMN for every
kilogram of body weight per day.
This means the substance would cost the average 86 kilogram man
$43,000 a day and the average 71 kilogram woman $35,000 a day.
Lead researcher University of NSW Professor David Sinclair says he
soon hopes to find a way to produce it more cheaply.
The compound is fast acting and could also benefit healthy people
by making them super charged.
Just a week after older mice were injected with the compound they
had improvements in their muscles that made them indistinguishable
from younger animals.
"It's something like a 60 year old being similar to a 20 year old
on some measures," says University NSW pharmacologist and
co-author Dr Nigel Turner.
Very old mice that were the equivalent of a human aged 85 also
benefited from the substance with their body improving to be like
that of a 40 year old.
"If those results stand, then ageing may be a reversible
condition, if it is caught early", says Professor Sinclair who is
based at Harvard Medical School.
Underpinning the breakthrough is the discovery that when there is
a communication breakdown between the mitochondria, the battery
pack of a cell, and the nucleus of the cell ageing accelerates.
A chemical called NAD is central to kick starting this cellular
communication process but it begins to decline as we age.
The only way to combat the decline in NAD is excessive calorie
restriction and intensive exercise.
In a research paper published in the journal Cell today the
researchers report they have found that NMN injected into an
animal body transforms into NAD to repair the broken communication
This compound mimics the effect of diet and exercise.
"It was shocking how quickly it happened," says Dr Nigel Turner.
"If the compound is administered early enough in the ageing
process, in just a week, the muscles of older mice were
indistinguishable from the younger animals," he said.
The research is also examining another molecule called HIF-1 that
also interferes with cellular communication and has a role in
This molecule is switched on in many cancers and researchers have
now found it also switches on as we age.
"We become cancer-like in our ageing process," says Professor
"This may explain why the greatest risk of cancer is age," he
Professor Sinclair has previously been behind research that found
resveratrol a substance found in red wine and certain nuts, made
an anti-ageing gene SIRT1 run faster.
This new compound, NMN, activates all seven of the sirtuin genes
implicated in longevity.
Further studies will test whether NMN leads to mice living longer
lives, whether it helps them lose weight or has any side effects.
Professor Sinclair stresses that although NMN is a naturally
occurring "I wouldn't advise anyone to take it until we know it is
safe, we wouldn't want any surprises".
Cell, Volume 155, Issue 7, 1624-1638, 19 December 2013
Declining NAD+ Induces a
Pseudohypoxic State Disrupting Nuclear-Mitochondrial
Communication during Aging
Ana P. Gomes, Nathan L. Price, Alvin J.Y. Ling, Javid J. Moslehi,
Magdalene K. Montgomery, Luis Rajman, James P. White, João S.
Teodoro, Christiane D. Wrann, Basil P. Hubbard, Evi M. Mercken,
Carlos M. Palmeira, Rafael de Cabo, Anabela P. Rolo, Nigel Turner,
Eric L. Bell, David A. Sinclair
Ever since eukaryotes subsumed the bacterial ancestor of
mitochondria, the nuclear and mitochondrial genomes have had to
closely coordinate their activities, as each encode different
subunits of the oxidative phosphorylation (OXPHOS) system.
Mitochondrial dysfunction is a hallmark of aging, but its causes
are debated. We show that, during aging, there is a specific loss
of mitochondrial, but not nuclear, encoded OXPHOS subunits. We
trace the cause to an alternate PGC-1a/ß-independent pathway of
nuclear-mitochondrial communication that is induced by a decline
in nuclear NAD+ and the accumulation of HIF-1a under normoxic
conditions, with parallels to Warburg reprogramming. Deleting
SIRT1 accelerates this process, whereas raising NAD+ levels in old
mice restores mitochondrial function to that of a young mouse in a
SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts
PGC-1a/ß-independent nuclear-mitochondrial communication
contributes to the decline in mitochondrial function with age, a
process that is apparently reversible.
Aussie Scientist David Sinclair Claims
IT sounds too good to be true, but a respected Australian
scientist believes he has invented a new class of superdrug that
could prevent cancer and Alzheimer's disease.
What's more, Professor David Sinclair says his drugs have the
potential to help some people enjoy a healthy life until the age
of 150. However, this needs further research.
A paper in the March 8 issue of the journal, Science, explains how
the drugs have the ability to switch on the body's defences
Three of the drugs are in human trials for the treatment of
specific illnesses such as type 2 diabetes and inflammatory bowel
disease, says the University of New South Wales geneticist.
Prof Sinclair is most excited about the potential to prevent
illness and hopes to prove the drugs will have a dual purpose of
treating and preventing disease at the same time.
"My research has been criticised because it sounds too good to be
true. This paper shows it is true," he says in a telephone
interview from Harvard Medical School, where he is based.
Prof Sinclair's drugs target the enzyme SIRT1, which is switched
on naturally by calorie restriction and exercise, but it can also
be enhanced through activators such as resveratrol in red wine.
He and his colleagues have developed 4000 synthetic activators.
Each one is 100 times more potent than a glass of red wine and the
best three are the ones in human trials.
"Our drugs can mimic the benefits of a healthy diet and exercise,
but there is no impact on weight," says Prof Sinclair, who
suggests the first medicine to be marketed could be for diabetes
in about five years.
Once a significant number of people are using the drugs, it will
be possible to assess other benefits.
"We can look at 10,000 people and see if they are healthier and
living longer than the general population."
In animal tests, overweight mice given synthetic resveratrol were
able to run twice as far as slim mice and they lived 15 per cent
"My prediction is that we will delay the onset of diseases and
will not have so many people becoming chronically sick in their
50s and 60s," says Prof Sinclair.
The hope is that people will live healthily into their hundreds.
METHODS AND COMPOSITIONS FOR
EXTENDING THE LIFE SPAN AND INCREASING THE STRESS RESISTANCE
OF CELLS AND ORGANISMS
[ PDF , 8
Also published as: US2012022013 // US2005267023
// US7977049 // WO2006086454 // WO2006086454 //
JP2012176962 // AU2010219395
The invention provides methods and compositions for modulating the
life span of eukaryotic and prokaryotic cells and for protecting
cells against certain stresses, e.g., heatshock. One method
comprises modulating the flux of the NAD+ salvage pathway in the
cell, e.g., by modulating the level or activity of one or more
proteins selected from the group consisting of NPT1, PNC1, NMA1
and NMA2. Another method comprises modulating the level of
nicotinamide in the cell.
BACKGROUND OF THE INVENTION
Physiological studies and, more recently, DNA array analysis of
gene expression patterns have confirmed that aging is a complex
biological process. In contrast, genetic studies in model
organisms have demonstrated that relatively minor changes to an
organism's environment or genetic makeup can dramatically slow the
aging process. For example, the life span of many diverse
organisms can be greatly extended simply by limiting calorie
intake, in a dietary regime known as caloric restriction (1-3).
How can simple changes have such profound effects on a complex
process such as aging? A picture is emerging in which all
eukaryotes possess a surprisingly conserved regulatory system that
governs the pace of aging (4,5). Such a regulatory system may have
arisen in evolution to allow organisms to survive in adverse
conditions by redirecting resources from growth and reproduction
to pathways that provide stress resistance (4,6).
One model that has proven particularly useful in the
identification of regulatory factors of aging is the budding
yeast, S. cerevisiae. Replicative life span in S. cerevisiae is
typically defined as the number of buds or "daughter cells"
produced by an individual "mother cell" (7). Mother cells undergo
age-dependent changes including an increase in size, a slowing of
the cell cycle, enlargement of the nucleolus, an increase in
steady-state NAD<+> levels, increased gluconeogenesis and
energy storage, and sterility resulting from the loss of silencing
at telomeres and mating-type loci (8-13). An alternative measure
of yeast life span, known as chronological aging, is the length of
time a population of non-dividing cells remains viable when
deprived of nutrients (14). Increased chronological life span
correlates with increased resistance to heat shock and oxidative
stress, suggesting that cumulative damage to cellular components
is a major cause of this type of aging (14,15). The extent of
overlap between replicative and chronological aging is currently
One cause of yeast replicative aging has been shown to stem from
the instability of the repeated ribosomal DNA (rDNA) locus (16).
This instability gives rise to circular forms of rDNA called ERCs
that replicate but fail to segregate to daughter cells.
Eventually, ERCs accumulate to over 1000 copies, which are thought
to kill cells by titrating essential transcription and/or
replication factors. (16-18). Regimens that reduce DNA
recombination such as caloric restriction or a fob1 deletion
extend replicative life span (17,19,20).
A key regulator of aging in yeast is the Sir2 silencing protein
(17), a nicotinamide adenine dinucleotide (NAD<+>)-dependent
deacetylase (21-24). Sir2 is a component of the heterotrimeric
Sir2/3/4 complex that catalyzes the formation of silent
heterochromatin at telomeres and the two silent mating-type loci
(25). Sir2 is also a component of the RENT complex that is
required for silencing at the rDNA locus and exit from telophase
(26,27). This complex has also recently been shown to directly
stimulate transcription of rRNA by Pol I and to be involved in
regulation of nucleolar structure (28).
Biochemical studies have shown that Sir2 can readily deacetylate
the amino-terminal tails of histones H3 and H4, resulting in the
formation of 1-O-acetyl-ADP-ribose and nicotinamide (21-23,29).
Strains with additional copies of SIR2 display increased rDNA
silencing (30) and a 30% longer life span (17). It has recently
been shown that additional copies of the C. elegans SIR2 homolog,
sir-2.1, greatly extend life span in that organism (31). This
implies that the SIR2-dependent regulatory pathway for aging arose
early in evolution and has been well conserved (4). Yeast life
span, like that of metazoans, is also extended by interventions
that resemble caloric restriction (19,32). Mutations that reduce
the activity of the glucose-responsive cAMP (adenosine
3'5'-monophosphate)-dependent (PKA) pathway extend life span in
wild type cells but not in mutant sir2 strains, demonstrating that
SIR2 is a key downstream component of the caloric restriction
In most organisms, there are two pathways of NAD+ biosynthesis
(see FIG. 1). NAD+ may be synthesized de novo from tryptophan or
recycled in four steps from nicotinamide via the NAD+ salvage
pathway. The first step in the bacterial NAD<+> salvage
pathway, the hydrolysis of nicotinamide to nicotinic acid and
ammonia, is catalyzed by the pncA gene product (33). An S.
cerevisiae gene with homology to pncA, YGL037, was recently
assigned the name PNC1 (SGD) (34). A nicotinate
phosphoribosyltransferase, encoded by the NPT1 gene in S.
cerevisiae, converts the nicotinic acid from this reaction to
nicotinic acid mononucleotide (NaMN) (35-38). At this point, the
NAD<+> salvage pathway and the de novo NAD<+> pathway
converge and NaMN is converted to desamido-NAD<+> (NaAD) by
a nicotinate mononucleotide adenylyltransferase (NaMNAT). In S.
cerevisiae, there are two putative ORFs with homology to bacterial
NaMNAT genes, YLR328 (39) and an uncharacterized ORF, YGR010
(23,39). We refer to these two ORFs as NMA1 and NMA2,
respectively. In Salmonella, the final step in the regeneration of
NAD<+> is catalyzed by an NAD synthetase (40). An as yet
uncharacterized ORF, QNS1, is predicted to encode a NAD synthetase
In yeast, null mutations in NPT1 reduce steady-state NAD<+>
levels by ~2-fold (23) and abolish the longevity provided by
limiting calories (19). One current hypothesis explaining how
caloric restriction extends replicative life span is that
decreased metabolic activity causes an increase in NAD<+>
levels, which then stimulate Sir2 activity (reviewed in Campisi,
2000 and Guarente, 2000).
Transcriptional silencing involves the heritable modification of
chromatin at distinct sites in the genome. Silencing is referred
to as long-range repression as it is promoter non-specific and
often encompasses an entire genomic locus (1',2'). In yeast these
silent regions of DNA, which are similar to the heterochromatin of
higher eukaryotes, are subject to a wide variety of modifications
(3'). Among the most well studied of these modifications is the
reversible acetylation of histones (reviewed in 4',5').
There are two classes of enzymes that affect the acetylation state
of histones: histone acetyltransferases (HATs) and the opposing
histone deacetylases (HDACs). Compared with more transcriptionally
active areas of the genome, histones within silent regions of
chromatin are known to be hypoacetylated, specifically on the
NH2-terminal tails of core histones H3 and H4 (6'). Three classes
of histone deacetylases have been described and classified based
on homology to yeast proteins. Proteins in class I (Rpd3-like) and
class II (Hda1-like) are characterized by their sensitivity to the
inhibitor trichostatin A (TSA) (7',8'). Studies using this
inhibitor have provided a wealth of information regarding the
cellular function of these proteins, including their involvement
in the expression of regulators of cell cycle, differentiation,
and apoptosis (reviewed in 9').
Yeast Sir2 is the founding member of Class III HDACs. Sir2-like
deacetylases are not inhibited by TSA and have the unique
characteristic of being NAD<+>-dependent (10'-13'). Proteins
of this class are found in a wide array of organisms, ranging from
bacteria to humans. At least two Sir2 homologues, yeast Hst2 and
human SIRT2, are localized to the cytoplasm and human SIRT1 has
recently been shown to target p53 for deacetylation (11',13'-15').
These results indicate that not all members of this family are
specific for histones or other nuclear substrates.
The term, silent information regulator (SIR), was first coined to
describe a set of non-essential genes required for repression of
the mating type loci (HML and HMR) in S. cerevisiae (16').
Silencing in yeast is also observed at telomeres and the ribosomal
DNA (rDNA) locus (2',17'). The formation of heterochromatin at
mating type loci and the poly(TG1-3) tracts of yeast telomeres is
mediated by a heterotrimeric complex of Sir2, Sir3 and Sir4
(18',19'). At the rDNA locus, Sir2 is part of the RENT (regulator
of nuleolar silencing and telophase exit) complex, which includes
Net1 and Cdc14 (20',21'). Of these proteins, Sir2 is the only
factor that is indispensable for silencing at all three silent
The yeast rDNA locus (RLN1) consists of 100-200 tandemly-repeated
9 kb units encoding ribosomal RNAs. A major cause of yeast aging
has been shown to stem from recombination between these repeats
(25'-27') which can lead to the excision of an extrachromosomal
rDNA circle (ERC). ERCs are replicated but they fail to segregate
to daughter cells, resulting in their exponential amplification as
cells divide. ERCs can accumulate to a DNA content greater than
that of the entire yeast genome in old cells and are thought to
kill cells by titrating essential transcription and/or replication
factors (28'). Although Sir2 silences Pol II-transcribed genes
integrated at the rDNA, there is evidence that its primary
function at this locus is to suppress recombination. Deletion of
SIR2 eliminates rDNA silencing and increases the frequency that a
marker gene is recombined out of the rDNA 10-fold (29'). This
results in increased ERC formation and a dramatic shortening of
life span (29',30').
Sir2 is a limiting component of yeast longevity. A single extra
copy of the SIR2 gene suppresses recombination and extends life
span by 40% (26',31',32'). Recently, it has been shown that SIR2
is essential for the increased longevity provided by calorie
restriction (31''), a regimen that extends the life span of every
organism it has been tested on. Moreover, increased dosage of the
Sir2 homologue sir2.1 has been shown to extend the life span of
the nematode C. elegans (33') and the nearest human homologue
SIRT1, has been shown to inhibit apoptosis through deacetylation
of p53 (34',35'). These findings suggest that Sir2 and its
homologues have a conserved role in the regulation of survival at
the cellular and organismal level.
Recently, a great deal of insight has been gained into the
biochemistry of Sir2-like deacetylases (reviewed by 36'). In
vitro, Sir2 has specificity for lysine 16 of histone H4 and
lysines 9 and 14 of histone H3 (10',12',13'). Although TSA
sensitive HDACs catalyze deacetylation without the need of a
cofactor, the Sir2 reaction requires NAD<+>. This allows for
regulation of Sir2 activity through changes in availability of
this co-substrate (10'-13'). Sir2 deacetylation is coupled to
cleavage of the high-energy glycosidic bond that joins the
ADP-ribose moiety of NAD<+> to nicotinamide. Upon cleavage,
Sir2 catalyzes the transfer of an acetyl group to ADP-ribose
(10',11',15',37'). The product of this transfer reaction is
O-acetyl-ADP-ribose, a novel metabolite, which has recently been
shown to cause a delay/block in the cell cycle and oocyte
maturation of embryos (38').
The other product of deacetylation is nicotinamide, a precursor of
nicotinic acid and a form of vitamin B3 (39'). High doses of
nicotinamide and nicotinic acid are often used interchangeably to
self-treat a range of conditions including anxiety,
osteoarthritis, psychosis, and nicotinamide is currently in
clinical trials as a therapy for cancer and type I diabetes (40').
The long-term safety of the high doses used in these treatments
has been questioned (41') and the possible effects of these
compounds at the molecular level are not clear.
SUMMARY OF THE INVENTION
In one embodidment, the invention provides methods for modulating
the life span of a cell or its resistance to stress, comprising
modulating the flux through the NAD+ salvage pathway in the cell.
The method may comprise increasing or extending the life of a cell
or increasing its resistance against stress, comprising increasing
the flux through the NAD+ salvage pathway in the cell. Modulating
the flux through the NAD+ salvage pathway may occur essentially
without changing steady state levels of NAD+ and NADH and
essentially by maintaining the NAD+/NADH ratio in the cell.
Increasing the flux through the NAD+ salvage pathway may comprise
increasing the level or activity of a protein selected from the
group consisting of NPT1, PNC1, NMA1 and NMA2. The method may
comprise introducing into the cell at least one nucleic acid
encoding a protein selected from the group consisting of NPT1,
PNC1, NMA1 and NMA2, or a nucleic acid comprising at least 5
copies of a gene. Alternatively, the method may comprise
introducing into the cell at least one protein selected from the
group consisting of NPT1, PNC1, NMA1 and NMA2. The method may
comprise contacting the cell with an agent that upregulates the
expression of a gene selected from the group consisting of NPT1,
PNC1, NMA1 and NMA2. The cell may live at least about 40% longer,
or at least about 60% longer.
The invention also provides methods for increasing the resistance
of the cell against stress, e.g., heat shock, osmotic stress, DNA
damaging agents (e.g., U.V.), and inadequate nitrogen levels,
comprising increasing the flux through the NAD+ salvage pathway in
In one embodiment, modulating the life span of a cell comprises
modulating silencing in the cell. Silencing may include telomeric
silencing and rDNA recombination.
The cell whose life span can be extended or who can be protected
against stress can be a eukaryotic cell, such as a yeast cell or a
prokaryotic cell, such as a bacterial cell. The cell can be in
vitro or in vivo.
In another embodiment, modulating the life span of a cell or its
resistance to stress comprises modulating the amount of
nicotinamide and/or the ratio of NAD:nicotinamide in the cell. The
ratio of NAD:nicotinamide may be modulated by a factor of at least
about 50%, 2, 3, 5, 10 or more. For example, reducing the life
span of a cell or rendering a cell more sensitive to stress may
comprise increasing the level of nicotinamide in the cell. This
may comprise contacting the cell with an amount of nicotinamide of
about 1 to 20 mM, preferably of about 2 to 10 mM. The level of
nicotinamide in a cell may also be increased by increasing the
level or activity of enzymes involved in the biosynthesis of
nicotinamide or by decreasing the level or activity of enzymes
that degrade or inactivate nicotinamide. Enzymes which directly or
indirectly inactivate nicotinamide include PNC1; nicotinamide
N-methyl transferase (NNMT and NNT1); NPT1, and human homologs
thereof; nicotinamide phosphoribosyltransferase (NAMPRT); and
optionally nicotinamide mononucleotide adenylyltransferase
(NMNAT-1 and 2); NMA1 and 2 and human homologs thereof.
On the contrary, extending the life span of a cell or rendering
the cell more resistant (i.e., less sensitive) to stress may
comprise decreasing the level of nicotinamide in the cell. This
may be achieved by decreasing the level or activity of enzymes
involved in the biosynthesis of nicotinamide or by increasing the
level or activity of enzymes that degrade or inactivate
nicotinamide. Accordingly, increasing lifespan or stress
resistance in a cell can be achieved by increasing the activity or
level of expression of a protein selected from the group
consisting of NPT1, PNC1, NMA1, NMA2, NNMT, NAMPRT, NMNAT-1, and
NMNAT-2. Increasing lifespan or stress resistance can also be
achieved by contacting the cell with nicotinamide riboside, an
NAD+ precursor, or a biologically active analog thereof or prodrug
thereof, and optionally increasing the protein level or activity
of nicotinamide riboside kinase, e.g., Nrk1 and Nrk2 (see,
Bieganowski et al. (2004) Cell 117:495).
The invention further provides methods for identifying compounds
that modulate the life span of a cell or its resistance to stress,
comprising (i) contacting a protein selected from the group
consisting of NPT1, PNC1, NMA1, NMA2, NNMT, NAMPRT, NMNAT-1, and
NMNAT-2 with a test compound for an amount of time that would be
sufficient to affect the activity of the protein; and (ii)
determining the activity of the enzyme, wherein a difference in
the activity of the enzyme in the presence of the test compound
relative to the absence of the test compound indicates that the
test compound is a compound that modulates the life span of the
cell or its resistance to stress. The method may further comprise
contacting a cell with the test compound and determining whether
the life span of the cell has been modulated. The method may also
further comprise contacting a cell with the test compound and
determining whether the resistance of the cell to stress has been
In another embodiment, the invention provides a method for
identifying a compound that modulates the life span of a cell or
its resistance to certain types of stresses, comprising (i)
contacting a cell or a lysate, comprising a transcriptional
regulatory nucleic acid of a gene selected from the group
consisting of NPT1, PNC1, NMA1, NMA2, NNMT, NAMPRT, NMNAT-1, and
NMNAT-2 operably linked to a reporter gene, with a test compound
for an amount of time that would be sufficient to affect the
transcriptional regulatory nucleic acid; and (ii) determining the
level or activity of the reporter gene, wherein a difference in
the level or activity of the reporter gene in the presence of the
test compound relative to the absence of the test compound
indicates that the test compound is a compound that modulates the
life span of the cell or its resistance to certain types of
stresses. The method may further comprise contacting a cell with
the test compound and determining whether the life span of the
cell has been modulated. The method may also further comprise
contacting a cell with the test compound and determining whether
the resistance of the cell to stress has been modulated.
Also provided herein are methods for identifying an agent, e.g., a
small molecule that modulates the nicotinamide level in a cell.
The method may comprise (i) providing a cell or cell lysate
comprising a reporter construct that is sensitive to the level of
nicotinamide in a cell; (ii) contacting the cell with a test
agent; and (iii) determining the level of nicotinamide in the cell
contacted with the test agent, wherein a different level of
nicotinamide in the cell treated with the test agent relative to a
cell not treated with the test agent indicates that the test agent
modulates the level of nicotinamide in the cell. The cell may
further comprise a vector encoding a fusion protein that can bind
to a DNA binding element operably linked to the reporter gene. The
fusion protein may comprise at least an NAD+ binding pocket of a
nicotinamide sensitive enzyme, e.g., a Sir2 family member, and a
heterologous polypeptide. The heterologous polypeptide may be a
transactivation domain of a transcription factor. The method may
further comprise contacting a cell with the test compound and
determining whether the life span of the cell or its resistance to
stress has been modulated.
Also within the scope of the invention are computer-assisted
methods for identifying an inhibitor of the activity of a Sir2
family member comprising: (i) supplying a computer modeling
application with a set of structure coordinates of a molecule or
molecular complex, the molecule or molecular complex including at
least a portion of a Sir2 family member comprising a C pocket;
(ii) supplying the computer modeling application with a set of
structure coordinates of a chemical entity; and (iii) determining
whether the chemical entity is an inhibitor expected to bind to or
interfere with the molecule or molecular complex, wherein binding
to or interfering with the molecule or molecular complex is
indicative of potential inhibition of the activity of the Sir2
family member. The chemical entity may be an analog of
nicotinamide. Another method for identifying an inhibitor of the
activity of a Sir2 family member comprises: (i) contacting a
protein of the Sir2 family comprising at least the C pocket with a
test compound for a time sufficient for the test compound to
potentially bind to the C pocket of the protein of the Sir2
family; and (ii) determining the activity of protein; wherein a
lower activity of the protein in the presence of the test compound
relative to the absence of the test compound indicates that the
test compound is an inhibitor of the activity of a Sir2 family
In addition, the invention provides methods for treating or
preventing diseases that are associated with aging or cell death
(e.g., apoptosis) in a subject or diseases that may benefit from
the effects of calorie restriction. A method may comprise
administering to a subject in need thereof an agent that increases
the flux through the NAD+ salvage pathway or reduces nicotinamide
levels or the ratio of nicotinamide/NAD+ in the cells susceptible
or subject to cell death. Diseases can be chronic or acute and
include Alzheimer's disease, Parkinson's disease, stroke,
myocardial infarction or a metabolic disease, such as insulin
resistance. The methods of the invention for extending life span
or increasing resistance to stress can also be used to reduce
aging, e.g., for cosmetic purposes. The agent can be administered
locally or systemically. Methods for extending life span or
increasing resistance to stress can also be used on cells, tissues
or organs outside of a subject, e.g., in an organ or tissue prior
The invention also provides methods for treating or preventing
diseases in which reducing the life span of cells or rendering
cells sensitive to stress is beneficial. Such diseases include
those in which cells are undesirable, e.g., cancer and autoimmune
diseases. Methods may also sensitize cells to killing by other
agents, e.g., chemotherapeutic agents.
The methods of the invention can also be used to modulate the
lifespan and stress resistance of organisms other than mammals.
For example, the method can be used in microorganisms and plants.
In particular, the methods of the invention permit to increase the
resistance of plants to high salt, drought or disease, e.g., by
treating these with a chemical that lowers nicotinamide levels or
by genetically modifying genes that modulate the NAD+ salvage
pathway or the level of nicotinamide in cells.
Also provided are diagnostic methods, e.g., a method for
determining the general health of a subject or whether a subject
has been exposed, e.g., unknowingly exposed, to a stress
condition. A diagnostic method may also be used for diagnosing the
presence or likelihood of developing cancer. A method may comprise
(i) providing a sample of cells or bodily fluid, e.g., blood or
serum, from a subject; and (ii) determining the level of
expression of a gene or level of protein or activity thereof
encoded thereby selected from the group consisting of NPT1, PNC1,
NMA1, NMA2, NNMT, NAMPRT, NMNAT-1, and NMNAT-2, wherein a higher
level of expression of a gene or the level of protein encoded
thereby or activity thereof relative to a control sample indicates
that the general health of the subject is not adequate, acceptable
or optimal. A diagnostic method may also comprise determining the
level of NAD+, NADH, nicotinamide or other intermediate compound
of the NAD+ salvage pathway. In one embodiment, the method
comprises determining the level of NAMPRT in serum of a subject.
METHODS AND KITS FOR MEASURING ENZYME
NICOTINAMIDE RIBOSIDE AND ANALOGUES
[ PDF ,
10 MB ]
Provided herein are sirtuin-modulating compounds and methods of
use thereof. The sirtuin-modulating compounds may be used for
increasing the lifespan of a cell, and treating and/or preventing
a wide variety of diseases and disorders including, for example,
diseases or disorders related to aging or stress, diabetes,
obesity, neurodegenerative diseases, cardiovascular disease, blood
clotting disorders, inflammation, cancer, and/or flushing. Also
provided are compositions comprising a sirtuin-modulating compound
in combination with another therapeutic agent.
0001] NICOTINAMIDE RIBOSIDE AND ANALOGUES THEREOF
 The Silent Information Regulator (SIR) family of genes
represents a highly conserved group of genes present in the
genomes of organisms ranging from archaebacteria to a variety of
eukaryotes (Frye, 2000). The encoded SIR proteins are involved in
diverse processes from regulation of gene silencing to DNA repair.
The proteins encoded by members of the SIR gene family show high
sequence conservation in a 250 amino acid core domain. A
well-characterized gene in this family is S. cerevisiae SIR2,
which is involved in silencing HM loci that contain information
specifying yeast mating type, telomere position effects and cell
aging (Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000). The
yeast Sir2 protein belongs to a family of histone deacetylases
(reviewed in Guarente, 2000; Shore, 2000). The Sir2 homolog, CobB,
in Salmonella typhimurium, functions as an NAD (nicotinamide
adenine dinucleotide)-dependent ADP-ribosyl transferase (Tsang and
Escalante- Semerena, 1998).
 The Sir2 protein is a class III deacetylase which uses NAD
as a cosubstrate (Imai et al., 2000; Moazed, 2001; Smith et al.,
2000; Tanner et al., 2000; Tanny and Moazed, 2001). Unlike other
deacetylases, many of which are involved in gene silencing, Sir2
is insensitive to class I and II histone deacetylase inhibitors
like trichostatin A (TSA) (Imai et al., 2000; Landry et al.,
2000a; Smith et al., 2000).
 Deacetylation of acetyl-lysine by Sir2 is tightly coupled
to NAD hydrolysis, producing nicotinamide and a novel acetyl-ADP
ribose compound (Tanner et al., 2000; Landry et al., 2000b; Tanny
and Moazed, 2001). The NAD-dependent deacetylase activity of Sir2
is essential for its functions which can connect its biological
role with cellular metabolism in yeast (Guarente, 2000; Imai et
al., 2000; Lin et al., 2000; Smith et al., 2000). Mammalian Sir2
homologs have NAD-dependent histone deacetylase activity (Imai et
al., 2000; Smith et al., 2000). Most information about Sir2
mediated functions comes from the studies in yeast (Gartenberg,
2000; Gottschling, 2000).
 Biochemical studies have shown that Sir2 can readily
deacetylate the amino- terminal tails of histones H3 and H4,
resulting in the formation of 1-Oacetyl-ADP- ribose and
nicotinamide. Strains with additional copies of SIR2 display
increased rDNA silencing and a 30% longer life span. It has
recently been shown that additional copies of the C. elegans SIR2
homolog, sir-2.1, and the D. melanogaster dSir2 gene greatly
extend life span in those organisms. This implies that the
,S'/i?2-dependent regulatory pathway for aging arose early in
evolution and has been well conserved. Today, Sir2 genes are
believed to have evolved to enhance an organism's health and
stress resistance to increase its chance of surviving adversity.
 Caloric restriction has been known for over 70 years to
improve the health and extend the lifespan of mammals (Masoro,
2000). Yeast life span, like that of metazoans, is also extended
by interventions that resemble caloric restriction, such as low
glucose. The discovery that both yeast and flies lacking the SIR2
gene do not live longer when calorically restricted provides
evidence that SIR2 genes mediate the beneficial health effects of
this diet (Anderson et al., 2003; Helfand and Rogina, 2004).
Moreover, mutations that reduce the activity of the yeast
glucose-responsive cAMP (adenosine 3'5'-monophosphate)-dependent
(PKA) pathway extend life span in wild type cells but not in
mutant sir2 strains, demonstrating that SIR2 is likely to be a key
downstream component of the caloric restriction pathway (Lin et
 The present invention is directed to nicotinamide riboside
and analogs thereof, including their use in methods of treating
diseases or conditions, such as diabetes/insulin resistance,
hyperlipidemia and obesity. It is believed that nicotinamide
riboside and its analogs directly or indirectly activate sirtuins,
such as the human protein SIRTl. For convenience, the compounds
disclosed herein are referred to as "sirtuin modulating
compounds"; however, Applicants do not intend this designation to
mean that the biological effects of these compounds are dependent
upon sirtuin modulation (activation).
 In certain embodiments of the invention, the invention is
directed to analogs of nicotinamide riboside, particularly
compounds that are metabolized, hydrolyzed or otherwise converted
to nicotinamide riboside in vivo...
EC number 220.127.116.11
CAS number 9032-70-6
In enzymology, a nicotinamide-nucleotide adenylyltransferase (EC
18.104.22.168) is an enzyme that catalyzes the chemical reaction
ATP + nicotinamide ribonucleotide \rightleftharpoons diphosphate +
Thus, the two substrates of this enzyme are ATP and nicotinamide
ribonucleotide, whereas its two products are diphosphate and NAD+.
This enzyme belongs to the family of transferases, specifically
those transferring phosphorus-containing nucleotide groups
(nucleotidyltransferases). The systematic name of this enzyme
class is ATP:nicotinamide-nucleotide adenylyltransferase. Other
names in common use include NAD+ pyrophosphorylase, adenosine
triphosphate-nicotinamide mononucleotide transadenylase, ATP:NMN
adenylyltransferase, diphosphopyridine nucleotide
pyrophosphorylase, nicotinamide adenine dinucleotide
pyrophosphorylase, nicotinamide mononucleotide
adenylyltransferase, and NMN adenylyltransferase. This enzyme
participates in nicotinate and nicotinamide metabolism. The human
version of this protein is NMNAT1.
As of late 2007, 11 structures have been solved for this class of
enzymes, with PDB accession codes 1EJ2, 1GZU, 1HYB, 1KKU, 1KQN,
1KQO, 1KR2, 1M8F, 1M8G, 1M8J, and 1M8K.
ATKINSON MR, JACKSON JF, MORTON RK (1961). "Nicotinamide
mononucleotide adenylyltransferase of pig-liver nuclei. The
effects of nicotinamide mononucleotide concentration and pH on
dinucleotide synthesis". Biochem. J. 80 (2): 318–23. PMC 1244001.
Dahmen W, Webb B, Preiss J (1967). "The deamido-diphosphopyridine
nucleotide and diphosphopyridine nucleotide pyrophosphorylases of
Escherichia coli and yeast". Arch. Biochem. Biophys. 120 (2):
440–50. doi:10.1016/0003-9861(67)90262-7. PMID 4291828.
Kornberg A and Pricer WE (1951). "Enzymatic cleavage of
diphosphopyridine nucleotide with radioactive pyrophosphate". J.
Biol. Chem. 191 (2): 535–541. PMID 14861199.
Cell Metab. 2011 October 5; 14(4): 528–536.
Nicotinamide mononucleotide, a key
NAD+ intermediate, treats the pathophysiology of diet- and
age-induced diabetes in mice
Jun Yoshino,* Kathryn F. Mills,* Myeong Jin Yoon, and Shin-ichiro
Type 2 diabetes (T2D) has become an epidemic in our modern
lifestyle, likely due to calorie-rich diets overwhelming our
adaptive metabolic pathways. One such pathway is mediated by
nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting
enzyme in mammalian NAD+ biosynthesis, and the NAD+-dependent
protein deacetylase SIRT1. Here we show that NAMPT-mediated NAD+
biosynthesis is severely compromised in metabolic organs by
high-fat diet (HFD). Strikingly, nicotinamide mononucleotide
(NMN), a product of the NAMPT reaction and a key NAD+
intermediate, ameliorates glucose intolerance by restoring NAD+
levels in HFD-induced T2D mice. NMN also enhances hepatic insulin
sensitivity and restores gene expression related to oxidative
stress, inflammatory response, and circadian rhythm, partly
through SIRT1 activation. Furthermore, NAD+ and NAMPT levels show
significant decreases in multiple organs during aging, and NMN
improves glucose intolerance and lipid profiles in age-induced T2D
mice. These findings provide critical insights into a potential
nutriceutical intervention against diet- and age-induced T2D.
Recent studies have raised an interesting possibility that various
physiological mechanisms that mediate metabolic adaptation have
evolved in response to nutritionally scarce conditions such as
famine and drought (Lazar, 2005). In our modern, sedentary
lifestyle with calorie-rich diets, such adaptive mechanisms could
be seriously overwhelmed, causing an epidemic of obesity and T2D
worldwide (Yach et al., 2006). In mammals, one such mechanism
comprises NAMPT-mediated NAD+ biosynthesis and the NAD+-dependent
protein deacetylase SIRT1 (Haigis and Sinclair, 2010; Imai, 2010;
Imai and Guarente, 2010). NAMPT-mediated NAD+ biosynthesis and
SIRT1 together play critical roles in regulating a variety of
biological processes that include metabolism, stress response,
cellular differentiation, and circadian rhythm, and also mediating
adaptive responses to limited energy intake, such as fasting and
diet restriction (Imai, 2010). For example, in skeletal muscle,
both nutritional deprivation and exercise increase Nampt
expression through the activation of AMP-activated protein kinase
(AMPK), enhancing NAD+ biosynthesis and SIRT1 activity (Canto et
al., 2010; Fulco et al., 2008). In pancreatic ß cells, both
NAMPT-mediated NAD+ biosynthesis and SIRT1 regulate
glucose-stimulated insulin secretion (GSIS) in response to glucose
availability (Moynihan et al., 2005; Revollo et al., 2007).
Additionally, in the liver and white adipose tissue (WAT), NAMPT
and SIRT1 comprise a novel transcriptional-enzymatic feedback loop
for the regulation of circadian rhythm, a powerful effecter for
metabolism (Imai, 2010).
How nutritional and environmental perturbations affect the system
dynamics of this NAMPT/NAD+/SIRT1-driven adaptive, systemic
regulatory network, named the “NAD World” (Imai, 2010), still
remains unclear. Here we show that HFD and aging compromise
NAMPT-mediated NAD+ biosynthesis, contributing to the pathogenesis
of T2D. Importantly, we also provide evidence that promoting NAD+
biosynthesis by using nicotinamide mononucleotide (NMN), a product
of the NAMPT reaction and a key NAD+ intermediate, could be an
effective intervention against diet- and age-induced T2D.