Tài liệu Báo cáo khoa học: Small molecule regulation of Sir2 protein deacetylases ppt

MINIREVIEW
Small molecule regulation of Sir2 protein deacetylases
Olivera Grubisha
1
, Brian C. Smith
2
and John M. Denu
1
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:
(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.
FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS 4607
In 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.
Small molecule regulation of Sir2 protein deacetylases O. Grubisha et al.
4608 FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS
NAD
+
for binding to Sir2. However, K
m
values for
NAD
+
typically fall between 10 and 100 lm, whereas
IC
50
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
IC
50
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 (inhibitors, activators, 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 (IC
50
of
 120 lm), whereas the worst inhibitors tested were
the three acid analogues NAMN, NAAD and nicotinic
acid, with IC
50
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
O. Grubisha et al. Small molecule regulation of Sir2 protein deacetylases
FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS 4609
component 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.

Small molecule regulation of Sir2 protein deacetylases O. Grubisha et al.
4610 FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS
SIRT1 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 K
ii
and K
is
of 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 IC
50
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 IC
50
value 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.
O. Grubisha et al. Small molecule regulation of Sir2 protein deacetylases
FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS 4611
complete 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 silico intestinal absorption test and exhibited favour-
able binding to a conserved hydrophobic pocket in the
NAD
+
binding site. Two of these compounds exhi-
bited IC
50
values 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] report 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
Small molecule regulation of Sir2 protein deacetylases O. Grubisha et al.
4612 FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS
assay from BIOMOL, Howitz and colleagues identified
several putative ySir2 and SIRT1 activating com-
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 K
m

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 conditions 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 vitro depended 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 organisms
(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 K
i
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
O. Grubisha et al. Small molecule regulation of Sir2 protein deacetylases
FEBS Journal 272 (2005) 4607–4616 ª 2005 FEBS 4613
upregulate 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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