In addition to silencing, Sir2 activity is linked to lifespan extension in yeast [7], worms [8] and flies [9].. The dependence of sirtuin activity on NAD+ has prompted investigations into
Trang 1Small molecule regulation of Sir2 protein deacetylases
Olivera Grubisha1, Brian C Smith2and John M Denu1
1 Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA
2 Department of Chemistry, University of Wisconsin, Madison, WI, USA
Introduction
The silent information regulator 2 (Sir2) family of
pro-teins (sirtuins) are class III histone⁄ protein deacetylases
(HDACs) [1] Members of this evolutionarily
con-served family include five homologues in yeast (ySir2
and Hst1–4) and seven in humans (SIRT1–7) [2,3],
with key roles in cellular processes such as gene
expres-sion, apoptosis, metabolism and ageing [4] The
found-ing member, yeast Sir2 (ySir2), was originally
identified as a trans-acting factor involved in
transcrip-tional repression of the silent mating type loci in yeast
[5] Now it is well established that ySir2 deacetylase
activity is required for silencing at telomeres, rDNA
and the silent mating type loci, and for maintaining
genome integrity [5,6] In addition to silencing, Sir2 activity is linked to lifespan extension in yeast [7], worms [8] and flies [9] SIRT1, the most extensively studied human Sir2 orthologue, localises to the nucleus where it negatively regulates damage-responsive Fork-head transcription factors [10–12] and p53 [13–15], promoting cell survival under stress SIRT1 also dis-plays tissue-specific roles including skeletal muscle differentiation [16] and fat mobilization in white adipocytes [17] In contrast to SIRT1, SIRT2, SIRT3 and SIRT5, no NAD+-dependent protein deacetylase activity has been reported for SIRT4, SIRT6 and SIRT7 The possibility remains that SIRT4, 6 and 7 exhibit specificity toward substrates other than those tested or that these proteins catalyse a distinct reaction
Keywords
Sir2; deacetylation; sirtuin; NAD; sirtinol;
splitomicin; resveratrol
Correspondence
J M Denu, University of Wisconsin,
Department of Biomolecular Chemistry,
1300 University Ave., Madison,
WI 53706–1532, USA
Fax: +1 608 262 5253
Tel: +1 608 265 1859
E-mail: jmdenu@wisc.edu
(Received 17 March 2005, revised 6 June
2005, accepted 8 June 2005)
doi:10.1111/j.1742-4658.2005.04862.x
The Sir2 family of histone⁄ protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetyl-ated substrates, which translates into an expanding range of physiological functions These roles include control of gene expression, cell cycle regula-tion, apoptosis, metabolism and ageing The dependence of sirtuin activity
on NAD+has spearheaded investigations into how these enzymes respond
to metabolic signals, such as caloric restriction In addition, NAD+ meta-bolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases
Abbreviations
CR, caloric restriction; ERCs, extrachromosomal rDNA circles; HDAC, histone ⁄ protein deacetylase; NADases, NAD + glycohydrolases; Npt1, nicotinate phosphoribosyltransferase; OAADPr, O-acetyl-ADP-ribose; PARPs, poly(ADP-ribose) polymerases; Sir2, silent information regulator 2.
Trang 2In support of the latter, SIRT6 was recently shown
to transfer the ADP-ribose moiety of NAD+ and
undergo mono-ADP-ribosylation [18]
Unlike class I and II HDACs, which activate a
water molecule for direct hydrolysis of the acetyl
group [1], class III HDACs require NAD+as a
cosub-strate for the deacetylation reaction [19–22] NAD+
and the acetylated lysine residue on the substrate react
in a 1 : 1 ratio to form deacetylated product,
nicotina-mide, and a novel metabolite 2¢-O-acetyl-ADP ribose
(OAADPr) (Fig 1) [23–26] The consumption of
NAD+ and the generation of OAADPr by class III
HDACs probably serve as a link between deacetylation
and other physiological processes Although the roles
of OAADPr are not yet known, microinjection of
OAADPr has been shown to inhibit oocyte maturation
and to block cell division in starfish blastomeres [27]
Furthermore, unidentified enzymes found in starfish,
yeast, and human cell extracts, are able to rapidly
metabolize OAADPr [27,28] This evidence suggests
that mechanisms exist to tightly control OAADPr
lev-els Therefore, it is possible that OAADPr may act as
a secondary messenger, a cofactor, or as a metabolic
intermediate that links deacetylation of target proteins
to other cellular pathways [29] In support of this view,
recent evidence suggests that OAADPr directly
regu-lates gene silencing in yeast [30] Elegant electron
micro-scopy studies showed that a complex consisting of
Sir2, Sir3 and Sir4 undergoes a supramolecular
rear-rangement in the presence of OAADPr The authors
hypothesize that OAADPr, the product of Sir2 histone
deacetylation, directly binds to one or more
constitu-ents in the complex resulting in structural
reorganiza-tion and the ability to establish silent chromatin
domains
The dependence of sirtuin activity on NAD+ has
prompted investigations into how these enzymes might
link the cellular energy state to processes such as gene expression, cell cycle regulation, apoptosis and ageing This review will evaluate recent discoveries concerning the physiological regulation of sirtuins by NAD+ metabolites and by enzymes in the NAD+salvage path-way In addition, we will cover the use and efficacy of small molecule inhibitors and activators of sirtuin activ-ity such as sirtinol, splitomicin and resveratrol with particular focus on the ability of these compounds to regulate Sir2-mediated lifespan extension
Physiological regulation The variety of important functions involving Sir2 enzymes underscores the need to understand the mech-anisms that regulate their physiological activity The requirement of NAD+ as a cosubstrate has led to the proposal that either intracellular NAD+ or NADH concentrations or a metabolic parameter such as the NAD+⁄ NADH ratio regulates Sir2 activity (reviewed
in [4,29,31]), effectively linking Sir2 activity to the metabolic status of cells Originally, caloric restriction (CR) in yeast was thought to increase the NAD+ lev-els, which would increase the activity of ySir2 and pro-mote its role in lifespan extension [32,33] However, there is little data to support the assertion that global changes in cellular NAD+ and NADH during CR would have a significant impact on ySir2 activity In yeast grown under aerobic conditions, concentrations
of NAD+ and NADH were reported to be approxi-mately 4 mm and 0.2 mm, respectively, yielding an NAD+⁄ NADH ratio of about 20 [34] Under caloric restriction, a condition that presumably activates Sir2, this ratio fluctuated less than twofold [35], due only to
a change in NADH levels NADH was reported to act
as a competitive inhibitor of Sir2 in vitro [35], leading
to a conclusion that NADH would compete with
Fig 1 Overview of the reaction catalysed
by Sir2 protein deacetylases.
Trang 3NAD+ for binding to Sir2 However, Km values for
NAD+ typically fall between 10 and 100 lm, whereas
IC50 values for NADH range from 11 to 28 mm [36]
Therefore, it is unlikely that NADH levels would reach
high enough concentrations to significantly inhibit Sir2
activity A dramatic drop in NAD+ levels would be
more likely to be a factor in Sir2 regulation, especially
if free intracellular NAD+concentrations were to fall
in the low micromolar range Such instances could
occur through activation of NAD+-consuming enzymes
such as poly(ADP-ribose) polymerases (PARPs),
NAD+glycohydrolases (NADases), or perhaps
mono-ADP-ribosyl transferases [37] An important caveat to
the aforementioned Sir2 studies is the fact that NAD+
and NADH levels were measured from whole cell
lysates and the possibility that microdomains of these
metabolites exist where ySir functions has not been
explored For instance, NAD+ synthesizing enzymes
might be a part of a Sir2-containing complex and these
enzymes may channel NAD+directly to Sir2, creating
a microdomain of high NAD+concentrations
specific-ally accessible to Sir2
Nicotinamide, a product of the Sir2 deacetylation
reaction, is a potent physiological inhibitor of Sir2
enzymes [36,38,39] In vitro, nicotinamide yields an
IC50 of 120 lm with several Sir2 homologues [36]
Originally, it was believed that nicotinamide bound to
an allosteric site and consequently inhibited Sir2
activ-ity [40] However, it was shown later that nicotinamide
inhibition arises from its ability to condense with a
high-energy enzyme–ADP ribose–acetyl-lysine
inter-mediate to reverse the reaction, reforming NAD+and
thereby inhibiting product formation [38,39]
Nicotin-amide acts as a classical noncompetitive product
inhi-bitor of the forward deacetylation reaction and was
shown in vivo to decrease gene silencing, increase
rDNA recombination and accelerate ageing in yeast
[40] Because nuclear nicotinamide levels are estimated
to be 10–150 lm [41], it is likely that nicotinamide
regulates Sir2 activity in vivo
By the same token, enzymes involved in NAD+
sal-vage regulate Sir2 function by modulating levels of
nicotinamide and other NAD+metabolites As
depic-ted in Fig 2A, the yeast NAD+salvage pathway
con-verts nicotinamide into NAD+ through four distinct
enzymatic steps Anderson et al showed that increased
dosage of several enzymes in the NAD+salvage
path-way increased ySir2-dependent silencing, albeit to
vary-ing extents [42] Most notably, overexpression of
nicotinamidase (Pnc1) rescued silencing at telomeres
and rDNA in the presence of exogenous nicotinamide
[43], whereas deletion of PNC1 had the opposite
effect [44] Although deletion of PNC1 did not change
cellular NAD+ levels [44], a 10-fold increase in nico-tinamide was observed [41] Therefore, the known up-regulation of PNC1 expression in response to heat and osmotic shock, and oxidative exposure ([43] and refs therein) would positively regulate ySir2 activity by reducing cellular nicotinamide levels Similarly, muta-tions in nicotinate phosphoribosyltransferase (Npt1),
an enzyme that converts nicotinic acid (vitamin B3) to nicotinic acid mononucleotide (NaMN), resulted in severe rDNA and telomere silencing defects, and a threefold reduction of intracellular NAD+ levels [44] The phenotype is more severe than that seen in a pnc1 deletion strain, probably because loss of Npt1 blocks the conversion of intracellular and environmental nico-tinic acid to NAD+ Overexpression of Npt1 led to enhanced Sir2-dependent silencing but did not alter NAD+ levels [42] Anderson et al suggested that increased dosage of NPT1 might increase local avail-ability of NAD+for ySir2 without detectable changes
in steady-state NAD+ levels These data support the idea that the NAD+ salvage pathway in yeast can regulate ySir2 activity by decreasing nicotinamide lev-els and increasing the flux through the pathway to increase NAD+ concentrations At this point, it is unclear whether the cellular pools of NAD+are distinct from those accessible to Sir2 As we suggested above, small global changes in NAD+may not be sufficient to alter Sir2 function, but instead, localized synthesis of NAD+(microdomains) at the site of Sir2 function may play a more significant role in controlling activity Recently, NAD+ analogues and salvage pathway intermediates were evaluated as possible direct regula-tors (inhibiregula-tors, activaregula-tors, substrates) of Sir2 activity This analysis showed that NAD+analogues, with sub-stitution at either the nicotinamide ring or the adenine base, are poor substrates for the Sir2 reaction [36] Furthermore, only nicotinamide displayed a level of inhibition consistent with a physiological role (IC50of
120 lm), whereas the worst inhibitors tested were the three acid analogues NAMN, NAAD and nicotinic acid, with IC50 values of 26–250 mm None of the metabolites tested yielded Sir2 activation These results are consistent with the proposal that changes in cellu-lar NAD+ and nicotinamide concentrations are likely
to be the greatest contributors to the physiological regulation of Sir2 enzymes
The NAD+salvage pathway in mammals, shown in Fig 2B, does not have an equivalent of nicotinamidase Pnc1 However, both nicotinamide and nicotinic acid are converted to NAD+ through different metabolic intermediates A recent report by Revollo et al [45] demonstrated that increased dosage of nicotinamide phosphoribosyltransferase (Nampt), the rate limiting
Trang 4component in mammalian NAD+ biosynthesis,
increased total cellular NAD+ levels by 40% and
enhanced the transcriptional regulation activity of
Sir2a, a mouse Sir2 orthologue Another study found
that overexpression of nicotinamide⁄ nicotinic acid
mononucleotide adenyltransferase (Nmnat1) or an increase in NAD+ concentrations protected injured axons in a Wallerian degeneration model [46] The protection depended on the presence of SIRT1, sug-gesting that an increase in Nmnat1 activity leads to
Fig 2 (A) NAD+salvage pathway in yeast (B) NAD+salvage pathway in mammals.
Trang 5SIRT1 activation, which consequently delays Wallerian
degeneration [46] These findings provide the first
insights into the physiological regulation of
mamma-lian Sir2 orthologues by metabolic pathways that
regu-late the levels of NAD+and its precursors Also, these
studies on mammalian sirtuins serve to confirm the
link between Sir2 enzymes and metabolic pathways,
which were originally demonstrated in yeast
Addi-tional evidence for the intimate connection between
metabolism and sirtuin activity comes from a host of
other observations Sir2 from Salmonella regulates
acetyl-CoA synthetase by direct lysine deacetylation of
an important catalytic residue [47] SIRT1 was shown
to promote fat mobilization in white adipocytes by
repressing PPAR-c [17] Recently, Rodgers et al [48]
reported that SIRT1 controls gluconeogenic⁄ glycolytic
pathways in liver in response to fasting signals through
the transcriptional coactivator PGC-1a
Small molecule sirtuin inhibitors
The importance of Sir2 deacetylases in a growing
num-ber of cellular processes has created the need for better
chemical tools to study Sir2 function In particular,
selective inhibitors and activators would allow
researchers to precisely dissect the roles of Sir2
homo-logues in each organism In addition, the involvement
of human Sir2 homologues in a variety of critical
cel-lular pathways makes them attractive drug targets For
example, the ability of SIRT1 to deactivate the p53
tumour suppressor protein suggests that SIRT1
inhibi-tors might act as anticancer agents [13–15]
Further-more, the capability of a-tubulin to serve as a
substrate of SIRT2 indicates that drugs that target
SIRT2 might regulate cell division, cell cycle and cell
motility [49]
Perhaps the simplest examples of Sir2 inhibitors are
nonhydrolysable analogues of NAD+, which compete
for coenzyme binding in the active site One such
example is carba-NAD+, which is a noncompetitive
inhibitor against NAD+ with inhibition constants Kii
and Kisof 210 and 170 lm, respectively [21,50]
How-ever, NAD+ analogues such as carba-NAD+ are
generally not cell-permeable Furthermore, these
com-pounds probably serve as inhibitors or substrates for a
variety of other NAD+-dependent enzymes Therefore,
other methods, such as forward chemical genetics,
have recently been used to screen for novel small
mole-cule Sir2 regulators
Forward chemical genetics is an approach employed
to screen a library of small organic molecules for their
ability to inhibit or enhance a known phenotype
Com-pounds that produce a desired effect are then assayed
in vitro to determine if they specifically target the pro-tein of interest Using this approach, Grozinger and colleagues screened a 1600-compound library for inhi-bition of ySir2-mediated silencing at the telomere [51] The screen was designed such that reporter gene expression from the telomere caused cell death Three inhibitors, A3, M15 and sirtinol, were identified, the later two containing a 2-hydroxy-1-napthaldehyde moi-ety (Fig 3A) Of these three, sirtinol was the most potent inhibitor overall, displaying IC50 values of 68 and 38 lm against ySir2 and SIRT2, respectively
A similar strategy was used by Bedalov and cowork-ers to uncover a new class of Sir2 inhibitors [52] Their screen was designed so that inhibition of ySir2-medi-ated telomeric silencing recovered normal cell growth Such a design advantageously eliminated cytotoxic compounds as false positives Out of 6000 compounds,
11 were capable of rescuing cell growth [52] Subse-quent screening for inhibition of silencing at the HMR and rDNA locus showed that only one of the 11 com-pounds, splitomicin (Fig 3A), was effective at all three loci Splitomicin inhibited ySir2 with an IC50value of
60 lm in vitro, and based on mapping of splitomicin-resistant Sir2 mutants, the authors postulated that splitomicin acted by preventing the binding of acetyl-ated lysine substrates to ySir2 However, it is import-ant to point out that the in vitro assays were performed on whole cell extracts of an hst2 deletion yeast strain rather than purified ySir2 Therefore,
Fig 3 (A) Known inhibitors of Sir2 deacetylases (B) Examples of known activators of Sir2 deacetylases.
Trang 6complete selectivity for ySir2 deacetylase activity
can-not be inferred from this data Further evaluation of
130 splitomicin analogues revealed the requirement for
an intact lactone ring, whereas the naphthalene ring
was dispensable for efficient ySir2 inhibition [53]
In a follow-up study using 100 splitomicin
ana-logues, Hirao et al identified dehydrosplitomicin and
compound 26 as selective inhibitors of Hst1 and ySir2,
respectively (Fig 3A) [54] However, compound 26
was not as potent as splitomicin in inhibiting ySir2 In
addition to studies in yeast, sirtinol and splitomicin
have been used as general sirtuin inhibitors in
mamma-lian cells [16,46,55] However, caution should be used
in examining these data as neither compound has been
extensively characterized as an selective inhibitor of
any of the mammalian Sir2 homologues, or been tested
for nonspecific effects in mammalian cells, in
partic-ular, their effects on other NAD+-consuming enzymes
In a different approach using in silico methodology,
Tervo et al discovered novel inhibitors of human
SIRT2, a more distantly related ySir2 homologue [56]
The authors identified 15 compounds that passed an
in silicointestinal absorption test and exhibited
favour-able binding to a conserved hydrophobic pocket in the
NAD+ binding site Two of these compounds
exhi-bited IC50values in the low micromolar range in vitro,
the efficacy of which has yet to be reported in vivo
It is important to emphasize that the Sir2 inhibitors
discovered to date only have potency in the
micro-molar level, comparable to that of nicotinamide In
addition, how these molecules inhibit Sir2 activity is
unknown It is possible that these compounds compete
for NAD+ binding with their aromatic rings serving
as nicotinamide or adenine mimics If this is the case,
then it is likely that they possess activity against other
NAD+binding enzymes This effect is seen with
nico-tinamide, which in addition to its Sir2 inhibitory
activ-ity, inhibits PARPs and acts as a substrate for
nicotinamidase and nicotinamide phosphorybosyl
transferase (reviewed in [57,58]) However, it is also
possible that these Sir2 inhibitors bind to the
acetyl-lysine peptide site, as suggested for splitomicin, or to
unknown allosteric sites on the enzyme Further
stud-ies evaluating the mechanism of inhibition are needed
to allow rational improvement of these compounds
Sir2 function in metabolism and ageing
ySir2-dependent silencing at the rDNA locus not only
maintains genome integrity but also extends lifespan in
yeast One cause of ageing stems from rDNA instability
[31,59] The rDNA locus consists of 100–200 tandem
repeats encoding ribosomal RNAs and homologous
recombination between these repeats results in the for-mation of extrachromosomal rDNA circles (ERCs), which accumulate in the mother cell, causing senes-cence Although the mechanism by which ERCs cause death is unknown, the rate at which these circular DNAs accumulate correlates with the yeast lifespan [60] A single extra copy of the SIR2 gene slows ERC formation and extends lifespan by 40%, presumably
by suppressing recombination [7,42,61] Conversely, deletion of SIR2 increases the frequency of rDNA recombination 10-fold [62] and shortens lifespan by 50% [7] Increased dosage of SIR2 orthologues in Caenorhabditis elegans and Drosophila extends lifespan
up to 50% in both organisms [8,9]
Another means of extending lifespan in yeast and other organisms is through caloric restriction [63,64] The mechanism by which CR increases replicative life span in yeast has been suggested to be Sir2-mediated [61,65] It was postulated that CR extends lifespan by causing NAD+ levels to rise or NADH levels to decrease, which, in turn, increases Sir2 activity In sup-port of this hypothesis, Lin et al [35] resup-port that CR leads to a twofold decrease in NADH, without any sig-nificant change in NAD+ The authors conclude that Sir2-mediated lifespan extension during CR results from decreased NADH levels [35] However, in vitro biochemical data indicate that NADH is a poor inhi-bitor of Sir2 deacetylases [36] and that such a small change would have at best a 5% stimulation of Sir2 activity Furthermore, rapidly ageing yeast were shown
to have increased NAD+ levels [42] Collectively, the reports on the levels of NAD+during CR suggest that NAD+ levels might not be a good indicator of ySir2 activity The involvement of Sir2 in lifespan extension during CR has been recently challenged Kaeberlein
et al suggest that Sir2 acts independently of pathways mediated by CR [66] They propose that senescence due
to ERC accumulation predominates over CR If ERC formation is suppressed, lifespan extension by CR is independent of Sir2 In the PSY316 strain used previ-ously to link CR to Sir2 [68], Kaeberlein et al demon-strated that overexpression of Sir2 does not increase life span [67] Clearly, further studies will be needed to explore the role of Sir2 enzymes in determining lifespan through CR, both in yeast and higher eukaryotes
Resveratrol activation of sirtuins Evidence implicating sirtuins in lifespan extension has motivated the hunt for small molecule sirtuin activa-tors that increase lifespan in yeast, with the potential promise of identifying such compounds for human use Utilizing a commercially available deacetylase activity
Trang 7assay from BIOMOL, Howitz and colleagues identified
several putative ySir2 and SIRT1 activating
pounds in a high-throughput screen [68] These
com-pounds included a few plant polyphenols, such as
resveratrol, fisetin and butein (Fig 3B) Of the
com-pounds tested, resveratrol, a molecule found in red
wine, exhibited the highest activation of SIRT1 by
lowering the Km for the acetylated substrate, without
affecting the overall turnover rate of the enzyme [68]
Given the reported cardioprotective, chemopreventive
and neuroprotective health benefits of resveratrol
(reviewed in [69]), the prospect of resveratrol-mediated
Sir2 activation was intriguing
In yeast, resveratrol treatment reduced rDNA
recombination by 60%, providing evidence of
resvera-trol-mediated ySir2 activation [68] Curiously, no effect
on ySir2-dependent transcriptional silencing at rDNA
was observed Growing yeast in the presence of
resve-ratrol increased lifespan up to 70%, whereas no
change in lifespan was observed in a sir2 deletion
strain, further supporting the hypothesis that
resvera-trol increased lifespan by activating ySir2 [68]
Addi-tion of resveratrol under CR condiAddi-tions did not cause
an additional increase in lifespan, leading the authors
to conclude that resveratrol and CR act through the
same pathway In C elegans and D melanogaster,
treatment with resveratrol extended lifespan by 14%
and 29%, respectively [70], but this effect was not
observed in organisms that lacked wild type copies of
ySir2 orthologues, dSir2 and Sir-2.1 Similar to results
obtained in yeast, the effects of CR and resveratrol on
lifespan extension in D melanogaster were not
addit-ive, leading the authors to conclude that resveratrol
extends lifespan through a mechanism related to CR
In contrast, Kaeberlein et al found no significant
increase in lifespan, telomeric silencing or rDNA
recombination with resveratrol treatment in three
different yeast strain backgrounds [67], including the
PSY316 strain used in the original study [68] The basis
for the discrepancy between studies has not been
resolved, but may be due to variability in growth
condi-tions In an effort to elucidate the mechanism of
res-veratrol activation, Kaeberlein et al and our lab
performed a series of biochemical studies and
independ-ently determined that resveratrol activation of SIRT1
in vitrodepended on the use of a nonphysiological
sub-strate [67,71] Specifically, the activation seen with
res-veratrol in vitro required the covalent attachment of a
fluorophore at the carboxyl-group of the acetyl-lysine
residue In addition, resveratrol was unable to
signifi-cantly activate ySir2 and SIRT2 in vitro suggesting that
resveratrol binds to a unique site within SIRT1
Although resveratrol activation of SIRT1 depended on
a specific fluorophore substrate in vitro, resveratrol might still directly affect SIRT1 activity in vivo For instance, resveratrol might induce a conformational change in SIRT1, thereby increasing the catalytic effi-ciency of the enzyme for specific protein substrates con-taining an aromatic residue, such as a tryptophan, at the equivalent position of the fluorophore-containing substrates This possibility has yet to be evaluated
In mammalian cells, resveratrol was reported to enhance SIRT1-dependent cellular processes such as axonal protection, fat mobilization, and inhibition of NF-jB-dependent transcription [17,46,55] In view of the possibility that the effect of resveratrol on SIRT1
is simply an in vitro phenomenon observed with fluor-escent peptides, it would be prudent to re-examine these in vivo studies and discern whether the observed activation of SIRT1 results from a direct interaction with resveratrol or through less direct mechanisms that are induced by resveratrol and indirectly impinge upon SIRT1-dependent processes For example, resveratrol’s known antioxidant activity [72] may induce redox sen-sitive processes, which in turn activate SIRT1 Alter-natively, resveratrol might act by scavenging reactive oxygen species generated by the mitochondria, a mech-anism known to increase lifespan in many orgmech-anisms (reviewed in [72]) Perhaps SIRT1 function is sensitive
to cellular oxidants and resveratrol offers protection from inactivation, with an apparent increase in SIRT1 activity Clearly, further studies will be needed to understand the molecular link between resveratrol and the apparent cellular activation of SIRT1
Mechanism-based activation Taking advantage of the unique mechanism of nicotin-amide inhibition, Sauve et al recently reported isonico-tinamide as an activator of Sir2 activity [41] Isonicotinamide was shown to directly compete with nicotinamide for binding Nicotinamide is a potent inhibitor of the Sir2 reaction because of its aforemen-tioned ability to rebind with the enzyme and react with
a high-energy intermediate, preventing deacetylation and regenerating starting materials [38,39] The basis for the observed activation is the relief of the inherent nicotinamide inhibition by competition with isonicotin-amide, which does not readily react with the enzyme intermediate Although the Ki for isonicotinamide was
68 mm, or about three orders of magnitude worse than nicotinamide binding, in vivo yeast studies showed that millimolar levels of isonicotinamide increased Sir2-dependent silencing of the telomeric URA3 gene These results suggest that the development of higher affinity nicotinamide antagonists may provide a means to
Trang 8upregulate cellular sirtuins However, great care will be
needed to avoid crossreactivity with other nicotinamide
utilizing enzymes, in particular, those involved in
NAD+salvage and synthesis
Conclusions
In summary, we suggest that small molecule regulation
of sirtuins involves the cellular balance of NAD+ to
nicotinamide, controlled by enzymes involved in
NAD+ synthesis or salvage Small global alterations
in NAD+levels would provide insufficient changes in
Sir2 activity, but microdomains of NAD+ produced
on location may be an effective regulatory mechanism
We predict that some of these NAD+ synthetic
enzymes might be components of sirtuin complexes,
channelling NAD+directly to Sir2 enzymes
Resveratrol was reported to be a general sirtuin
acti-vator; however, recent reports question the validity of
that proposal and that resveratrol-dependent lifespan
increases are mediated directly by ySir2 activation
Although mammalian SIRT1 appears to be activated
by resveratrol treatment, the mechanistic basis for this
cellular phenomenon remains to be elucidated
Small molecule inhibitors (such as splitomicin and
sirtinol) were identified based on phenotypic screening
for compounds that phenocopy a ySir2 yeast deletion
So far, these compounds only inhibit at the
micro-molar level, and a full evaluation of their selectivity for
other sirtuins has not been determined Future rational
inhibitor design and direct high-throughput screening
against all sirtuins, particularly the mammalian
homo-logues, undoubtedly will lead to the development of
highly selective and potent inhibitors These
com-pounds will provide an essential tool to uncover the
cellular functions of these enzymes and may lead to
therapeutics that target individual sirtuins
Acknowledgements
This work was supported by NIH Grant GM065386
(to J.M.D.) and by NIH Biotechnology Training
Grant NIH 5 T32 G08349 (to B.C.S.)
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