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http://en.wikipedia.org/wiki/Tumor_necrosis_factor-alpha
(TNF-alpha) is an
important factor for inflammation, but too much is not a
good thing, it
seems. Diseases such as
http://en.wikipedia.org/wiki/Lupus_erythematosus
may result in later years from elevated inflammation.
Females tend to have
more such diseases, purportedly involving their greater
sensitivity to
inflammatory substances. CR appears to counter the
age-associated increase
in inflammatory TNF-alpha, as documented in (1). "Sirtuin
activators mimic caloric restriction and delay ageing in
metazoans", is the title of (2)r. Now another paper
(3) has examined both TNF-alpha and the human sirtuin,
SIRT1. Unlike, it
appears, reveratrol, new CR mimetic compounds generated by
an Asian company
called "S*BIO PTE LTD, Singapore" can suppress TNF-alpha.
Other good things
can occur due to the new compounds increasing or decreasing
SIRT1, it seems.
"However, representatives of these compound families need to
be further
optimized in hit-to-lead programs comprising medicinal
chemistry efforts to
address important issues such as toxicity and specificity."

1. Dirks AJ, Leeuwenburgh C.
Tumor necrosis factor alpha signaling in skeletal muscle:
effects of age and
caloric restriction.
J Nutr Biochem. 2006 Aug;17(8):501-8. Epub 2005 Dec 1. Review.
PMID: 16517142

2. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar
M, Sinclair D.
Sirtuin activators mimic caloric restriction and delay
ageing in metazoans.
Nature. 2004 Aug 5;430(7000):686-9. Epub 2004 Jul 14.
Erratum in: Nature.
2004 Sep 2;431(7004):107.
PMID: 15254550

3. Nayagam VM, Wang X, Tan YC, Poulsen A, Goh KC, Ng T,
Wang H, Song HY, Ni
B, Entzeroth M, Stunkel W.
SIRT1 Modulating Compounds from High-Throughput Screening as
Anti-Inflammatory and Insulin-Sensitizing Agents.
J Biomol Screen. 2006 Nov 12; [Epub ahead of print]
PMID: 17099246

The nicotinamide adenine dinucleotide (NAD(+))-dependent
protein deacetylase
SIRT1 has been linked to fatty acid metabolism via
suppression of peroxysome
proliferator-activated receptor -gamma (PPAR--gamma) and to
inflammatory
processes by deacetylating the transcription factor
NF-kappaB. First,
modulation of SIRT1 activity affects lipid accumulation in
adipocytes, which
has an impact on the etiology of a variety of human
metabolic diseases such
as obesity and insulin-resistant diabetes. Second,
activation of SIRT1
suppresses inflammation via regulation of cytokine
expression. Using
high-throughput screening, the authors identified compounds
with SIRT1
activating and inhibiting potential. The biological activity
of these
SIRT1-modulating compounds was confirmed in cell-based
assays using mouse
adipocytes, as well as human THP-1 monocytes. SIRT1
activators were found to
be potent lipolytic agents, reducing the overall lipid
content of fully
differentiated NIH L1 adipocytes. In addition, the same
compounds have
anti-inflammatory properties, as became evident by the
reduction of the
proinflammatory cytokine tumor necrosis factor-alpha
(TNF-alpha). In
contrast, a SIRT1 inhibitory compound showed a stimulatory
activity on the
differentiation of adipocytes, a feature often linked to
insulin
sensitization.

... RESULTS
Identification of SIRT1 activators and inhibitors
An HTS campaign using a total of 147,000 compounds was
carried out and primary hits selected. We defined a SIRT1
activator
by a signal increase of 150% compared to the control
plate, as described under Materials and Methods, whereas
inhibitors were regarded as primary hits when their inhibitory
activity was higher then 50%. Hits from the HTS were retested
in hit confirmation assays to obtain average IC50 or EC50
values.
Tables 1 and 2 show the structure and properties of some of the
SIRT1 activators identified.

Table 2. Increase of Fluorescent Signal by
SIRT1 activating compounds
===================
Compound (at 10 µM)---% Signal Increase Relative to Signal
Window
===================
1 228±71
2 216.5±61
3 233±13
===================
Shown are average values (± standard deviation) from 2
independent
experiments.

Because the compound resveratrol has been identified by
other groups as a potent agonist of SIRT1 activity in cell-based
models and in vitro screens, we set up cell-based models,
which helped to address potency and biological activity for
selected compounds. SIRT1 has been shown to target PPAR--gamma,
leading to deacetylation in differentiating adipocytes and
promoting
lipolysis in white adipocytes.16 We have tested the
newly discovered SIRT1 activators in the well-known cellbased
adipogenesis differentiation assay. In line with the published
report on resveratrol effects in this system, we were able
to demonstrate with some of our compounds a dose-dependent
fat mobilization, as shown in Figure 1 (representative data are
shown for compound 3). Macroscopically, the intensity of Red
Oil staining decreases in samples, which have received a higher
dose of the compound (Fig. 1A). Quantification of the results
obtained by dye extraction and absorbance reading using a
spectrophotometer are described in detail in the Materials and
Methods section and are shown in Figure 1B. The histological
picture confirms this finding, whereby the size and number
of intracellular cytosolic fat deposits are reduced in a
dosedependant
manner (Fig. 1C). These results suggest that the
compounds identified with SIRT1-activating properties have a
significant effect on fat mobilization in differentiated
adipocytes,
and thus these compounds are anticipated to have antiobesity
and/or antidiabetic properties.
Anti-inflammatory in vitro properties of SIRT1 activators
The transcription factor NF-?B has been linked to inflammatory
processes, and SIRT1 has been recently described to
inhibit the transactivation potential of the RelA/p65 subunit.13
Because biological activity of our SIRT1 activators could be
confirmed in matured adipocytes, we established an ELISAbased
assay to monitor TNF-alpha modulation comprising the
human leukemia cell line THP-1, which is well known to produce
and release TNF-alpha and other cytokines upon stimulation
with LPS (as described in the Materials and Methods section).
As summarized in Table 3, compounds with SIRT1 activating
potential exert strong suppression of TNF-alpha release at
concentrations
between 20 and 60 µM. Resveratrol did not show a
significant biological effect in this assay at comparable
concentrations
(Fig. 2A). Histograms of the dose-response experiments
for the activator compounds 1 to 3 are shown in Figure 2B. Cell
viability was controlled by Trypan blue exclusion for all
experiments
shown and did not reveal any cytotoxic effects for the
compounds tested.

Table 3. ELISA-Based Quantification of TNF-alpha
Suppression by SIRT1 Activator Compounds 1 to 3
================================
Compound Control 20 µM 60 µM
================================
1 205.5±20.8 116.4±20.7 60.9±3.3
2 248.3±4.4 107.7±1.4 50.6±5.4
3 324.9±39 103.7±16.2 52.6±12.7
================================
The amount of TNF-alpha was measured as pg/mL after
treatment of THP-1
cells at the indicated
concentrations (shown as the average±standard deviation).
ELISA = enzymelinked
immunosorbent assay; TNF-alpha = tumor necrosis factor-alpha.

Inhibitors of SIRT1 stimulate lipogenesis in 3T3L1 cells
The HTS campaign led to the discovery of potent inhibitors of
SIRT1 (a representative is shown in Figure 3A, compound 4),
which were studied in the same biological assay as described
above. The tetrahydrocarbazole compound 4 from the initial
screen was found to be unstable and to decompose into an
amide (5) as the major product. The chemical structure of
compound 5 was confirmed by isolation from decomposed
compound 4 and comparison of its analytical data (nuclear
magnetic resonance [NMR], LC/MS, and high-performance
liquid chromatography/ultraviolet [HPLC/UV]) with those of
authentic samples of compound 5. Compound 5 was found to be
a potent SIRT1 inhibitor and was the major active component of
compound 4. During the period of preparing this manuscript,
Napper et al.19 also published that compound 5 was a potent
SIRT1 inhibitor. Their results were in line with ours.
PPAR-gamma activation is a result of direct interaction with
agonists
or, alternatively, based on the relief of a transcriptional
block due
to SIRT1 inhibition.16 One obvious cellular change during the
course of differentiation from a fibroblast-like state to a
fully
matured fat cell is the increase in the number of lipid
deposits.
Known PPAR-gamma agonists, such as the insulin sensitizer
rosiglitazone,
strongly enhance adipogenesis and differentiation, and we
used this known compound as a reference for biological effects
of SIRT1 inhibitors.26 As shown in Figure 3B, rosiglitazone
potently induces cellular differentiation at 100 nM, as becomes
evident by an increase in the number of lipid deposits stained
with Red Oil O. A 2-fold increase in the lipid content was
observed for the SIRT1 inhibitor compound 4, although the
concentration
applied was 20 and 50 µM. Quantification was carried
out by absorbance reading after Red Oil O extraction (Fig. 3C).
This result confirms that modulation of SIRT1 activity leads
to an opposite biological effect depending on the use of either
specific activators or inhibitors.
SIRT1 inhibitor counteracts suppression of TNF-alpha
As enzymatic activity can be regulated by either agonists or
antagonists, we carried out a proof-of-concept experiment for
our activators and the potent SIRT1 inhibitor. By blocking
SIRT1 function in THP-1 cells with the specific inhibitor
(compound
4) prior to treatment with the SIRT1 activator (compound
5), we observed a lack of TNF-alpha suppression when
compared with the activator alone (Fig. 4A,B). The optimal
time points for these compound treatments (pretreatment with
SIRT1 inhibitor), followed by treatment with the SIRT1 activator
and subsequent LPS-mediated TNF-alpha release, were determined
empirically. This result demonstrates that the catalytic
activity of SIRT1 can be regulated in opposing directions by
either specific activators or inhibitors.
We propose the binding mode for this type of SIRT1
inhibitor compounds by homology modeling, as described under
As shown in Table 4, all compounds
except the inactive 11, 13, and 16 could be docked into the
NAD+-binding site of the SIRT1 homology model with a binding
mode that explained the observed structure-activity relation.
Compound 4, which is unstable and is degraded into the amide
compound 5 under assay conditions, did not dock either. All
docked compounds had some affinity to SIRT1, and all docked
in a conformation within 12 kJ/mol of the global energy minima.
These are reasonable energies for bioactive conformations.27 The
predicted binding mode for the most active compound 5 is
displayed
in Figure 4C. R1 docks into a small hydrophobic pocket
formed by the backbone of Val412 and the side chains of Phe297,
Ile347, Phe366, and Ile411. The indol nitrogen forms a hydrogen
bond to Arg274. The ring system is positioned between the side
chains of Phe273, His363, and Val445. The amide side chain falls
into a hydrophilic pocket formed by the side chains of Arg274,
Gln345, His363, Ser442, and the backbone nitrogen of Lys444.
The amide forms hydrogen bonds to Gln345 and Lys444.
The less active compound 12, with a hydroxyl group at R1,
and compound 10, without any R1 substituent, are lacking the
TNF-alpha is one of the key cytokine mediators involved in the
inflammatory response and is used as a marker for many
inflammatory disorders.29 The biological importance of TNF-alpha
inhibition in the treatment of inflammatory disorders such as
rheumatoid arthritis, Crohn's disease, and ulcerative colitis
became apparent with the discovery and use of infliximab, a
monoclonal antibody directed against TNF-alpha.30 It has
also been
reported that NF-?B binding elements in the murine and human
TNF-alpha gene promoters mediate TNF-alpha expression in
response
to LPS treatment.31,32 Increasing the activity of SIR proteins,
especially SIRT1, could be an effective new approach in the
dysregulation of proinflammatory factors such as TNF-alpha.
Here we show that quinoxaline-based potent activators of
SIRT1, discovered from a high-throughput screen, decrease
TNF-alpha after LPS induction, as quantified by a sensitive
ELISA
assay. Resveratrol is a weak activator of SIRT1 function,
thereby
providing a precedented standard for comparative studies with
these more potent activators. These compounds are at least
10-fold more potent in the in vitro suppression of TNF-alpha
release
compared to resveratrol. Another important function of SIRT1 in
vivo is gene silencing, and 1 of the targets regulated by SIRT1
encodes the nuclear receptor PPAR-gamma, which plays a
central role
in lipid metabolism and adipocyte differentiation. We therefore
evaluated the quinoxaline compounds in a cell-based adipocyte
differentiation system. PPAR-gamma is the driving force of lipid
accumulation in matured adipocytes, and resveratrol has been
reported to enhance fat mobilization, an effect that could
be confirmed
with the more potent SIRT1 agonists in our assays.
The effect of SIRT1 activation is reversible by using a specific
inhibitor, which we have identified in our HTS campaign
(as shown in Table 4). This inhibitor compound is a
tetrahydrocarbazole
and is similar to a recently published indole
showing SIRT1 inhibition.19 SIRT1 is an enzyme whose activity
can be modulated with either agonists or antagonists,
and Figure 4 illustrates a case whereby the effect of a SIRT1
antagonist counteracts the TNF-alpha-suppressing potential of an
agonist. We studied the SIRT1 inhibitors in our cell-based
lipogenesis
assay as well and could show that down-regulation of
PPAR-gamma activity with a SIRT1 inhibitor leads to fat
accumulation,
a frequently observed effect seen in the treatment with
insulin-sensitizing agents such as rosiglitazone. The
intracellular
accumulation of lipids is a hallmark for the insulin-sensitizing
effect because it parallels an increased uptake of free fatty
acids, which have been associated with insulin resistance, and
fine regulation of the cellular lipid content may be dependent
on the specific therapeutic indication addressed (e.g.,
obesity or
type II diabetes).33
Many in vitro assays make use of artificial substrates, which
contain a fluorophore or modified peptides, which carry only 1
residue such as an acetyl-group. In vivo, multiple modifications
occur on correctly folded proteins at the same time, and it is
conceivable that compounds such as resveratrol and the
activators
presented in this study have a specific mode of action. We
have not further investigated this mechanism of SIRT1
activation,
but it is tempting to speculate that resveratrol-like compounds,
which increase SIRT1 activity, may in fact be allosteric
effectors leading to stabilization of substrate binding. It
is not
known whether this allosteric mode of action will direct SIRT1
to low-affinity targets in vivo. This idea is supported by 2
recently published reports on the mechanism of SIRT1 activation
by resveratrol.34,35
In summary, in an HTS campaign against human recombinant
SIRT1 using a library comprising 147,000 compounds,
we have successfully identified 2 classes of compounds,
activators
and inhibitors. We confirmed a set of quinoxalins with
strong activating potential on recombinant SIRT1. Compounds,
such as the SIRT1 activators 1 to 3 and the
tetrahydrocarbazole-based
SIRT1 inhibitor compound 4 presented here, have the
ability to modulate the activity of SIR proteins such as SIRT1.
They may therefore provide a new means of treating obesity
and related metabolic disorders such as atherosclerosis,
hypertension,
type II or non-insulin-dependent diabetes mellitus,
pancreatitis, hypercholesterolemia, hypertriglyceridaema, and
hyperlipidemia, as well as a variety of inflammatory diseases.
However, representatives of these compound families need to
be further optimized in hit-to-lead programs comprising
medicinal
chemistry efforts to address important issues such as toxicity
and specificity.