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J. Biol. Chem., Vol. 276, Issue 42, 38837-38843, October 19, 2001
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From the
Received for publication, July 18, 2001, and in revised form, July 31, 2001
The yeast transcriptional repressor Sir2p
silences gene expression from the telomeric, rDNA, and silent
mating-type loci and may play a role in higher order processes such as
aging. Sir2p is the founding member of a large family of
NAD-dependent deacetylase enzymes, named the sirtuins.
These proteins are conserved from prokaryotes to eukaryotes, but most
remain uncharacterized, including all seven human sirtuins. A reverse
chemical genetic approach would be useful in identifying the biological
function of sirtuins in a wide variety of experimental systems, but no
cell-permeable small molecule inhibitors of sirtuins have been reported
previously. Herein we describe a high throughput, phenotypic screen in
cells that led to the discovery of a class of sirtuin inhibitors. All three compounds inhibited yeast Sir2p transcriptional silencing activity in vivo, and yeast Sir2p and human SIRT2
deacetylase activity in vitro. Such specific results
demonstrate the utility and robustness of this screening methodology.
Structure-activity relationship analysis of the compounds identified a
key hydroxy-napthaldehyde moiety that is necessary and sufficient for
inhibitory activity. Preliminary studies using one of these compounds
suggest that inhibition of sirtuins interferes with body axis formation
in Arabidopsis.
In yeast, the Sir2 family of proteins functions in transcriptional
regulation, cell cycle progression, DNA-damage repair, and aging (1,
2). Sir2p deacetylates histones in an NAD-dependent manner
(reviewed in Ref. 3), which is thought to account for both its
transcriptional silencing activity at the telomeric, rDNA, and silent
mating-type loci (reviewed in Ref. 1), as well as its ability to
regulate lifespan in response to metabolic rates (reviewed in Ref. 2).
Deacetylation of histones is a known mechanism for causing chromatin
condensation and transcriptional silencing; indeed, the loci regulated
by Sir2p are hypoacetylated (4), and deletion of Sir2 results in a
dramatic increase in the acetylation of the histones at these sites
(5). Furthermore, it is believed that Sir2p regulates aging in yeast by
forming condensed chromatin structures at the rDNA locus. This
decreases recombination rates, thereby decreasing the formation of
toxic extrachromasomal rDNA circles, which shorten lifespan (reviewed in 2). Therefore, according to this hypothesis, increasing Sir2p activity or overexpressing Sir2p leads to an increased lifespan in
yeast. Interestingly, lifespan can also be extended by glucose deprivation, and this is postulated to be mediated by the
NAD-dependence of Sir2p. Decreasing glucose intake drops metabolic
rates, resulting in the oxidation of NADH to NAD. Increasing the amount
of available NAD increases Sir2p activity, thereby preventing the
formation of toxic extrachromasomal rDNA circles. The critical
role of NAD levels in this process is evident by the fact that
mutations in an NAD biosynthetic pathway in yeast leads to a decrease
in lifespan that is not rescued by mutation in the glucose-sensing
pathways or overexpression of Sir2p (6). Thus, the metabolic and
oxidative state of the cell may be linked directly to transcriptional
regulation of certain genes via Sir2p, which in turn may regulate lifespan.
Sir2p represents the founding member of a large family of
NAD-dependent deacetylases, termed the sirtuins (7, 8).
These enzymes are highly conserved from prokaryotes to eukaryotes, with five sirtuins in yeast and seven in humans. The sirtuins contain a
conserved catalytic domain of ~275 amino acids, and mutation of the
highly conserved residues in this domain abrogates enzymatic activity
(7, 9). Although Sir2p and yeast HST1p appear to be involved directly
in transcriptional silencing, the biological functions of the other
sirtuins remain unknown, though recently a Sir2p homolog in
Caenorhabditis elegans was shown to regulate lifespan in
this organism, as well, albeit via a different mechanism (10). Several
yeast and human sirtuins are not localized to the nucleus (11-13),
suggesting that these proteins may have diverse roles in the cell aside
from transcriptional regulation. Indeed, one Salmonella
sirtuin was found to be competent to function in the cobalamine
biosynthetic pathway (14), and thus some of these enzymes may have
small molecules, rather than proteins, as substrates.
One approach to dissecting the biological function of sirtuins in a
variety of biological systems is reverse chemical genetics. Rather than
deleting or mutating the sirtuin genes in a particular system, a
cell-permeable small molecule inhibitor of sirtuins can be used to
block sirtuin activity. Such an inhibitor would permit fine temporal
regulation of sirtuin inhibition, and thus the function of sirtuins in
essential or developmental processes could be studied. Furthermore,
these inhibitors should be easily transferable to model organisms or
systems in which it is extremely difficult to specifically delete
genes. Indeed, inhibitors of the family of histone deacetylases,
HDACs,1 have greatly
accelerated the characterization of the cellular function of these
proteins. For example, inhibition of HDAC activity by the nanomolar
inhibitor trichostatin A (TSA) produces a dramatic morphological change
in tumor cell lines, which subsequently was shown to be caused by an
HDAC-dependent transcriptional regulation of gelsolin, an
actin-binding protein (15). Thus, a general sirtuin inhibitor could be
used to refine our understanding of yeast sirtuins and initiate
investigations into the function of insect, vertebrate, and human
sirtuins. Thus far, however, no cell-permeable sirtuin inhibitors have
been reported. A non-hydrolyzable NAD analog (carbanicotamide adenine
dinucleotide) does inhibit sirtuin activity in vitro (16),
but such molecules are not cell-permeable and undoubtedly inhibit other
NAD-dependent enzymes, as well. Thus, these NAD analogs
would not be suitable for in vivo studies.
Herein we describe the identification of a class of cell-permeable
small molecule inhibitors of sirtuin NAD-dependent
deacetylase activity from a high throughput cell-based screen of 1600 unbiased compounds. The primary screen was for inhibitors of
Sir2p-mediated silencing of a URA3 reporter gene integrated into a
telomeric locus. This yeast strain can grow in the presence of
5-fluoroorotic acid (5-FOA), but the addition of an inhibitor of
Sir2p will result in expression of the URA3 gene and death in the
presence of FUra. Three compounds of 1600 scored positively in this
screen, and two of these possessed substructures derived from
2-hydroxy-1-napthaldehyde. All three compounds inhibit Sir2p
transcriptional silencing in vivo, in the context of
different Sir2p complexes and at different chromosomal domains. These
compounds inhibit yeast Sir2p, as well as human SIRT2 activity in
vitro, demonstrating that they inhibit enzymatic activity directly
and function as general inhibitors of sirtuins. Interestingly,
2-hydroxy-1-napthaldehye and other compounds containing this moiety
also inhibit Sir2p activity in vivo and in vitro,
suggesting that these small molecules represent a new class of
inhibitors of sirtuins. Preliminary studies with one these inhibitors
(sirtinol; Sir two inhibitor
napthol) suggest that sirtuins do not regulate
global histone acetylation levels in mammalian cells and that they may
be involved in body-axis formation during plant development.
Yeast Strains
The genotype for the TEL::URA3 strain (UCC1001) is as
follows: MAT Small Molecules
The compounds that were screened were derived from two
libraries. The first was purchased from ChemBridge Corporation (San Diego, CA), and the second, an Institute of Chemistry and Cell Biology diversity set, consisted of compounds synthesized at the Harvard Institute of Chemistry and Cell Biology (ICCB; Harvard University) using diversity-oriented synthesis (17). Compound A3
(8,9-dihydroxy-6H-(1)benzofuro[3,2-c]chromen-6-one) was
synthesized by Hua Miao (ICCB). Compounds M15
(1-[(4-methoxy-2-nitro-phenylimino)-methyl]- naphthalene-2-ol) and
Sirtinol
(2-[(2-hydroxy-naphthalen-1- ylmethylene)-amino]-N-(1-phenyl-ethyl)-benzamide) were purchased directly from ChemBridge. 2-Hydroxy-1-naphthaldehyde, 2-hydroxy-1-naphthoic acid, 4-methoxy-2-nitroaniline, and trichostatin A were purchased from Sigma, whereas
2-amino-N-(1-phenyl-ethyl)-benzamide was synthesized by
Travis Dunn (Harvard University).
In Vivo Yeast Screen
General Procedures--
Several freshly streaked colonies of the
desired yeast strain were resuspended in 1 ml of YPDA medium
(YPD/0.003% adenine hemisulfate) by vortexing, diluted in 50 ml of
YPDA, and vortexed again. A stock solution of 15% 5-FOA in
Me2SO was used to produce 0.4-0.8% 5-FOA solution
for the TEL::URA3, rDNA::URA3, and
HML::URA3 strains. An equivalent percentage of
Me2SO was used for the untreated samples to account for
cytotoxicity. A multidropper apparatus (Labsystems) was used to add
yeast to clear bottom 96-well or 394-well plates in volumes of 40-100
µl. Although this density of yeast in the well was not visible
initially, a wild-type strain typically saturated the bottom of the
well after 24-48 h of growth at room temperature. To test the dose
response on the three strains, 100 µl of the various yeast strain
suspensions were transferred to a 96-well plate, and 0.5 µl of the
compounds or Me2SO were added. Growth was monitored by
visual inspection or by the A600 of the cultures
using a Wallac spectrometer.
ICCB Diversity Set Compound Library
A collection of 400 compounds, called an ICCB diversity set of
compounds, was screened for activity. These compounds were contributed
by members of the Harvard Institute of Chemistry and Chemical Biology
(ICCB; Harvard University), and were aliquoted into 96-well plates at
dilutions between 10 and 20 mM. For this screen, 100 µl of the TEL::URA3 yeast suspensions ± 5-FOA were transferred to 96-well plates. 100 nl of 10-20 mM compound
stocks were manually pin-transferred to these plates, and thus the
compounds were tested at concentrations of 10-20 µM.
Growth was monitored by visual inspection and scored after 1, 2, and 3 days.
ChemBridge Library
The screens tested 1200 compounds of plates from the ICCB
ChemBridge Library (plates 1-3). An automated robot pin-transferred 40 nl of compound to each well of the plates. For this screen, 40 µl of
the yeast suspensions, ± 5-FOA, were transferred to 384-well plates.
40 nl of 5 mg/ml stocks of compounds were pin-transferred to the
plates, and thus these were tested at a concentration of ~10
µM. Growth was monitored by visual inspection and
scored after 1, 2, and 3 days.
DNA Constructs and Mutagenesis
The bacterial expression construct for GST-SIRT2 (amino
acids 18-340) in the pGex4T3 vector (Amersham Pharmacia
Biotech) was generously provided by M. Finnin and N. Pavletich
(Memorial Sloan-Kettering Cancer Research Center, New York).
Protein Expression and Purification
BL21-(DE3)LysS cells were transformed with the GST-SIRT2 (amino
acids 18-340)/pGex4T3 construct. Cells were grown to an
A600 of 0.3 in NCZYM medium (Life Technologies, Inc.)
and induced for 5 h with 0.5 mM
isopropyl-1-thio- Immunoprecipitation of HDAC1 from HeLa Cells
HeLa cells were lysed in JLB containing a complete protease
inhibitor mixture (Roche Molecular Biochemicals). Lysis
proceeded for 15 min at 4 °C, after which the cellular debris was
pelleted by centrifugation at 14,000 rpm for 5 min. Endogenous HDAC1
was purified from the supernatant by incubation with anti-HDAC1
antibody (Sigma) for 30 min and then precipitation with protein G beads (Life Technologies, Inc.) for 45 min at 4 °C. The beads were washed three times with JLB at 4 °C and resuspended in HD buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 10%
glycerol) for the histone deacetylase assays.
In Vitro Histone Deacetylation Assays
1.5 µg of recombinant human GST-SIRT2 (amino acids 18-340) or
0.5 µg of recombinant yeast Sir2p were incubated for 2 h at 30 °C in 50 µl of assay buffer (50 mM Tris-HCl,
pH 8.8, 4 mM MgCl2, 0.2 mM
dithiothreitol) (9), with or without 50 µM NAD and
acetylated HeLa histones (1000 cpm), purified by acid extraction. HDAC
activity was determined by scintillation counting of the ethyl
acetate-soluble [3H]acetic acid (19).
Antibodies and Western blot analysis
Antibodies to tetra-acetylated histones H3 and H4 were purchased
from Upstate Biotechnology (Lake Placid, NY), and antibodies to
acetylated Analysis of Cell Morphology
100 × 105 HeLa cells were plated onto
gelatin-coated coverslips and treated with 1% Me2SO, 10 µM sirtinol, 25 µM sirtinol, or 1 µM TSA for 24 h. Cells were fixed in 4%
formaldehyde and viewed with a light microscope.
Analysis of Effect of E8 on Arabidopsis Development
Seeds (Arabidopsis thaliana ecotype Lansberg erecta
(Ler)) were surface-sterilized by treatment with 70%
ethanol for 1 min and then 50% Clorox/0.1% Tween 20 for 8 min
and washed 6 times with sterile water. The seeds were air-dried on
sterile filter paper in a laminar flow hood for 3 h.
Murashige and Skoog (MS) medium (pH 5.7; Sigma) (20) containing 0.8%
PhytoAgar (Life Technologies, Inc.) was pipetted into 96-well, flat
bottom microtiter plates. Me2SO (99.9%; EM Science) or
sirtinol solutions in Me2SO were pipetted into the warm,
liquid agar and thoroughly mixed. The agar was allowed to set for 30 min prior to manually arraying the seeds into each well with a
moistened, sterile pipette tip (4-6 seeds per well). Plates were
sealed and incubated for 3 days in the dark at 5 °C to break
dormancy (stratification). Plates were incubated thereafter at 25 °C
under continuous white fluorescent light, and seedling development was
measured in terms of days of growth after transfer to 25 °C.
For unstained whole-mount preparations, plants were placed in FluorSave
reagent (CalBiochem) on glass slides and sealed with a coverslip. For
staining of the vasculature, plants were fixed in acetic acid:95%
ethanol (3:1) for 1 h, incubated in 25% formaldehyde for 2 h, dehydrated for 1 h in 95% ethanol, stained for 1 min in 1%
safranin-O (Sigma) in 95% ethanol, hydrated through an ethanol series
to water, and mounted on slides with FluorSave (21).
Screening for Compounds That Derepress Telomeric
Genes--
A high throughput, cell-based URA3 reporter-based screen
was used to identify candidate compounds that interrupt the Sir2p silencing pathway at the telomeres (1). To assess telomeric silencing,
the yeast strain (TEL::URA3) containing a URA3 reporter gene
inserted near the telomeres was used. Addition of a small molecule
inhibitor of Sir2p should derepress the URA3 gene, resulting in death
in the presence of 5-FOA. A parallel screen was conducted in the
absence of 5-FOA to identify small molecules that were simply cytotoxic
(Fig. 1). As a control, a strain with a
URA3 reporter gene integrated into a transcriptionally active internal chromosomal region (non-TEL::URA3) was tested for growth in
the presence of 5-FOA (see Fig. 1B; note that in the absence
of growth, the media in the well is clear, whereas growth
results in a layer of yeast covering the bottom of the well, and thus
the well appears opaque).
Two libraries, with a total of 1600 compounds, were screened for small
molecules that derepress telomeric silencing. Compounds were
pin-transferred to a 96- or 384-well clear bottom plate containing yeast in YPDA media, with or without 0.4% 5-FOA. This method
transferred 40 or 100 nl of the 10 to 20 mM stock solutions
into a volume of 40 or 100 µl. Thus, this screen was conducted at a
concentration of 10 to 20 µM. An example of a positive
from this screen is shown in Fig. 1B. Twelve hits were
identified in the initial screen, three of which were verified as
reproducible positives in a subsequent retest of these compounds in the
same assay. The structures of these compounds are shown in Fig.
1C. Note that all of these compounds are planar and
aromatic, similar in structure to the adenine and nicotinamide moieties
of NAD. Furthermore, two of the three compounds (sirtinol and M15)
contain substructures derived from 2-hydroxy-1-napthaldehyde.
Dose Response for in Vivo Inhibition of Sir2p-mediated Silencing at
Three Loci--
Sir2p can repress transcription at the telomeric,
silent mating-type, and rDNA loci, as a member of the
Sir2·-3·-4 complex at the first two loci and as a member of
the Net1·cdc14·Sir2 complex for the rDNA locus (reviewed in Ref.
1). To ensure that the three compounds specifically targeted Sir2p and
did not simply disrupt its interactions with other proteins, the
compounds were resynthesized or purchased, and the new stocks were
tested for depression of URA3 reporter genes at the telomeric,
HM, and rDNA loci. All three compounds inhibited growth in the
three strains of yeast in the presence of 5-FOA, even after more than 3 days of growth (Fig. 2). A3 and M15
inhibited growth at 100 µM, whereas sirtinol inhibited
growth at 25 µM. Thus, these compounds appear to inhibit
Sir2p transcriptional silencing activity directly.
Inhibition of Yeast Sir2p and Human SIRT2--
The ability of
these compounds to abrogate silencing at several chromosomal domains
controlled by Sir2p suggested that they directly inhibit Sir2p
deacetylase activity. To confirm this, the three compounds were tested
for their ability to inhibit NAD-dependent histone
deacetylase activity of purified recombinant yeast Sir2p in
vitro. Furthermore, these compounds were assessed for their ability to generally inhibit sirtuin deacetylase activity by testing them with the catalytic domain of human SIRT2 (amino acids 18-340). A3
inhibited yeast Sir2p and human SIRT2 at IC50 values of
~70 and 45 µM, respectively (Table
I), whereas sirtinol was slightly more potent against the human sirtuin, with IC50 values of
70 and 40 µM. M15 did not inhibit either enzyme
particularly well. Unfortunately, M15 was not very soluble in aqueous
media and precipitated out at concentration higher than 50 µM. Thus, it is possible that M15 can inhibit these
enzymes but at concentrations higher than solubility allows.
Alternatively, M15 may act on a common unidentified component in the
Sir2p complexes.
Structure-Activity Relationship Analysis--
Sirtinol and M15 are
synthesized by coupling 2-hydroxy-1-naphthaldehyde to either
2-amino-N-(1-phenyl-ethyl)-benzamide or 4-methoxy-2-nitroaniline, respectively (Fig.
3A). Thus, it is possible that
the 2-hydroxy-1-naphthaldehyde moiety is primarily responsible for the
inhibitory activity of these compounds. To test this hypothesis, the
individual fragments of sirtinol and M15 (2-hydroxy-1-naphthaldehyde,
2-amino-N-(1-phenyl-ethyl)-benzamide and
4-methoxy-2-nitroaniline) were tested for their ability to inhibit
SIRT2 activity in vitro at 75 µM (Fig.
3B). Sirtinol potently inhibited SIRT2 at 75 µM, whereas 2-hydroxy-1-naphthaldehyde partially inhibited activity. The amine monomers did not significantly reduce activity. TSA, a specific nanomolar inhibitor of HDACs, did not inhibit sirtuin activity. Thus, 2-hydroxy-1-naphthaldehyde did inhibit
sirtuin deacetylase activity in vitro but at much higher concentrations than required for sirtinol.
2-Hydroxy-1-naphthaldehyde was also tested for its ability to inhibit
Sir2p transcriptional silencing in yeast, at the telomeric, HM, and
rDNA loci (Fig. 3C). This compound was able to activate transcription of the URA3 reporter at 50 µM in all cases
but became cytotoxic at 200 µM.
Inhibition of HDAC1 Activity--
A3, sirtinol, and
2-hydroxy-1-naphthaldehyde were tested for their ability to inhibit
human HDAC1 (Table II) to assess
their specificity for sirtuin deacetylases. A3 is the only
compound that significantly inhibits HDAC activity at 50 µM. This is somewhat surprising, because it does not
resemble any known HDAC inhibitors (22). Sirtinol and
2-hydroxy-1-naphthaldehyde did not affect HDAC1 activity at 50 and 100 µM, respectively, suggesting that they specifically
inhibit sirtuin deacetylase activity.
Effect of Sirtinol Treatment on Mammalian Cells--
Treatment of
mammalian cells with the potent HDAC inhibitor TSA causes acetylation
of histones (15) and
To determine whether sirtuins are involved in the regulation of the
global acetylation state of histones or tubulin, primary fibroblast
cells were treated with 10 and 50 µM sirtinol, as well as
1 µM TSA, for 24 h (Fig. 4A). The cells
were harvested and lysed, and equal amounts of total cellular protein
were separated by SDS-polyacrylamide gel electrophoresis and analyzed
for acetylation levels of histones and Effect of Sirtinol Treatment on Arabidopsis
Development--
Arabidopsis seeds were germinated and
grown for 4 days on agar containing 1% Me2SO, 25, 50, and
100 µM sirtinol. Plants were visually inspected under a
dissecting light microscope. Seedlings grown in the presence of
Me2SO alone developed normally, with a long hypocotyl
(seedling stem) and primary root apparent by 3 days of growth. In the
case of plants treated with sirtinol, the hypocotyl was significantly
shorter and thicker, and the primary root was completely absent (Fig.
5A). This phenotype was
apparent at all sirtinol concentrations, though was most severe
(shortest hypocotyls and smallest cotelydons) at 100 µM.
All plants that germinated in sirtinol exhibited this phenotype in two
independent experiments.
Similar phenotypic alterations in the formation of the apical-basal
body axis (which collectively refers to the hypocotyl and primary root
structures) have been observed in the Arabidopsis mutant
MONOPTEROS (24, 25). In this case, vascularization of the
seedling is also severely affected, and thus only the central vascular
strands are apparent. To assess the state of the vascular system in the
seedlings treated with sirtinol, plants were stained and visualized
under a light microscope. Although Me2SO-treated plants
have distinct veins apparent in the hypocotyl and cotyledons after 5 days of growth, sirtinol-treated plants have distinct veins only
apparent in the hypocotyl (Fig. 5B). Note, however, that the
cotyledons of the treated plants are somewhat smaller and might develop
normal veins at a much slower rate than untreated seedling. Thus,
sirtinol inhibits body axis formation and vascularization in growing plants.
Herein we described a high-throughput, phenotypic screen in cells
using 1600 unbiased small molecules. The assay was aimed to uncover
cell-permeable inhibitors of yeast and human sirtuins; indeed it led to
the identification of a class of molecules that specifically inhibit
yeast Sir2p and human SIRT2. The initial screen identified inhibitors
of Sir2p silencing activity in yeast, and this was followed by several
subsequent secondary assays. First, the three compounds identified in
the primary screen were tested for their ability to inhibit Sir2p
transcriptional silencing in the context of different Sir2p complexes
and at different chromosomal domains. All three compounds scored
positively in this assay, suggesting that they directly inhibited Sir2p
transcriptional silencing activity. Second, these compounds were tested
for their ability to inhibit yeast Sir2p and human SIRT2 activity
in vitro. Two of the three (A3 and sirtinol) are inhibitors
of both enzymes, with IC50 values ranging from 40 to 70 µM. Third, these two compounds were tested for their
ability to inhibit HDAC1 histone deacetylase activity in
vitro. Although sirtinol had no effect on HDAC1 activity, A3 does
weakly inhibit it, suggesting that it may not specifically inhibit
sirtuin deacetylase activity. Thus, sirtinol is the first reported
cell-permeable inhibitor of the sirtuin class of deacetylases. Furthermore, because it is competent to inhibit sirtuin enzymes from
two different species, it is likely to be a general sirtuin inhibitor
and thus transferable between a number of systems.
Screening Methodology--
This yeast-based reporter gene assay
provides a platform for screening for small molecule modulators of
Sir2p and possibly of other transcriptional regulators. The screen is
high throughput, requires minimal labor, is easy to score for
positives, and does not require specialized equipment. Furthermore, the
screen gave remarkably specific results, as is evident by the fact that
all three hits functioned in secondary assays. It is interesting to note that though the screen was for inhibitors of yeast Sir2p, both A3
and sirtinol were more potent inhibitors of human SIRT2, thus further
demonstrating the versatility of this screening methodology. Finally,
because it is a cell-based screen, it is selective for compounds that
are cell-permeable and therefore more useful in vivo.
Identification of a Class of Sirtuin Inhibitors--
Two of the
compounds (sirtinol and M15) identified in the primary screen were
derived by coupling 2-hydroxy-1-naphthaldehyde to an amine.
2-Hydroxy-1-naphthaldehyde alone also inhibited human SIRT2 activity,
though not as well as sirtinol. Subsequent testing of other
2-hydroxy-1-naphthaldehyde derivatives from the ChemBridge library
revealed that a subset of these inhibited SIRT2 in vitro deacetylase activity at approximate IC50 values of 50-70
µM, as well (data not shown). Thus, it appears that the
naphthaldehyde group makes important contacts with the enzyme active
site and is primarily responsible for the inhibitory activity of these molecules. It may be possible to produce more potent inhibitors by
making analogs of these 2-hydroxy-1-naphthaldehyde derivatives. An even
more exciting possibility is that it will be possible to generate
specific inhibitors of the different sirtuin homologs by modifying this
basic structure.
Preliminary Studies of Effects of Sirtinol Treatment--
This
general inhibitor of sirtuin enzymes can be applied to the study of the
biological function of sirtuins in a variety of systems and species.
Thus far, the in vivo function of sirtuins has only been
well understood in yeast, and even in this case there are several
sirtuins that have not been characterized. Unfortunately, because of
the lack of characterization of sirtuins in mammals, there is no direct
method to test sirtinol for its ability to inhibit sirtuins in human
cells. Attempts to create an artificial system in which a reporter gene
was repressed by the catalytic domain SIRT2 were unsuccessful, perhaps
because SIRT2 does not function in transcriptional regulation, or the
recombinant protein was unable to recruit essential associated
proteins. However, given that sirtinol can penetrate the yeast cell, it
is expected that it can enter human cells, as well. Furthermore,
because sirtinol inhibits both yeast Sir2p and human SIRT2 in
vitro, it may be competent to inhibit all sirtuin enzymes, given
the high degree of conservation of the catalytic domains. Unlike TSA,
treatment of human primary fibroblasts with sirtinol did not cause
global changes in acetylation of histones and tubulin, nor did it
induce a morphological changes in the HeLa tumor cell line. This
provides further evidence that sirtinol is selective for sirtuin
deacetylases and does not affect HDACs and suggests that sirtuins
perform very different functions than HDACs in vivo.
Preliminary work with sirtinol on Arabidopsis,
which contain at least two sirtuins (GenBankTM accession
numbers 2656026 and 9955507), suggests that these proteins play
a critical role in apical-basal body axis development and vascularization. Seedlings grown in sirtinol have thick, short hypocotyls and no primary roots, and the vein system was abnormal in
the cotelydons. This morphology is very similar to that observed in
MONOPTEROS mutants (24, 25). The MONOPTEROS
protein is a member of a family of transcriptional regulators, several
of which have been shown to regulate expression of auxin-responsive genes (24, 26). Auxin is a plant hormone that is involved in
vascularization and elongation of the hypocotyl and primary root.
Inhibition of auxin transport through the plant by certain compounds
results in seedlings with shortened hypocotyls and roots or lacking in
the primary root structure altogether (27, 28). Interestingly,
seedlings treated with these auxin transport inhibitors still possess
normal vein development in the cotyledons but not in the first true
leaves (28). Thus, the mutation of MONOPTEROS and the
phenotype produced by treatment with sirtinol do not match exactly that
observed by auxin transport inhibition but do suggest that these
operate on a related or the same pathway. At this stage, it is unclear
whether the observed phenotype is due solely to the inhibition of
sirtuin activity, though it does provide an interesting avenue to
explore with respect to the transcriptional regulation of basal-apical
body axis formation in plants. Sirtuins may control the synthesis of
auxin or auxin-transporting proteins, or they may regulate the cellular
transcriptional response to auxin.
Future Directions--
Reverse chemical genetics using small
molecule modulators of proteins can be used to probe the cellular
functions of proteins in different systems. Such inhibitors can be used
in a temporally defined manner and are applicable in a variety of
experimental systems. Fine temporal control has not been possible with
even the best characterized sirtuin, yeast Sir2p, because no
temperature-sensitive alleles have been reported. Thus, inhibitors of
sirtuins will allow us to refine our understanding of, for example,
yeast Sir2p, by comparing the immediate effects of a loss of Sir2p
function with those steady-state cellular changes observed in
yeast strains in which Sir2 has been deleted. A particularly powerful
approach for assessing the resulting effect of sirtuin inhibition, by
analogy to studies of HDACs, is global transcriptional profiling (29). Furthermore, these small molecules can be used to initiate studies of
sirtuin function in a wide variety of experimental systems, ranging
from yeast to human cell cultures, to organisms such as zebrafish
and Arabidopsis.
Sirtuin proteins in yeast and bacteria have diverse biological
functions, including the regulation of transcription, aging, the
control of the cell cycle, DNA-damage repair, and metabolism (reviewed
in Ref. 1) (14). With seven human sirtuins (8), it is expected that the
roles of these proteins in mammalian cells are equally diverse, but
thus far these proteins have not been characterized. The identification
of a class of small molecule inhibitors of the sirtuin family of
deacetylases will now allow for a broad investigation of the functions
of these proteins and the cellular pathways in which they are involved.
We thank Mary Kay Pflum and
Christian Hassig for critical reading of the manuscript.
*
This work was supported in part by NIGMS, National
Institutes of Health Grant GM38627 (to S. L. S.). Research at the
Harvard Institute of Chemistry and Chemical Biology is supported by the NCI, the Keck Foundation, Merck KGOA and Merck & Co. S. L. S. is an
Investigator at the Howard Hughes Medical Institute. H. E. B. is
supported by a post-doctoral fellowship from the Jane Coffin Childs
Memorial Fund for Medical Research (sponsored by Merck & Co.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Chemistry and Chemical Biology, Harvard University, 12 Oxford St.,
Cambridge, MA 02138. Tel.: 617-495-5318; Fax: 617-495-0751; E-mail:
sls@slsiris.harvard.edu.
Published, JBC Papers in Press, August 1, 2001, DOI 10.1074/jbc.M106779200
The abbreviations used are:
HDAC, histone
deacetylase;
TSA, trichostatin A;
GST, glutathione
S-transferase;
5-FOA, 5-fluoroorotic acid;
ICCB, Harvard
Institute of Chemistry and Cell Biology;
HM, homothallic
mating-type locus;
JLB, Jurkat lysis buffer.
Identification of a Class of Small Molecule
Inhibitors of the Sirtuin Family of NAD-dependent
Deacetylases by Phenotypic Screening*
,,,
Department of Chemistry and Chemical
Biology, the Howard Hughes Medical Institute, Harvard
University, Cambridge, Massachusetts 02138 and the
Harvard Institute of Chemistry and Cell Biology and the
§ Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52 lys2-801 ade2-101
trp
63 his3200 leu2-1 TEL adh4::URA3. The genotype for the
HML::URA3 strain (UCC3515) is as follows MAT
ura3-52 lys2-801 ade2-101 trp63
his3200 leu2-1 HML::URA3. The genotype for
non-TEL::URA3 strain (UCC1003), where the URA3 reporter is
inserted in an internal chromosomal domain, is as follows:
MAT ura3-52 lys2-801 ade2-101
trp63 his3200 leu2-1
adh4::URA3. These strains were obtained from
D. Gottshling (Fred Hutchinson Cancer Research Center, Seattle, WA).
The genotype for the rDNA::URA3 strain is as follows:
MAT his3 200 leu2- ura3-167 rDNA::Ty1-URA3. The control
strain for this is non-rDNA::URA3, where the URA3 gene is
inserted into an unknown site outside of the rDNA locus, has the
following genotype:: MAT his3 200 leu2- ura3-167
???::Ty1-URA3. These strains were obtained
from J. D. Boeke (Johns Hopkins University).
-D-galactopyranoside (Sigma) at room
temperature. Cells were lysed in 50 mM Tris-HCl, pH 8, 200 mM NaCl, 5 mM dithiothreitol, and 1 mg/ml
lysozyme (Sigma) with a freeze-thaw cycle. 40 units of DNase I
(Promega) were added to the solution and incubated for 20 min on ice.
The lysate was clarified by centrifugation at 14,000 rpm for 15 min,
and the incubated was clarified with 1.5 ml of glutathione-agarose
beads (Amersham Pharmacia Biotech) at 4 °C for 1 h. The beads
were washed twice with phosphate-buffered saline, and the protein was
eluted by incubation with 10 mM glutathione (Sigma) in
JLB (50 mM Tris-HCl, pH 8, 150 mM NaCl,
10% glycerol, 0.5% Triton X-100) for 1 h at 4 °C and twice
with 10 mM glutathione, JLB for 30 min. This solution was
then dialyzed overnight against JLB and stored at 
70 °C. Expression and purification of recombinant yeast Sir2 protein has been described previously (18).
-tubulin were purchased from Sigma. 1.2 × 106 primary foreskin fibroblast cells were plated in 6 ml of DMEM/10% FBS media, and treated with 0.5%
Me2SO, 10 µM sirtinol, 50 µM sirtinol, or 1 µM TSA for 24 h. Cells were
trypsinized, harvested, and lysed in 75 µl of JLB. The protein
concentrations of the lysates were determined by a Bradford assay, and
a normalized amount of protein for each sample was separated by
SDS-polyacrylamide gel electrophoresis and analyzed by Western blot.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (57K):
[in a new window]
Fig. 1.
Screen for small molecule inhibitors of yeast
Sir2p. A, a yeast strain containing a URA3
reporter gene integrated into a transcriptionally silent telomeric
region was used (TEL::URA3). Sir2p represses URA3 expression
at this locus, and thus a small molecule inhibitor of Sir2p should
allow expression of URA3, resulting in cell death in the presence of
5-FOA. B, a strain with constitutively expressed URA3
(non-TEL::URA3) does not grow in the presence of
5-FOA, whereas the TEL::URA3 strain does. Upon treatment with
one of the compounds from the library (A3), the
TEL::URA3 strain does not grow in the presence of 5-FOA.
Growth is not affected in the absence of 5-FOA, demonstrating that A3
is not cytotoxic. C, three small molecules
(sirtinol, A3, and M15) tested
positively in this screen.
View larger version (77K):
[in a new window]
Fig. 2.
Sirtinol, A3, and M15 inhibit Sir2p
transcriptional silencing at three loci in yeast. The three
positives from the primary screen were tested for their ability to
inhibit Sir2p-mediated transcriptional silencing at the telomeric locus
(TEL::URA3), silent mating-type locus
(HML::URA3), and rDNA locus
(rDNA::URA3). Cultures were visually inspected for
growth, and growth was quantitated by taking the
A600 of the wells (data not shown). A3 and M15
inhibited growth in the three strains for at least 3 days at 100 µM, whereas sirtinol was effective at 25 µM.
Inhibition in vitro of yeast Sir2p and human SIRT2 activity by A3,
sirtinol, and M15
View larger version (34K):
[in a new window]
Fig. 3.
Structure-activity relationship
analysis. A, sirtinol and M15 are synthesized by
coupling 2-hydroxy-1-napthaldehyde with an amine. B, assay
for inhibition of SIRT2 activity in vitro. Sirtinol,
2-hydroxy-1-napthaldehyde, and the amine moieties of sirtinol and M15
were incubated with purified, recombinant human GST-SIRT2 (amino acids
18-340), in the presence of 50 µM NAD and tritiated
acetylated histones. Enzymatic activity was monitored by determining
the amount of released tritiated acetic acid by scintillation counting.
Each experiment was performed in duplicate. Sirtinol potently inhibited
SIRT2 activity, whereas 2-hydroxy-1-naphthaldehyde moderately inhibited
activity at this concentration. C,
2-hydroxy-1-naphthaldehyde was tested for its ability to inhibit
Sir2p-mediated transcriptional silencing at the telomeric locus
(TEL::URA3), mating locus
(HML::URA3), and rDNA locus
(rDNA::URA3). 2-Hydroxy-1-naphthaldehyde inhibited
growth in the presence of 5-FOA at 50 µM.
Effect of A3, sirtinol, and 2-hydroxy-1-naphthaldehyde on HDAC1
activity
-tubulin, as well as morphological changes
resulting from the rearrangement of the actin cytoskeleton produced by
increased expression of the actin-binding protein gelsolin (23). To
assess the effect of sirtinol treatment on cell morphology, HeLa cells
were treated for 24 h with 10 and 50 µM sirtinol, as
well as 1 µM TSA, and visualized under a light microscope. Treatment with TSA caused HeLa cells to change from a
rounded morphology to a flattened morphology (Fig.
4B), whereas treatment with
sirtinol did not produce this effect. These studies provide further
evidence that sirtinol does not affect HDAC activity and suggests that
sirtuins are involved in processes distinct from those of HDACs in
mammalian cells.
View larger version (57K):
[in a new window]
Fig. 4.
Inhibition of HDACs and sirtuins does not
produce the same effect in mammalian cells. A,
inhibition of sirtuins does not result in a morphological change in
HeLa cells. HeLa cells were treated with 10 µM sirtinol,
25 µM sirtinol, or 1 µM TSA for 24 h.
Cells were fixed in 4% formaldehyde and viewed with a light
microscope. Treatment with TSA resulted in a dramatic morphological
change, in which cells spread from their normally rounded phenotype.
Treatment with sirtinol did not produce this effect. B,
inhibition of sirtuins does not result in global acetylation of
histones or tubulin. Primary foreskin fibroblasts were treated with 10 µM sirtinol, 50 µM sirtinol, or 1 µM TSA for 24 h. Cells were harvested and lysed, and
the total protein content was normalized. The amount of acetylated
histones H3 and H4 and -tubulin in each sample was assessed by
Western blot analysis using antibodies to tetra-acetylated histone H3
and H4 (Upstate Biotechnology) and acetylated -tubulin (Sigma). TSA
caused robust acetylation of all three proteins, whereas sirtinol had
no effect. NT, not treated.
-tubulin by Western blot.
Treatment with 1 µM TSA caused robust acetylation of
histones H3 and H4, as well as -tubulin, whereas treatment with
sirtinol did not produce any of these effects.
View larger version (37K):
[in a new window]
Fig. 5.
Treatment with sirtinol inhibits body-axis
formation and vascularization in Arabidopsis.
Arabidopsis seedlings were germinated in the
presence of 1% Me2SO, 25, 50, and 100 µM
sirtinol (A). After 4 days of growth in the presence of
sirtinol, the seedlings lack primary roots and have shortened and
thickened hypocotyls. Staining of seedlings after 5 days of growth
revealed that sirtinol treatment also blocks the formation of the
vascular system in the cotyledons these plants (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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M. T. Borra, F. J. O'Neill, M. D. Jackson, B. Marshall, E. Verdin, K. R. Foltz, and J. M. Denu Conserved Enzymatic Production and Biological Effect of O-Acetyl-ADP-ribose by Silent Information Regulator 2-like NAD+-dependent Deacetylases J. Biol. Chem., April 5, 2002; 277(15): 12632 - 12641. [Abstract] [Full Text] [PDF]
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A. Bedalov, T. Gatbonton, W. P. Irvine, D. E. Gottschling, and J. A. Simon Identification of a small molecule inhibitor of Sir2p PNAS, December 18, 2001; 98(26): 15113 - 15118. [Abstract] [Full Text] [PDF]
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