ArticlePDF Available

Abstract and Figures

Flavonoids are polyphenolic secondary metabolites synthesized by plants and fungus with various pharmacological effects. Due to their plethora of biological activities, they have been studied extensively in drug development. They have been shown to modulate the activity of a NAD+-dependent histone deacetylase, SIRT6. Because SIRT6 has been implicated in longevity, metabolism, DNA-repair, and inflammatory response reduction, it is an interesting target in inflammatory and metabolic diseases as well as in cancer. Here we show, that flavonoids can alter SIRT6 activity in a structure dependent manner. Catechin derivatives with galloyl moiety displayed significant inhibition potency against SIRT6 at 10 µM concentration. The most potent SIRT6 activator, cyanidin, belonged to anthocyanidins, and produced a 55-fold increase in SIRT6 activity compared to the 3-10 fold increase for the others. Cyanidin also significantly increased SIRT6 expression in Caco-2 cells. Results from the docking studies indicated possible binding sites for the inhibitors and activators. Inhibitors likely bind in a manner that could disturb NAD+binding. The putative activator binding site was found next to a loop near the acetylated peptide substrate binding site. In some cases, the activators changed the conformation of this loop suggesting that it may play a role in SIRT6 activation.
This content is subject to copyright. Terms and conditions apply.
1
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
www.nature.com/scientificreports
Natural polyphenols as sirtuin 6
modulators
Minna Rahnasto-Rilla1,2, Jonna Tyni2, Marjo Huovinen2, Elina Jarho2, Tomasz Kulikowicz1,
Sarangan Ravichandran3, Vilhelm A. Bohr1, Luigi Ferrucci1, Maija Lahtela-Kakkonen2 &
Ruin Moaddel1
Flavonoids are polyphenolic secondary metabolites synthesized by plants and fungus with various
pharmacological eects. Due to their plethora of biological activities, they have been studied
extensively in drug development. They have been shown to modulate the activity of a NAD+-dependent
histone deacetylase, SIRT6. Because SIRT6 has been implicated in longevity, metabolism, DNA-repair,
and inammatory response reduction, it is an interesting target in inammatory and metabolic diseases
as well as in cancer. Here we show, that avonoids can alter SIRT6 activity in a structure dependent
manner. Catechin derivatives with galloyl moiety displayed signicant inhibition potency against SIRT6
at 10 µM concentration. The most potent SIRT6 activator, cyanidin, belonged to anthocyanidins, and
produced a 55-fold increase in SIRT6 activity compared to the 3–10 fold increase for the others. Cyanidin
also signicantly increased SIRT6 expression in Caco-2 cells. Results from the docking studies indicated
possible binding sites for the inhibitors and activators. Inhibitors likely bind in a manner that could
disturb NAD+ binding. The putative activator binding site was found next to a loop near the acetylated
peptide substrate binding site. In some cases, the activators changed the conformation of this loop
suggesting that it may play a role in SIRT6 activation.
Flavonoids are a large family of naturally occurring polyphenolic compounds that provide important health ben-
ets and help to protect against cancer, cognitive decline, diabetes, heart disease, and obesity.
Chemically avonoids contain a een-carbon skeleton consisting of two benzene rings (A and B) linked via
a heterocyclic pyran ring (C) (Fig.1A). ey are synthesized via the phenylpropanoid pathway, representing a
rich source of metabolites in plants. e chemical nature of avonoids depends on their structural class, degree of
hydroxylation, other substitutions and conjugations, and the degree of polymerization. Flavonoids are classied in
ve major structural classes including avan-3-ols, avanones, avones, avonols, and anthocyanidins. Catechins
share a general avan-3-ol structure whereas avanones, avones and avonols include a carbonyl group on position
4. Flavones, avonols and anthocyanidins also contain a double bond between positions 2 and 3.
The antioxidant property of flavonoids may be mediated by many mechanisms including inhibition of
enzymes involved in free radical generation and subsequently suppression of reactive oxygen species (ROS).
Sirtuins (SIRTs) is an enzyme family that can modulate ROS levels notably during calorie restriction, which has
been shown to enhance lifespan for several organisms. SIRTs are nicotinamide adenine dinucleotide (NAD+)
dependent histone deacetylases that catalyze the removal of acetyl group from lysine residue. Among the
seven-membered mammalian sirtuin family, SIRT6 deacetylates histone 3 lysine 9 (H3K9)1,2 and 56 (H3K56)3
and also displays mono-ADP ribosyltransferase4 and deacylase activities5. ese functions of SIRT6 are involved
in the regulation of many genes including stress responses. SIRT6 decient cells display sensitivity to oxida-
tive stress and a reduced capacity for DNA repair6,7 and SIRT6 knockout mice show many hallmarks of prema-
ture aging. Adversely, male mice overexpressing SIRT6 have a signicantly longer lifespan than their wild-type
counterparts8,9. SIRT6 also plays an important role in controlling glucose and lipid metabolism, regulating the
expression of multiple glycolytic and lipid genes involved in cellular response10,11. ese diverse functions of
SIRT6, highlights its importance in aging and protecting many cellular functions. erefore, compounds that
1Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland,
21224, USA. 2School of Pharmacy, University of Eastern Finland, P.O.Box 1627, 70210 Kuopio, Finland. 3Advanced
Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research
Inc, Fredrick, Maryland, 21701, USA. Minna Rahnasto-Rilla and Jonna Tyni contributed equally to this work.
Correspondence and requests for materials should be addressed to M.L.-K. (email: maija.lahtela-kakkonen@uef.)
or R.M. (email: Moaddelru@grc.nia.nih.gov)
Received: 10 May 2017
Accepted: 19 February 2018
Published: xx xx xxxx
OPEN
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
2
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
can regulate SIRT6 activities are considered as promising therapeutics for age-related diseases including cancer,
diabetes, neurodegenerative diseases and metabolic disorders.
Resveratrol is a widely studied polyphenolic12 antioxidant and is known to increase the deacetylation activity
of SIRT1 through a mechanism that is still not fully understood. So far only a few compounds have been identi-
ed to regulate the deacetylation activity of SIRT6. For example, long-chain free fatty acids stimulated deacetyl-
ation13 activity, and polyphenols, specically quercetin and luteolin, were shown to increase SIRT6 deacetylation
activity at high concentrations, while also inhibiting deacetylation activity at low concentrations14. In addition,
the known sirtuin inhibitor EX-52715 and a group of peptides and pseudopeptides16 were reported as SIRT6
inhibitors, but they did not exhibit selectivity towards SIRT6. Quinazolinedione derivatives, were also recently
discovered17,18 to inhibit SIRT6 deacetylation activity. To the best of our knowledge, this is the rst work that
shows rational structure activity relationship (SAR) for SIRT6 to compare inhibition and activation of SIRT6
deacetylation activity using chemically diverse compounds. e present study evaluated the dierences in chem-
ical features between SIRT6 inhibitors and SIRT6 activators. Molecular docking was also carried out to discover
their binding sites on SIRT6, and to identify major interactions occurring on the enzyme active site with inhibi-
tors and activators. is represents an expansion of the chemical spectrum of SIRT6 modulators. Here we showed
that anthocyanidins strongly increase SIRT6 deacetylation activity in vitro. Moreover, the most potent activator,
cyanidin, up-regulated SIRT6 protein expression on the human colon adenocarcinoma Caco-2 cells.
Results
Flavonoids modulate SIRT6 deacetylation activity in vitro. A set of avonoids (Fig.1B; TableS1) and
phenolic acids (TableS2) were tested using recently developed HPLC-based SIRT6 assay14,19,20 with two substrate
concentrations by determining the level of deacetylated peptide H3K9. SIRT6 activity was determined at multiple
concentrations of the tested compounds (Fig.2), to determine whether they modulated (increased/decreased)
SIRT6 deacetylation activity. e developed assay was carried out in the presence of GST-tagged SIRT6, NAD+
and H3K9Ac with tested polyphenols.
e observed deacetylation activity of avonoids is shown in Fig.2. Of the tested compounds, catechins
showed inhibition of the deacetylation activity of SIRT6, whereas anthocyanidins (Fig.2C) increased the
deacetylation activity. Flavonoids can inhibit or activate deacetylase activity of SIRT6 depending on the concen-
tration. e inhibition of compounds 4 and 5 was signicant at a concentration of 10 µM (Table1; Suppl. Fig.S1),
Figure 1. General scaold of avonoids (A). e structures of the most potent SIRT6 modulators are displayed,
and additional structures are available in supplementary TablesS1 and S2. e yellow background represents
inhibitors, gray represents inhibitors and activators and blue represents activators (B).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
3
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
while compounds 1618 more than doubled SIRT6 deacetylase activity. eir respective IC50 and EC50 were
determined (Table1; Supp. Fig.S1). Maximal activation was determined at saturating concentrations of the tested
compounds and indicates the maximal eect of the tested compounds in the in vitro as say.
Catechins. Catechins and epicatechins are stereoisomers that results in dierent inhibition towards SIRT6.
Catechins with trans conguration (catechins) are more potent SIRT6 inhibitors than cis stereoisomeric analogs
(epicatechins). (+)-Enantiomer (1) showed only 26% inhibition at 100 µM concentration towards SIRT6 but for
()-enantiomer (2) the inhibition was 54%. ()-Gallocatechin was a weak inhibitor, similar to compound 1.
e galloyl moiety on the carbon 3 seems to signicantly increase deacetylation activity of SIRT6. is was also
observed in case of compounds 4, 5 and 8 in which inhibition exceeded 60% whereas compounds 6 and 7 showed
less than 10% inhibition towards SIRT6. Compound 9 showed moderate inhibition towards SIRT6. e most
potent inhibitors, 4 and 5 displayed IC50 values of 2.5 µM and 5.4 µM, respectively (Table1).
Figure 2. SIRT6 modulation by selected avonoids. Inhibition % at the 100 µM concentration (A), at the 10 µM
concentration (B) of avonoids. Black bars indicate 60% inhibition. Activation in the presence of 100 µM
avonoids (C). Black bars represent more than 2 -fold activation. e data is presented as mean ± SD, (n = 3).
Compound IC50 value (µM)
4 ()-Catechin gallate 2.5 ± 0.03
5 ()-Gallocatechin gallate 5.4 ± 0.04
Compound EC50 value
(µM) Maximal activation
(fold)
13 Luteolin14 270 ± 25 6.1
14 Kaempferol n.d 3.0
15 Quercetin14 990 ± 250 10
16 Myricetin 404 ± 20 7.7
17 Cyanidin 460 ± 20 55
18 Delphinidin 760 ± 200 6.3
Table 1. Dose response data of modulators. Data are presented as mean ± SD, (n = 3).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
4
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
Flavanones. e carbonyl group at position 4 in compounds 10 and 11 did not improve the inhibitory activity
towards SIRT6 compared to compound 1. O-Glycosylation occurs primarily on position 5 and 7 on the A ring
and, C-glycosylation primarily occurs on position 6 and 8 on the A-ring. Glycosylation by glucose on these car-
bons displayed weaker inhibition activity compared to the compounds with basic avonoid scaold (data not
shown).
Flavones and avonols. e hydroxyl group on the carbon 3 improved the inhibition potency of compound 13
compared with 12. Compound 13 was a weak inhibitor and it increased deacetylation with maximal activation of
6-fold although at higher concentrations. Both avonols 14 and 15 were inhibitors as well as activators depending
on the concentration of compound. Surprisingly, compound 16 with the hydroxyl group on the position 5 (R3)
increased SIRT6 activity.
Anthocyanidins. Two anthocyanidins (17 and 18) were tested and both showed an increase in deacetylase activ-
ity for SIRT6. Compound 17 was signicantly more eective producing 55-fold maximal activation (Table1)
compared to the other activators with maximal activation of 3–10 -fold. In general, EC50 values of activators
varied from 270 µM of compound 13 to 990 µM of compound 15. Compound 18 with three hydroxyl groups (R1,
R2 and R3) displayed weaker activity against SIRT6 than compound 17. e in vitro SIRT6 deacetylation activity
for cyanidin (17) and delphinidin (19) was also determined by western blot analysis using the core histones and
determining the remaining levels of histone H3 acetylated on lysine 9 (Suppl. Fig.S2). Both compounds increased
deacetylation activity 2.5 fold at 100 µM.
Isoavones. ese compounds are structurally similar to estrogens and are also known as phytoestrogens. Two
isoavones (19 and 20) were tested and both were weak SIRT6 inhibitors but compound 20 was also able to acti-
vate deacetylation of SIRT6. e methoxy moiety seems to improve slightly the inhibition potency toward SIRT6
although the result was ambiguous, since the inhibition potency of compound 20 was same level as compounds
10 and 14.
Phenolic acids. A set of phenolic acids (gallic acid derivatives), which is another main class of plant polyphenols,
were also included in the study. Although compounds 22 and 23 increased slightly SIRT6 activation, overall phe-
nolic acids (2227) were weaker modulators than avonoids.
Cyanidin up-regulates SIRT6 and FoxO3α protein expression and downregulates Twist1 and
GLUT1 expression in Caco-2 cells. In order to assess the eects of the most potent activator on SIRT6
expression, Caco-2 cells at passages 30–40 were exposed to DMSO (control) or various concentrations of com-
pound 17 (12.5–200 µM) for 24 h. Aer the treatment, conditions of the cells were evaluated under a light micro-
scope (Fig.3A). Cells treated with 12.5 µM to 100 µM of compound 17 were similar to control cells, whereas
at 200 µM compound 17 precipitated out of solution. Immunoblotting analysis of total Caco-2 protein lysates
(Fig.3B, Suppl. Fig.S3) demonstrated that compound 17 was eective in a dose-dependent manner aer 24 hour
Figure 3. Caco-2 cells aer cyanidin treatment and expression of SIRT6 protein. Cells were exposed to 0.5%
DMSO (control) or various concentration of compound 17 (n = 5) for 24 h. (A) Representative light microscopy
images of Caco-2 cells aer control (Cnt), 100 µM cyanidin or 200 µM cyanidin treatment. (B) Immunoblotting
analysis of SIRT6 protein. SIRT6 protein levels (MW: 39 kDa) were normalized relative to α-tubulin (MW:
50 kDa) and quantication is represented as fold change respect to control. Values are expressed as mean ± SEM
of four independent experiment (*p values < 0.05; one way-ANOVA). SIRT6 expression were determined by
immunoblotting (B).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
5
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
exposure, while cells treated by 50–100 µM concentration showed signicantly increased SIRT6 expression with
3.5 fold up-regulation. In addition, the eect of compound 17 on the expression levels of the transcription factor
forkhead box O-3α (FoxO3α), Twist-related protein 1 (Twist1) and glucose transporter (GLUT1) were studied
at 50–200 µM concentrations, and subsequently Caco2 lysates were analyzed by immunoblotting. Compound
17 enhanced the protein expression of FOXO3 signicantly (Fig.4A), but downregulated Twist1 (Fig.4B) and
GLUT1 (Fig.4C) expression at 100 µM concentration. Full-length blots are presented in Supplementary Fig.S4.
The binding sites of avonoids in SIRT6. To study the interactions, molecular docking studies of avonoids
were performed in the binding sites of the human SIRT6 (PDB entry:3ZG6, resolution 2.2 Å)5. e research showed
that the inhibitors bound quite close to the binding site of nicotinamide (NAM) moiety of NAD+. e most potent
inhibitor, compound 4 (Fig.5A) occupied partially the peptide substrate binding site and subsequently prevented the
active histidine (His131) to orient towards NAD+ for reaction. Compound 4 formed interactions with residues Asn2,
Ser8, Ala11, Phe62, and Glu187. However, most commonly the binding pose of inhibitors corresponded to the pose of
compound 5 (Fig.5B). is site resembles the binding site of co-crystallized inhibitor Ex-527 in SIRT1.
e activity of SIRT6 inhibitors was increased in vitro when the hydroxyl group at position 3 (Fig.1A) was
replaced by a galloyl moiety. e overall comparison of compounds 3 and 5 revealed that compound 5 can occupy
a larger volume of the inhibitor binding pocket than compound 3 (Suppl. Fig.S5). A closer investigation showed
that compound 5 can form additional interactions within the binding site involving the following residues: Pro60,
Phe62, Phe80, Phe84 and Leu184 (Suppl. Fig.S6). e pose comparison of compounds 6 and 8 also showed the
importance of galloyl moiety, as it ensured the interaction to Leu184 which is located deep in the pocket while
the other moieties of compound 6 could interact with other parts of the inhibitor binding pocket (Suppl. Fig.S7).
A comparison of compounds 3 and 7 together with compounds 5 and 9 was carried out to examine how the
conguration of the galloyl moiety aected the inhibition potency. Compound 7 did not reach as deep into the
binding pocket as did compound 3 (Suppl. Fig.S8). Although the position of compounds 9 and 5 were similar
in the inhibitor pocket, compound 5 formed more interactions with residues Phe62 and Phe84 (Suppl. Fig.S9).
e additional carbonyl group (ring C; Fig.1A) in compounds 10 and 11 did not result in additional interactions
when compared to compound 1. Although there was no major dierence in the binding poses of compounds 12
and 13 at the inhibitor binding site, compound 13 could form more interactions than compound 12 in majority of
the poses. Interestingly, the methoxy moiety in compound 20 did not contribute any additional interactions in the
docking studies compared to compound 19. Phenolic acids (compounds 22–27), on the other hand, occupied only
a limited volume of the inhibitor binding pocket (Suppl. Fig.S10), resulting in decreased interactions, which may
explain their poor inhibitory potency. Compounds binding to the putative inhibitor/activator binding sites using 2D
interaction diagrams are presented in Supplementary FiguresS11–S16 and S17–S20, respectively.
e activator binding site was discovered with SiteMap. SiteMap uses dierent scoring functions to assess the
found sites. One of these functions is SiteScore, which evaluates if the site is likely to bind a drug or not. Scores
over 1.0 are dened to be promising drug-binding sites, and sites having scores under 0.8 most likely will not bind
drugs. e putative activator site had a SiteScore of 1.003, and was located close to the β6/α6 loop region (Fig.5).
All activators formed interactions at the β6/α6 loop region with Trp186 and/or Glu187. Some of the activators
had additional interactions with Gly156, Asp185 and Asp188. e most potent activator, compound 17 (Fig.5C)
formed all of these interactions except for the interaction with Asp188. Unlike the other activators, compounds
16, 17 and 18 interacted with Asp185 at the activator binding site, which may be responsible for their increased
activity (Suppl. Fig.S21).
Figure 4. Cyanidin (compound 17) eect on the expression of FoxO3α (A), Twist1 (B) and GLUT1 (C)
protein. Caco-2 cells were treated with DMSO (white bar) or 50 and 100 µM compound 17 (grey bars) for 24 h.
FoxO3α, Twist1 and GLUT1 expression were quantied and normalised with α-tubulin or H3. Data represent
the mean ± SEM of three independent experiments,*p < 0.05, **p < 0.01 and ***p < 0.001 between the
indicated groups.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
6
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
Figure 5. SIRT6 and locations of binding sites of activators (light turquoise), inhibitors (yellow), peptide
substrates (blue) and for NAD+ (brownish gray). Close-up view of the interactions of best inhibitors, compound
4 (A) and compound 5 (B) and best activator, compound 17 (C). e best activators compound 17 (D) and
compound 18 (E) induce changes on the β6/α6 loop and the orientation of Trp186 and Glu187 similar to
known activators oleic acid (F) and linoleic acid (G). Interactions: Yellow dashes indicate hydrogen-bonding,
dark green dashes indicate π-π stacking and light purple dash indicates salt bridge (interaction to Asp185). Pink
residues and loops indicate the original residue and loop orientation in the protein structure before inhibitor or
activator binding.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
7
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
Some of the activators changed the orientation of Trp186 and/or Glu187 and some altered the conformation of
β6/α6 loop. Two of these were the most potent activators, compounds 17 and 18 (Fig.5E). Similarly, the docking
of oleic acid (Fig.5F) and linoleic acid (Fig.5G), known SIRT6 activators, also resulted in changes in the Trp186
and/or Glu187 orientation and/or β6/α6 loop conformation. ese results suggest that changes in the Trp186
and/or Glu187 orientation and β6/α6 loop conformation could be factors that are involved in the activation of
SIRT6. Subsequently, an impact analysis of the key activator residues were carried out (Suppl. TablesS3 and S4).
e results demonstrate that only residue Asp188 is not conserved and can accommodate a wide range of substi-
tutions (Suppl. TableS3).
Dual modulators, such as compound 14, could form interactions with the same amino acid residues that
interacted with the most potent activator/inhibitors with their respective binding sites. Compound 14 had similar
interactions with Pro60, Phe62, Val113 and Leu184 as did the potent inhibitor, compound 5, at the inhibitor site
(Suppl. Fig.S22). It also had interactions with, Glu187 and Asp188, as did the most potent activators, compounds
17 and 18, at the putative activator site. Compound 14 also induced a minor change on the conformation of β6/α6
loop, but it did not change the orientation of Trp186, nor did it form an interaction with Trp186 (Suppl. Fig.S22).
Discussion
Among sirtuins, SIRT6, has been implicated in aging and age-related diseases, but its physiological role is not
completely understood. e extent to which increased SIRT6 activation aects these disease conditions is still
unclear; it might oer a protective mechanism or, alternatively, represent part of the disorder process. Although
there is considerable evidence that SIRT6 is a tumor suppressor, the eect is double-edged since it can also inac-
tivate tumor suppressor proteins FoxO3a and p53. To further examine these opposite roles of SIRT6, there is a
denite need for novel potent SIRT6 modulators, for both inhibitors and activators. ese modulators make it
possible to study the physiological role and therapeutic potential of SIRT6.
Several SIRT6 inhibitors have been reported previously16,17 and the most potent compounds have been dis-
covered against the acylation activity of SIRT621,22. SIRT6 preferentially removes long chain fatty acyl lysine in
vitro compared to the deacetylation of target substrates5. SIRT6 has been shown to eciently deacetylate lysines
9 (H3K9) and 56 (H3K56) on the H3 sequence in vivo2,3. H3K9 is the specic regulation site of chromatin at
telomeres1,23,24 while the acetylation status of H3K56 controls DNA damage response and genomic stability3,25.
In the present study, catechins exhibited inhibition activity against SIRT6 catalyzed H3K9Ac deacetylation.
Catechins are a major component of green tea and in recent years many health benets associated with the con-
sumption of green tea have been reported. Green tea has been suggested to reduce ROS production and subse-
quently exhibit protective role against oxidative stress mediated diseases. Interestingly, catechins have also been
demonstrated to protect cells against oxidative stress and DNA damage by increasing the activity of SIRTs. Tao et
al. 2015 reported that (-)-epigallocatechin-3-gallate induced oxidative stress in cancer cells but it had protective
role in normal cells, which was linked to the increased SIRT3 activity26. In addition, (-)-epigallocatechin-3-gallate
has been reported to extend the lifespan in rats, which was consequence of activation of SIRT1 and protection
against oxidative stress27. More studies are needed to reveal the role ()-epigallocatechin-3-gallate may play with
sirtuins in oxidative stress.
Interestingly, the most prominent activators for SIRT6 among the avonoids were the anthocyanidins, the
universal plant pigment, responsible for the red, purple, and blue color in many fruits, vegetables and owers.
e most potent compound in the class of anthocyanidins, cyanidin, signicantly increased the deacetylation
activity of SIRT6. It is most abundant in red berries including bilberry, raspberry and cranberry. Studies have
suggested that anthocyanidins, including cyanidin, may play important roles in helping to reduce the risk of many
age-related diseases. e eect has been linked to their protective eect against oxidative stress, which results in
the decreased production of ROS and nitrogen species2830. Cell culture and in vivo studies of anthocyanidins and
their glycosylated counterparts (anthocyanins) revealed anticarcinogenic properties against colon, skin, and lung
cancer. While laboratory studies have provided some insight into how anthocyanins may work, the exact mecha-
nism for how these compounds prevent cancer is unclear. us far studies in a variety of cancer cells revealed that
anthocyanins activate detoxifying enzymes, prevent cancer cell proliferation, induce cancer cell apoptosis and
have anti-inammatory and antiangiogenic eects31,32. To the best of our knowledge, this is the rst study that
showed the up-regulation of SIRT6 in colon adenocarcinoma Caco-2 cells treated by cyanidin.
Additionally, cyanidin aected the expression levels of SIRT6 associated genes such as FoxO3α, Twist1 and
GLUT1. FoxO3α belongs to the family of forkhead box transcription factors that play important roles in regu-
lating the expression of genes involved in cell growth, proliferation, dierentiation, and longevity. Deregulation
of FoxO3 is involved in tumorigenesis. Previous studies reported that FoxO3α gene is regulated by SIRT6 which
forms a complex with FoxO3α in the nucleus, and further induces the expression of genes involved in antiox-
idation33. Embryonic transcription factors Twist1 and glucose transporter GLUT1 are overexpressed in many
tumors. SIRT6 suppress cell proliferation via Twist1, which is also a key factor in the promotion of metasta-
sis of cancer cells34. SIRT6 regulates the expression of many glycolytic genes via the hypoxia inducible factor-1
(HIF1)-alpha pathway10. SIRT6 was recently shown to regulate metabolic reprogramming in cancer cells via
metabolic signaling pathways decreasing the expression of glycolytic genes, including GLUT1 and HIF1-alpha
and decreasing glucose uptake and lactate formation by cells.
Sirtuins are involved in a number of central physiological processes, and their activity are likely regulated by
endogenous signaling pathways in a tissue-specic and signal-dependent manner at various levels. Sirtuins can
be regulated by many mechanisms, including transcriptionally and post-translationally by changing the stability,
activity, localization or degradation of the protein. In addition, protein complex formation with other binding
partner proteins and changes in NAD+ availability may play an important role in regulation of sirtuin functions
and expression. Many enzymes such as nicotinamide phosphoribosyltransferase (NAMPT) are involved in NAD+
synthesis, which is upregulated by AMP-activated protein kinase (AMPK)35. Polyphenols such as resveratrol36 and
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
8
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
anthocyanins37 have been reported to activate AMPK, an eect that may be mediated by SIRT1. us, the acti-
vation of AMPK by polyphenols such as cyanidin may be one possible mechanism to upregulate SIRT6 expres-
sion in Caco-2-cells. Alternatively, recently it has been demonstrated that activation of PPARγ via rosiglitazone
increases SIRT6 expression38. Moreover, anthocyanins have been shown to induce PPARγ expression39, and in a
separate study it was demonstrated that anthocyanin-rich berries increased PPARγ activity as well40. ese stud-
ies demonstrate that the observed increase in SIRT6 expression may result from changes in multiple pathways.
Molecular docking studies were carried out to identify interactions occurring between diverse avonoid
classes and SIRT6.e results revealed existence of diverse possible binding sites for inhibitors (yellow region) and
activators (turquoise region) (Fig. 5). e binding site of most of the inhibitors was situated close to the binding
site of known sirtuin inhibitors, Ex-527 and NAM41,42. e site was located at highly conserved region at the cle
between two domains, a large Rossman fold domain and a smaller zinc binding domain41. e cle also forms
the pocket for the acetylated sirtuin substrate (blue region Fig.5) and for the co-factor, NAD+ (brownish gray
region). e most potent inhibitor, compound 4, could partially occupy the binding site of the acetylated sirtuin
substrate. Inhibitors with a galloyl moiety, such as compound 5, had more interactions with the binding pocket.
is may explain the improved potency of these compounds. e conguration of the benzene ring B (Fig.1A)
also seems to have impact on the interactions and poses. For smaller inhibitors, it might be more important than
for the larger ones to reach partially the acetylated substrate binding site to gain inhibition activity.
Contrary to the inhibitors, activators bound outside of the cle and formed interactions to the β6/α6 loop
which is a part of a stable antiparallel three-stranded β sheet motif forming the acetyl-lysine binding tunnel. e
importance of this β6/α6 loop area for substrate binding for all sirtuins has been discussed previously43,44. us,
the activators might improve the binding of acetylated substrates by inducing conformational changes in the β6/
α6 loop when binding to the putative activator site. Known activators, oleic and linoleic acids bound between the
inhibitor binding site and the N-terminal tail. ese fatty acids as well as anthocyanidins induced changes in the
β6/α6 loop conformation or orientation of residues in this loop. e observed dual role of some avonoids might
be explained by their ability to bind both inhibitor and activator sites. Based on the docking results, these dual
modulators can also form interactions with the same amino acid residues as do the best activators and inhibitors.
Taken together, the observed dual role of some avonoids, such as compound 14, might be explained by their
ability to bind multiple sites and form similar interactions as did the most potent modulators. However, the eect
of these compounds is concentration dependent and thus, the mechanism of modulation might be more complex
than with the activators and inhibitors. erefore, it can be dicult to evaluate the reason for dual role of some
avonoids with docking or other studies.
To predict the possibility of the key residues at activator site (Gly156, Asp185, Trp186, Glu187 and Asp188) to
have an impact on SIRT6 function or structure, an in silico based mutation analysis was carried out. e predic-
tions (Suppl. TablesS3 and S4) show that these residues might be important SIRT6 function or structure. Future
experiments, should target these residues for increased activity.
Previously, it has been demonstrated that some avonoids14,4550, including quercetin and luteolin, were SIRT6
modulators. is study included a larger array of molecular diverse structures of polyphenols and identied other
classes of avonoids with robust SIRT6 activity. Additionally, this study demonstrated that dierent classes of
avonoids can either inhibit or activate the deacetylation activity of SIRT6. Moreover, the eect was found to be
dependent on the avonoid subclass: catechins showed inhibition, anthocyanidins activation and avonones and
avonols both showed inhibition and activation of SIRT6. Molecular modeling studies, also revealed discrete
putative binding sites for both inhibitors and activators.
Methods
Materials. Acetylated histone H3(K9) peptide (residues 1–21) (H3K9Ac) was from AnaSpec (USA). Fetal
bovine serum (FBS), Novex 10–20% gradient gels, anti-GLUT1-IgG (PA5–16793), anti-SIRT6-IgG (PA517215)
and anti-rabbit-IgG (mouse) Horseradish peroxidase (HRP)-conjugated secondary antibody (G21234) were
from Life Technologies (UK). Anti- FoxO3α-IgG (SAB3500508), anti-α-Tubulin (T5168)-IgG1, anti-Twist1-IgG
(SAB2106420), NAD+, formic acid and compounds were from Sigma Aldrich (USA). Anti-mouse-IgG (rab-
bit) HRP-conjugated secondary antibody (ab97046) was from Abcam (UK). Dulbecco modied Eagle medium
(DMEM) and non-essential amino acids were from Lonza (Belgium). Rabbit anti-acetyl H3K9 antibody and
puried chicken core histones (13–107) were from Millipore (USA). Enhanced chemiluminescence (ECL) prime
western blotting detection reagents were from Amersham BioSciences (UK). Penicillin/Streptomycin was from
EuroClone (Italy).
e human SIRT6 expression vector hSIRT6-pGEX-6P3 was kindly provided by Prof. Katrin Chua (Stanford,
USA). Recombinant GST-tagged SIRT6 was produced by fermentation in E. coli BL21(DE3)-pRARE. e pro-
duction was done at +16 °C with 0.1 mM IPTG for 20 h and the soluble overexpressed protein was puried on
glutathione agarose (Sigma, Saint Louis, USA).
Radioimmunoprecipitation assay (RIPA) lysis buer was prepared in 50 mM Tris-HCl buer (pH = 8.0) con-
sisting of 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 5 mM EDTA, 0.1% SDS.
SIRT6 deacetylation assay. e in vitro assay was carried out as previously described14. Briey, 0.6 µl of
compounds (100 µM) in DMSO and DMSO (control) were incubated for 30 min with GST-SIRT6 (3 µg/well),
H3K9Ac (40 µM for activation/200 µM) and 500 µM NAD+ in Tris Buer [25 mM, pH 8.0] at +37 °C. DMSO
concentration was 1% in all samples. Control samples for compounds without NAD+ or SIRT6 were carried out.
e deacetylation reaction was terminated by adding 6 µl of cold 10% formic acid and centrifuged for 15 min.
e samples were analyzed by reversed-phase HPLC. e formation of deacetylated product (H3K9) and sub-
strate (H3K9Ac) peaks was monitored and subsequently quantied by measuring area under the curve. e
dose response was determined for compounds 4, 5, 13, 15, 16, 17 (Fig.1B) and 18 (0.5 dilutions from 1000 µM).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
9
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
Maximal activation for the most potent activators was determined by maximal eect indicating the maximal
increase in activation for the tested compounds. Experiments were repeated in triplicate, and IC50/EC50 values
were calculated using Graph Pad Prism Soware version 6 (California, USA).
HPLC. Chromatographic separation of H3K9/H3K9Ac was achieved on a Zorbax Eclipse XDB-C18 column
(4.6 mm × 50 mm, 1.8 µ Agilent, Santa Clara, CA, USA) using a Shimadzu prominence system (Shimadzu, Japan)
consisting of a CBM-20A, LC-20AB binary pumps, SIL-20AC-HT autosampler and DGU-20A3 degasser. Mobile
phase consisted of water with 0.02% formic acid (eluent A) and acetonitrile with 0.02% formic acid (eluent B). e
gradient of eluent B was set up as follows: 0–2.0 min 0%; 2.0–10 min 0–8%; 10–10.1 min 8–80%; 10.1–12 min 80%;
12–12.1 min 80–0%; 15 min 0%. Flow rate was 0.9 ml/min, run time 15 min and injection volume 20 µl. HPLC system
was coupled to a 5500 QTRAP equipped with Turbo V electrospray ionization source (TIS)® (Applied Biosystems,
Foster City, CA, USA). Data were acquired and analyzed using Analyst version 1.5.1 (Applied Biosystems). Positive
electrospray ionization data were acquired using multiple reactions monitoring (MRM). TIS instrumental source
settings for temperature, curtain gas, ion source gas 1 (nebulizer), ion source gas 2 (turbo ion spray), entrance poten-
tial and ion spray voltage were 550 C, 20 psi, 60 psi, 50 psi, 10 V and 5500 V, respectively. TIS compound parame-
ter settings for declustering potential, collision energy, and collision cell exit potential were 231 V, 45 V, and 12 V,
respectively, for H3K9Ac and 36 V, 43 V and 12 V, respectively for H3K9. e standards were characterized using the
following MRM ion transitions: H3K9Ac (766.339 760.690) and H3K9 (752.198 746.717).
Western blot analysis. Western blot analysis was carried out as previously described, with slight modi-
cations20. Briey, 100 µM cyanidin or dephinidin and DMSO control were incubated for 30 min in the presence
of 3 µg of puried recombinant GST-SIRT6, 1.25 µg puried chicken core histones, and 500 µM NAD+ in 25 mM
Tris-HCl, pH 8.0 at +37 °C. e reaction was stopped with Laemmli (sample buer) and separated by SDS-PAGE
using 10–20% gradient gels and transferred onto polyvinylidene diuoride (PVDF) membranes. H3K9 acetyl-
ation was detected with rabbit anti-acetyl H3K9 antibody followed by anti-rabbit HRP-conjugated secondary
antibody. Membranes were stripped and re-probed with rabbit anti-histone H3 antibody. Chemiluminescent
signal detection and image acquisition were carried out using ECL prime western blotting detection reagents.
Molecular modeling. In docking studies, Maestro 11.0.015 was used (Small-Molecule Drug Discovery Suite
2016-4, Schrödinger, LLC, New York, NY, 2016).
Protein structure preparation. SIRT6 protein structure was downloaded from RCSB Protein Data Bank (PDB ID:
3ZG6)5. e structure was prepared using Protein Preparation Wizard with default settings (assign bond orders,
add hydrogens, create zero-order bonds to metals, create disulde bonds). Hydrogen bonds were assigned using
PROPKA (pH 7.4) and waters having less than 3 H-bonds to non-waters were removed (default setting). Protein
was minimized using OPLS3 force eld (heavy atom converging RMSD 0.30 Å). Myristoyl peptide was removed.
Ligand preparation. 3D-structures for linoleic acid, oleic acid and compounds 127 were generated using Maestro
and prepared with LigPrep using OPLS3 forceeld. Compounds were ionized at target pH 7.4, desalted and tautom-
ers were generated using Epik. For stereoisomer generation, chiralities were determined from 3D structure.
Binding site detection. Inhibitor binding site was determined based on the binding site of SIRT1 inhibitor Ex-527
in crystal structure of SIRT1 (PDB ID 4I5I)51. SIRT6 and SIRT1 structures were overlaid, and binding site corre-
sponding to Ex-527 in SIRT1 was determined for SIRT6. In this study, Maestro SiteMap was used to determine
the activator binding site. At least 10 site points per reported site was set to be required, and shallow binding sites
were also detected. Other settings were kept as default (identify top-ranked potential receptor binding sites, use
more restrictive denition of hydrophobicity, use standard grid and crop site maps at 4 Å from nearest site point).
Docking. Ligands were docked with Maestro Induced Fit (Version 3.1, Glide Grid generation version 5.1) which
docks ligands to the dened receptor with Glide, then Prime Renement processes predened amount of best
scoring poses and relaxes the receptor structure. e ligands are redocked with Glide and the best scoring poses
are shown. Herein, the grid center of inhibitor binding site was in the middle of Ile59, Phe62, Val68, Asn112 and
Ile183, close to the binding site of nicotinamide. e grid center for the activator site (for control fatty acids and
SIRT6 activators) was in the center of Ser8, Gly156, Lys158 and Glu187. Other settings were kept as default (grid
enclosing box size 26 Å on a side, ring conformation sampling with energy window of 2.5 kcal/mol, penalize
nonpolar conformation of amide bonds, receptor and Van der Waals scale of 0.5 Å, rene residues within 5.0 Å
of ligand poses, redock within 30.0 kcal/mol of the best structure and use Standard Precision docking). e nal
poses selected for interaction comparison were among the best three scoring poses.
In silico mutation analysis. e functional impact of the key SIRT6 residues at the putative activator site
was assessed using SIFT (http://si.bii.a-star.edu.sg/)52, PROVEAN (http://provean.jcvi.org/index.php)53 and
PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)54. PROVEAN and SIFT employ sequence similarity based
methods to identify homologous sequences and use the sequence conservation to calculate impact score(s).
PolyPhen-2 also uses sequence-based information but, in addition uses 3D structure based predictive features.
SIRT6 genomic and residue information were obtained from Ensembl transcript (ENST00000337491.6), and
UniProt (ID: Q8N6T7-1) respectively. In silico alanine type scanning was used to assess the functional impor-
tance of the key residues. e amino acid substitutions were either neutral (Ala), or other residues that possess
drastically dierent chemical properties. e results (Suppl. TableS3) predict, whether residues are likely to be
important either for the function or structure of SIRT6.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
10
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
Cell culture and treatments. Caco-2 cells (passage 30–40) were cultured in DMEM with 10% FBS, 1%
nonessential amino acids, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin at +37 °C.
Cells were cultured 14 days before the treatments. For the immunoblotting, cells were seeded on 24-well plates
(1 × 105 cells/well). Aer 24 hours, cells were treated with 0.5% DMSO (control) or various concentrations (12.5–
200 µM) of compound 17 for 24 h.
Preparation of total cell fraction for immunoblotting. Aer treatment, medium was discarded and
cells were washed twice with ice-cold PBS. RIPA buer was added to the cells and incubated for 30 min. Cell sus-
pensions were collected and centrifuged (13,000 rpm, 20 min +4 °C). Supernatant containing the proteins were
aliquoted and stored at 80 °C. Sample protein concentrations were measured with Bradford Assay (Bio-Rad
DCTM Protein Assay).
Immunoblotting. Immunoblotting was performed according to standard protocols from four independent
experiments. Briey, protein samples (18 µg/sample) were separated by SDS-PAGE using 10–20% gradient gels
and transferred onto PVDF membranes. Membranes were blocked in 3% non-fat dry milk and further incu-
bated with primary rabbit anti- FoxO3α (1:2000), anti-GLUT1 (1:1000), anti-H3, anti-SIRT6 (1:2000), anti-Twist
(1:2000) anti-α-tubulin (1:8000) antibodies overnight at +4 °C. HRP-conjugated secondary antibodies (goat
anti-rabbit, goat anti-mouse) were incubated for 1 hour at room temperature and proteins were detected using
ELC prime western blotting system. Densitometric analysis of protein bands was carried out using ImageJ 1.32
soware and the data were normalized by α-tubulin (loading control).
References
1. Michishita, E. et al. SIT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nat. 452, 492–96 (2008).
2. awahara, T. L. et al. SIT6 lins histone H3 lysine 9 deacetylation to NF-appaB-dependent gene expression and organismal life
span. Cell 136, 62–74 (2009).
3. Michishita, E. et al. Cell cycle-dependent deacetylation of telomeric histone H3 lysine 56 by human SIT6. Cell Cycle 8, 2664–66
(2009).
4. Mao, Z. et al. SIT6 promotes DNA repair under stress by activating PAP1. Sci. 332, 1443–46 (2011).
5. Jiang, H. et al. SIT6 regulates TNF-alpha secretion through hydrolysis of long-chain fatty acyl lysine. Nat. 496, 110–13 (2013).
6. Lombard, D. B., Schwer, B., Alt, F. W. & Mostoslavsy, . SIT6 in DNA repair, metabolism and ageing. J Intern Med 263, 128–41
(2008).
7. Lombard, D. B. Sirtuins at the breaing point: SIT6 in DNA repair. Aging (Albany NY) 1, 12–16 (2009).
8. an, Y. et al. e sirtuin SIT6 regulates lifespan in male mice. Nat. 483, 218–21 (2012).
9. oichman, A. et al. SIT6 overexpression improves various aspects of mouse healthspan. J Gerontol A Biol Sci Med Sci 72, 603–15
(2016).
10. Zhong, L. et al. e histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 140, 280–93 (2010).
11. Sebastian, C. et al. e histone deacetylase SIT6 is a tumor suppressor that controls cancer metabolism. Cell 151, 1185–99 (2012).
12. Howitz, . T. et al. Small molecule activators of sirtuins extend saccharomyces cerevisiae lifespan. Nat. 425, 191–96 (2003).
13. Feldman, J. L., Baeza, J. & Denu, J. M. Activation of the protein deacetylase SIT6 by long-chain fatty acids and widespread
deacylation by mammalian sirtuins. J Biol Chem 288, 31350–56 (2013).
14. ahnasto-illa, M., oola, T., Jarho, E., Lahtela-aonen, M. & Moaddel, . N-acylethanolamines bind to SIT6. Chembiochem
17, 77–81 (2016).
15. oonen, P. et al. Studying SITt6 regulation using H356 based substrate and small molecules. Eur J Pharm Sci 63, 71–76 (2014).
16. oonen, P. et al. Peptides and pseudopeptides as SIT6 deacetylation inhibitors. ACS Med Chem Lett 3, 969–74 (2012).
17. Sociali, G. et al. Quinazolinedione SIT6 inhibitors sensitize cancer cells to chemotherapeutics. Eur J Med Chem 102, 530–39 (2015).
18. Parenti, M. D. et al. Discovery of novel and selective SIT6 inhibitors. J Med Chem 57, 4796–804 (2014).
19. ahnasto-illa, M., Lahtela-aonen, M. & Moaddel, . Sirtuin 6 (SIT6) activity assays. Methods Mol Biol 1436, 259–69 (2016).
20. a hnasto-illa, M. et al. e identication of a SIT6 activator from brown algae Fucus distichus. Mar Drugs 6, https://doi.
org/10.3390/md15060190 (2017).
21. Liu, J. & Zheng, W. Cyclic peptide-based potent human SIT6 inhibitors. Org Biomol Chem 14, 5928–35 (2016).
22. He, B., Hu, J., Zhang, X. & Lin, H. iomyristoyl peptides as cell-permeable Sirt6 inhibitors. Org Biomol Chem 12, 7498–502 (2014).
23. Tennen, . I., Bua, D. J., Wright, W. E. & Chua, . F. SIT6 is required for maintenance of telomere position eect in human cells.
Nat Commun 2, 433 (2011).
24. Tennen, . I. & Chua, . F. Chromatin regulation and genome maintenance by mammalian SIT6. Trends Biochem. Sci 36, 39–46
(2011).
25. Yuan, J., Pu, M., Zhang, Z. & Lou, Z. Histone H3-56 acetylation is important for genomic stability in mammals. Cell Cycle 8,
1747–53 (2009).
26. Tao, L., Par, J. Y. & Lambert, J. D. Dierential prooxidative eects of the green tea polyphenol, ()-epigallocatechin-3gallate, in
normal and oral cancer cells are related to dierences in sirtuin 3 signaling. Mol Nutr Food es 59, 203–11 (2015).
27. Niu, Y. et al. e phytochemical, EGCG, extends lifespan by reducing liver and idney function damage and improving age-
associated inammation and oxidative stress in healthy rats. Aging Cell 12, 1041–49 (2013).
28. He, J. & Giusti, M. M. Anthocyanins: natural colorants with health-promoting properties. Annu. ev Food Sci Technol 1, 163–87
(2010).
29. Smeriglio, A., Barreca, D., Bellocco, E. & Trombetta, D. Chemistry, pharmacology and health benets of anthocyanins. Phytother es
30, 1265–86 (2016).
30. umar, S. & Pandey, A. . Chemistry and biological activities of avonoids: an overview. Sci. 2013, 162750 (2013).
31. Wang, L. S. & Stoner, G. D. Anthocyanins and their role in cancer prevention. Cancer Lett 269, 281–90 (2008).
32. Lin, B. W., Gong, C. C., Song, H. F. & Cui, Y. Y. Eects of anthocyanins on the prevention and treatment of cancer. Br J Pharmacol
174, 1226–43 (2016).
33. Wang, X. X. et al. SIT6 protects cardiomyocytes against ischemia/reperfusion injury by augmenting FoxO3α-dependent
antioxidant defense mechanisms. Basic es Cardiol 111, 13 (2016).
34. Han, Z., Liu, L., Liu, Y. & Li, S. Sirtuin SIT6 suppresses cell proliferation through inhibition of Twist1 expression in non-small cell
lung cancer. Int J Clin Exp Pathol 7, 4774–81 (2014).
35. Satoh, A., Stein, L. & Imai, S. e role of mammalian sirtuins in the regulation of metabolism, aging and longevity. Handb Exp
Pharmacol 206, 125–62 (2011).
36. Lan, F., Weiel, ., Cacicedo, J. & Ido, Y., esveratrol-induced AMP-activated protein inase activation is cell-type dependent:
Lessons from basic research for clinical application. Nutr. 9, https://doi.org/10.3390/nu9070751 (2017).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
www.nature.com/scientificreports/
11
SCIeNtIfIC RepoRTS | (2018) 8:4163 | DOI:10.1038/s41598-018-22388-5
37. Guo, H., Liu, G., Zhong, ., Wang, D. & Xia, M. Cyanidin-3-o-b-glucoside regulates fatty acid metabolism via an amp-activated
protein inase-dependent signaling pathway in human HepG2 cells. Lipids Heal. Dis 11, 10 (2012).
38. Yang, S. J. et al. Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone increases Sirt6 expression and
ameliorates hepatic steatosis in rats. PLoS ONE 6, https://doi.org/10.1371/journal.pone.0017057 (2011).
39. Xia, M. et al. Anthocyanins induce cholesterol eux from mouse peritoneal macrophages. J. Biol. Chem 280, 36792–801 (2005).
40. Seymour, E. M. et al. Blueberry intae alters seletal muscle and adipose tissue peroxisome proliferator-activated receptor activity
and reduces insulin resistance in obese rats. J.Med Food 14, 1511–18 (2011).
41. Yuan, H. & Marmorstein, . Structural basis for sirtuin activity and inhibition. J Biol Chem 287, 42428–35 (2012).
42. Nguyen, G. T., Schaefer, S., Gertz, M., Weyand, M. & Steegborn, C. Structures of human sirtuin 3 complexes with ADP-ribose and
with carba-NAD+ and ST1720: binding details and inhibition mechanism. Acta Crystallogr D Biol Crystallogr 69, 1423–32 (2013).
43. Pan, P. W. et al. Structure and Biochemical Functions of SIT6. J Biol Chem 286, 14575–87 (2011).
44. Saunders, B. D., Jacson, B. & Marmorstein, . Structural basis for sirtuin function: what we now and what we don’t. Biochim
Biophys Acta. 1804, 1604–16 (2010).
45. Trinh, Q. & Le, L. An investigation of antidiabetic activities of bioactive compounds in Euphorbia hirta Linn using molecular
docing and pharmacophore. Med Chem es 23, 2033–45 (2014).
46. Singh, N., avichandran, S., Spelman, ., Fugmann, S. & Moaddel, . e identication of a novel SIT6 modulator from Tr igonella
foenum-graecum using ligand shing with protein coated magnetic beads. J Chromatogr B Anal. Technol Biomed Life Sci 968,
105–11 (2014).
47. Nguyen, V., T., Tran, N., Nguyen, D. & Le, L. An in silico study on antidiabetic activity of bioactive compounds in Euphorbia
hymifolia Linn. Springerplus 5, 1359 (2016).
48. Yasuda, M., Wilson, D., Fugmann, S. & Moaddel, . Synthesis and characterization of Tt6 protein coated magnetic beads:
identication of a novel inhibitor of SIT6 deacetylase from medicinal plant extracts. Anal Chem 83, 7400–407 (2011).
49. Singh, N., avichandran, S., Norton, D., Fugmann, S. & Moaddel, . Synthesis and characterization of a SIT6 open tubular column:
predicting deacetylation activity using frontal chromatography. Anal Biochem. 436, 78–83 (2013).
50. avichandran, S. et al. Pharmacophore model of the quercetin binding site of the SIT6 protein. J Mol Graph Model. 49, 38–46
(2014).
51. Zhao, X. et al. e 2.5 Å crystal structure of the sirt1 catalytic domain bound to nicotinamide adenine dinucleotide (NAD+) and an
indole (EX527 analogue) reveals a novel mechanism of histone deacetylase inhibition. J Med Chem 56, 963–69 (2013).
52. umar, P., Henio, S. & Ng, P. C. Predicting the eects of coding non-synonymous variants on protein function using the SIFT
algorithm. Nat. Protoc. 4, 1073–81 (2009).
53. Choi, Y. & Chan, A. P. POVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels.
Bioinforma. 31, 2745–47 (2017).
54. Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–49 (2010).
Acknowledgements
e authors thank Sari Ukkonen for her skillful technical assistance and Biocenter Kuopio for the facilities, the
CSC-IT Cen-ter of Science Limited for providing soware license for the Schrodinger soware package. is
research was supported in part by the Intramural Research Program of the National Institute on Aging, NIH. e
authors thank Academy of Finland (grant no. 269341), Orion – Farmos Research Foundation, the Foundation
of Saastamoinen, Finnish Cultural Foundation for nancial support and with federal funds from the National
Cancer Institute, National Institutes of Health, under contract HHSN 261200800001E. e authors are solely
responsible for the contents of this study, which may not represent the ocial views of the National Institutes
of Health or policies of the Department of Health and Human Services. ere is no mention of trade names,
commercial products, or organizations implying endorsement by the US government.
Author Contributions
M.R.-R., J.T., M.L.-K. and R.M. conceived and designed the study. M.R.-R., J.T., M.H., T.K. and S.R. conducted
experiments and analyzed the results. M.L.-K. and R.M. contributed the interpretation and analysis of the data.
M.R.-R., J.T., M.H., E.J., T.K., S.R., V.A.B., L.F., M.L.-K. and R.M. contributed to the writing of the manuscript. All
authors reviewed and approved the nal version of manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-22388-5.
Competing Interests: e authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© e Author(s) 2018
Content courtesy of Springer Nature, terms of use apply. Rights reserved
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com

Supplementary resource (1)

... Inhibitors, on the other hand, are likely to bind in a way that disrupts NAD + binding. Cyanidin is indicated as the strongest SIRT6 activator [80]. Other activators include: Nacetylamines [81], icariin [82], ergothioneine [83]. ...
... Other activators include: Nacetylamines [81], icariin [82], ergothioneine [83]. Significant inhibitory power for SIRT6 activity has been shown for galloylated catechins [80]. Compounds that will exhibit inhibitory activity on SIRT6 activity include quercetin, vitexin [84], peptides containing netioacetylated lysine [85] and EX-527 [86]. ...
Article
Full-text available
Sirtuins, in mammals, are a group of seven enzymes (SIRT1-SIRT7) involved in the post-translational modification of proteins-they are considered longevity proteins. SIRT6, classified as class IV, is located on the cell nucleus; however, its action is also connected with other regions, e.g., mitochondria and cytoplasm. It affects many molecular pathways involved in aging: telomere maintenance, DNA repair, inflammatory processes or glycolysis. A literature search for keywords or phrases was carried out in PubMed and further searches were carried out on the ClinicalTrials.gov website. The role of SIRT6 in both premature and chronological aging has been pointed out. SIRT6 is involved in the regulation of homeostasis-an increase in the protein's activity has been noted in calorie-restriction diets and with significant weight loss, among others. Expression of this protein is also elevated in people who regularly exercise. SIRT6 has been shown to have different effects on inflammation, depending on the cells involved. The protein is considered a factor in phenotypic attachment and the migratory responses of macrophages, thus accelerating the process of wound healing. Furthermore, exogenous substances will affect the expression level of SIRT6: resveratrol, sirtinol, flavonoids, cyanidin, quercetin and others. This study discusses the importance of the role of SIRT6 in aging, metabolic activity, inflammation, the wound healing process and physical activity.
... Anthocyanidins can also greatly increase SIRT6 deacetylation activity in vitro. Moreover, cyaniding can upregulate SIRT6 protein expression in human colon adenocarcinoma Caco-2 cells (Rahnasto-Rilla et al., 2018). Interestingly, Khan et al. (2021) proposed that SIRT activators can be investigated to determine their effectiveness of alleviating COVID-19 symptoms. ...
Article
Full-text available
Living and eating healthiness have been the topic of interest for the elderly and their caregivers. Functional foods with balanced nutrition and anti-aging properties were considered and researched continuously. This review aimed to summarize and discuss the aging process and functional foods from natural sources for the elderly using studies conducted between 1996 and 2023 that focus on their effectiveness to alleviate aging symptoms. Aging is a complex process with various mechanisms including oxidative stress, hormonal system, activity of the enzyme sirtuin, and intestinal microbiota. The aging process leads to many disorders in the elderly such as Alzheimer's, Parkinson's, and non-communicable diseases. Additionally, it was found that balanced nutrients and energy along with the consumption of natural antioxidants from vitamins, phytochemicals, and sirtuin activator foods were effective at slowing down aging and extending an individual's lifespan. Recently, innovative research into foods for the elderly has focused on their sensory perception and whether they consist of the necessary nutrients. Therefore, this review might support a future perspective product of the food and pharmaceutical industries because of the different requirements of the elderly in society.
... Moreover, a significant proportion of papillary thyroid cancers harbor NTRK1 somatic rearrangements that generate chimeric oncogenes with constitutive tyrosine kinase activity, as reviewed by Pierotti and Greco [262]. Several other computational studies have investigated the effects of EGCG on other cancer-related factors including trypsin [263], proteasome [264], PTP1B phosphatase [265], glutathione S-transferase (GST P1-1) [266], or the epigenetic modulators DNMT [267], HDAC [57], and sirtuin-6 (SIRT6) [268], as recently reviewed [269,270]. ...
Article
Full-text available
Cellular signaling pathways involved in the maintenance of the equilibrium between cell proliferation and apoptosis have emerged as rational targets that can be exploited in the prevention and treatment of cancer. Epigallocatechin-3-gallate (EGCG) is the most abundant phenolic compound found in green tea. It has been shown to regulate multiple crucial cellular signaling pathways, including those mediated by EGFR, JAK-STAT, MAPKs, NF-κB, PI3K-AKT-mTOR, and others. Deregulation of the abovementioned pathways is involved in the pathophysiology of cancer. It has been demonstrated that EGCG may exert anti-proliferative, anti-inflammatory, and apoptosis-inducing effects or induce epigenetic changes. Furthermore, preclinical and clinical studies suggest that EGCG may be used in the treatment of numerous disorders, including cancer. This review aims to summarize the existing knowledge regarding the biological properties of EGCG, especially in the context of cancer treatment and prophylaxis.
... It is well known that RES, PD and CUR exert their pharmacological effects by regulating multiple targets and metabolic pathways [40,46]. The literature review from 2006 to 2021 pointed out that flavonoids frequently interact with SIRT-1 [52,53] and SIRT-3 [54] , followed by SIRT-6 [55].RES is the most widely studied natural product that can activate AMPK by multiple mechanisms, such as the activation of SIRT-1. Preliminary studies show that RES by activating (SIRT-1), can protect against the detrimental effects of oxidative stress and promotes neuronal development [56] .Likewise, RES, PD is usually considered a potential SIRT-1 activator and the pharmacological mechanisms of PD could involve members of the SIRT protein family, among which SIRT-1 plays a prevalent role [57,58] .CUR can modulate the activity of Sirtuins and facilitate adaptation to certain cellular settings, including stress factors. ...
Article
Full-text available
Sirtuins (SIRTs), a family of NAD +-dependent deacetylases, are involved in the regulation of physiological functions such as aging and inflammation. They are able to catalyze metabolic reactions, thus regulating several cellular processes, such as energy metabolism, stress response, cell survival and apoptosis, DNA repair, tissue regeneration and neuronal signaling. The present article examines the interaction of three polyphenols, as Resveratrol, Polydatin and Curcumin, with Sirtuins family. The data obtained through a computational analysis, by Molecular Docking and Molecular Dynamics approaches, shows that these natural compounds are able to bind the active site of Sirtuins involved in numerous biochemical signaling. Moreover, the results highlight that Polydatin bind all the considered SIRT proteins showing an excellent docking capability in terms of Binding Energies scores and estimation of Inhibition Constant Ki. Moreover, by the study of Dynamic Simulation (RMSF, RMSD, protein-ligand interactions, timeline simulation in the range of 100 ns) and Repeatability Docking tests, Polydatin appear more stable than Curcumin when binds SIRT-3 rather than SIRT-5 protein.
... Polyphenols of the anthocyanin family, which are found in berries, are most compatible with the human physiology [94]. This class of polyphenols exhibits its anti-inflammatory properties by activating AMPK through activation of sirtuins [95,96], inhibiting inflammasome formation [97,98], and promoting proliferation of the gut bacterium Akkermansia muciniphila [74]. Multiple studies have determined the minimum therapeutic dose of polyphenols to be 150 to 500mg per day depending on the patient's condition and genetics [99,100]. ...
Article
Diabetes is a prevalent disease that affects millions of people around the world with severe and costly complications, such as neuropathy, retinopathy, nephropathy, peripheral vascular disease, ischemic heart disease, and cerebrovascular disease. It has been determined that prolonged state of hyperglycemia causes oxidative and nitrosamine stress and ischemia to the tissues, which leads to inflammation that the immune system cannot resolve. Because of this, diabetic patients live in a state of chronic inflammation. Current treatment of diabetes primarily revolves around glycemic control and lifestyle modifications; and while modern diabetic drugs continue to improve, nutritional aspects usually lag in the overall management of the disease. However, well optimized nutrition can not only minimize the source of inflammation and decrease the existing chronic inflammation, but it can also help the immune system to transition into the resolution and repair state. Such anti-inflammatory diet should abide by the following rules: 1) caloric restriction; 2) consumption of adequate amount of proteins; 3) decreased consumption of saturated fats; 4) increased consumption of unsaturated fats, especially omega-3 fatty acids; 5) increased intake of fermented fiber; 6) increased intake of polyphenols; 7) decreased consumption of both arachidonic acid and linoleic acid. It is important to mention that it might not be easy for the patient to switch their diet to the new one and maintain it over the long period of time. However, if done correctly, it might be a relatively cheap and effective addition to the standard medical treatment of diabetes.
... Nitro-fatty acids including nitrooleic acid and nitro-conjugated linoleic acid have been shown to activate the deacetylase activity of SIRT6 through binding to the hydrophobic crevice of the SIRT6 active site [106]. Galloflavin and ellagic acids, the most common polyphenols in berries, have been shown to activate the deacetylase activity of SIRT6, possibly through a direct interaction with G6 and D188 residues [107]. Structure-activity relationship analysis has led to the identification of the 2-(1-benzofuran-2-yl)-N-(diphenylmethyl) quinoline-4-carboxamide (also named 12q) compound as a very potent SIRT6 activator with an EC 50 of 5.35 ± 0.69 µM in an in vitro deacetylase assay. ...
Article
Full-text available
Sirtuin 6 (SIRT6) is an NAD-dependent deacetylase/deacylase/mono-ADP ribosyltransferase, a member of the sirtuin protein family. SIRT6 has been implicated in hepatic lipid homeostasis and liver health. Hepatic lipogenesis is driven by several master regulators including liver X receptor (LXR), carbohydrate response element binding protein (ChREBP), and sterol regulatory element binding protein 1 (SREBP1). Interestingly, these three transcription factors can be negatively regulated by SIRT6 through direct deacetylation. Fatty acid oxidation is regulated by peroxisome proliferator activated receptor alpha (PPARα) in the liver. SIRT6 can promote fatty acid oxidation by the activation of PPARα or the suppression of miR-122. SIRT6 can also directly modulate acyl-CoA synthetase long chain family member 5 (ACSL5) activity for fatty acid oxidation. SIRT6 also plays a critical role in the regulation of total cholesterol and low-density lipoprotein (LDL)-cholesterol through the regulation of SREBP2 and proprotein convertase subtilisin/kexin type 9 (PCSK9), respectively. Hepatic deficiency of Sirt6 in mice has been shown to cause hepatic steatosis, inflammation, and fibrosis, hallmarks of alcoholic and nonalcoholic steatohepatitis. SIRT6 can dampen hepatic inflammation through the modulation of macrophage polarization from M1 to M2 type. Hepatic stellate cells are a key cell type in hepatic fibrogenesis. SIRT6 plays a strong anti-fibrosis role by the suppression of multiple fibrogenic pathways including the transforming growth factor beta (TGFβ)-SMAD family proteins and Hippo pathways. The role of SIRT6 in liver cancer is quite complicated, as both tumor-suppressive and tumor-promoting activities have been documented in the literature. Overall, SIRT6 has multiple salutary effects on metabolic homeostasis and liver health, and it may serve as a therapeutic target for hepatic metabolic diseases. To date, numerous activators and inhibitors of SIRT6 have been developed for translational research.
... Given these results, there may be a benefit to enhancing SIRT6 activity, specifically the ribosylase activity. Molecules that impact SIRT6 activity, as well as its major co-enzyme NAD + , have been identified and hold potential as future methods of anti-aging interventions (Li et al, 2017;Rahnasto-Rilla et al, 2018;Rajman et al, 2018). Further refining the search for interventions that specifically target the mADPr activity of SIRT6 may yield more specific therapeutics to improve lifespan and healthspan. ...
Article
Full-text available
Natural compounds with pharmacological activity, flavonoids have been the subject of an exponential increase in studies in the field of scientific research focused on therapeutic purposes due to their bioactive properties, such as antioxidant, anti-inflammatory, anti-aging, antibacterial, antiviral, neuroprotective, radioprotective, and antitumor activities. The biological potential of flavonoids, added to their bioavailability, cost-effectiveness, and minimal side effects, direct them as promising cytotoxic anticancer compounds in the optimization of therapies and the search for new drugs in the treatment of cancer, since some extensively antineoplastic therapeutic approaches have become less effective due to tumor resistance to drugs commonly used in chemotherapy. In this review, we emphasize the antitumor properties of tangeretin, a flavonoid found in citrus fruits that has shown activity against some hallmarks of cancer in several types of cancerous cell lines, such as antiproliferative, apoptotic, anti-inflammatory, anti-metastatic, anti-angiogenic, antioxidant, regulatory expression of tumor-suppressor genes, and epigenetic modulation.
Article
Natural pigments are important sources for the screening of bioactive lead compounds. This article reviewed the chemistry and therapeutic potentials of over 570 colored molecules from plants, fungi, bacteria, insects, algae, and marine sources. Moreover, related biological activities, advanced extraction, and identification approaches were reviewed. A variety of biological activities, including cytotoxicity against cancer cells, antioxidant, anti-inflammatory, wound healing, anti-microbial, antiviral, and anti-protozoal activities, have been reported for different pigments. Considering their structural backbone, they were classified as naphthoquinones, carotenoids, flavonoids, xanthones, anthocyanins, benzotropolones, alkaloids, terpenoids, isoprenoids, and non-isoprenoids. Alkaloid pigments were mostly isolated from bacteria and marine sources, while flavonoids were mostly found in plants and mushrooms. Colored quinones and xanthones were mostly extracted from plants and fungi, while colored polyketides and terpenoids are often found in marine sources and fungi. Carotenoids are mostly distributed among bacteria, followed by fungi and plants. The pigments isolated from insects have different structures, but among them, carotenoids and quinone/xanthone are the most important. Considering good manufacturing practices, the current permitted natural colorants are: Carotenoids (canthaxanthin, β-carotene, β-apo-8'-carotenal, annatto, astaxanthin) and their sources, lycopene, anthocyanins, betanin, chlorophyllins, spirulina extract, carmine and cochineal extract, henna, riboflavin, pyrogallol, logwood extract, guaiazulene, turmeric, and soy leghemoglobin.
Article
SIRT6 has emerged as a novel therapeutic target for a variety of diseases. In this study, a total of 102 pyrazolo [1,5-a]quinazoline derivatives were designed and synthesized. The result revealed that 2-methyl-N-(4-phenoxy-phenyl)pyrazolo [1,5-a]quinazoline-5-amine (21q) was the most active compound by structure-activity relationship study, which significantly enhanced SIRT6 defatty-acylation activity with an EC1.5 value of 1.85±0.41 μM and EC50 value of 11.15±0.33 μM. The biological activity of 21q was further verified by differential scanning fluorimetry assay (DSF) and surface plasmon resonance assay (SPR). Molecular docking showed that the pyrazolo [1,5-a]quinazoline of 21q formed a hydrogen bond with Val115 and four π- π interactions with Phe64, Phe82 and Phe86. 21q can significantly improve the thermal stability of SIRT6 protein and inhibit the PI3K/Akt signaling pathway in mouse embryonic fibroblasts (MEFs), thereby inhibiting the proliferation of MEFs. Collectively, we discovered a new potent SIRT6 activator, which can be taken as a lead compound for later studies.
Article
Full-text available
Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol’s effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD⁺ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50-100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD⁺ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical setting.
Article
Full-text available
Brown seaweeds contain many bioactive compounds, including polyphenols, polysaccharides, fucosterol, and fucoxantin. These compounds have several biological activities, including anti-inflammatory, hepatoprotective, anti-tumor, anti-hypertensive, and anti-diabetic activity, although in most cases their mechanisms of action are not understood. In this study, extracts generated from five brown algae (Fucus dichitus, Fucus vesiculosus (Linnaeus), Cytoseira tamariscofolia, Cytoseira nodacaulis, Alaria esculenta) were tested for their ability to activate SIRT6 resulting in H3K9 deacetylation. Three of the five macroalgal extracts caused a significant increase of H3K9 deacetylation, and the effect was most pronounced for F. dichitus. The compound responsible for this in vitro activity was identified by mass spectrometry as fucoidan.
Article
Full-text available
Herbal medicines have become strongly preferred treatment to reduce the negative impacts of diabetes mellitus (DM) and its severe complications due to lesser side effects and low cost. Recently, strong anti-hyperglycemic effect of Euphorbia thymifolia Linn. (E. thymifolia) on mice models has reported but the action mechanism of its bioactive compounds has remained unknown. This study aimed to evaluate molecular interactions existing between various bioactive compounds in E. thymifolia and targeted proteins related to Type 2 DM. This process involved the molecular docking of 3D structures of those substances into 4 targeted proteins: 11-β hydroxysteroid dehydrogenase type 1, glutamine: fructose-6-phosphate amidotransferase, protein-tyrosine phosphatase 1B and mono-ADP-ribosyltransferase sirtuin-6. In the next step, LigandScout was applied to evaluate the bonds formed between 20 ligands and the binding sites of each targeted proteins. The results identified seven bioactive compounds with high binding affinity (<−8.0 kcal/mol) to all 4 targeted proteins including β-amyrine, taraxerol, 1-O-galloyl-β-d-glucose, corilagin, cosmosiin, quercetin-3-galactoside and quercitrin. The pharmacophore features were also explained in 2D figures which indicated hydrophobic interactions, hydrogen bond acceptors and hydrogen bond donors forming between carbonyl oxygen molecules of those ligands and active site residues of 4 targeted protein.Graphical abstractEuphorbia thymifolia Linn. is a small prostrate herbaceous annual weed that can positively impact on reducing hyperglycemic effect. In order to clearly understand about molecular level of the its bioactive compounds, in silico approach is performed
Article
Full-text available
The extension in human lifespan in the last century results in a significant increase in incidence of age related diseases. It is therefore crucial to identify key factors that control elderly healthspan. Similar to dietary restriction, mice overexpressing the NAD+ dependent protein deacylase SIRT6 (MOSES) live longer and have reduced IGF-1 levels. However, it is as yet unknown whether SIRT6 also affects various healthspan parameters. Here, a range of age related phenotypes was evaluated in MOSES mice. In comparison to their wild-type (WT) littermates, old MOSES mice showed amelioration of a variety of age-related disorders, including: improved glucose tolerance, younger hormonal profile, reduced age-related adipose inflammation and increased physical activity. The increased activity was accompanied with increased muscle AMP-activated protein kinase (AMPK) activity. Altogether, these results indicate that overexpression of SIRT6 in mice retards important aspects of the aging process and suggest SIRT6 to be a potential therapeutic target for the treatment of a set of age-related disorders.
Article
Full-text available
SIRT6, a member of the NAD(+)-dependent class III deacetylase sirtuin family, has been revealed to play important roles in promoting cellular resistance against oxidative stress. The formation of reactive oxygen species (ROS) and oxidative stress are the crucial mechanisms underlying cellular damage and dysfunction in cardiac ischemia/reperfusion (I/R) injury, but the role of SIRT6 in I/R-induced ROS and oxidative stress is poorly understood. In this study, by using heterozygous SIRT6 knockout (SIRT6+/−) mice and cultured neonatal cardiomyocyte models, we investigated how SIRT6 mediates oxidative stress and myocardial injury during I/R. Partial knockout (KO) of SIRT6 aggravated myocardial damage, ventricular remodeling, and oxidative stress in mice subjected to myocardial I/R, whereas restoration of SIRT6 expression by direct cardiac injection of adenoviral constructs encoding SIRT6 reversed these deleterious effects of SIRT6 KO in the ischemic heart. In addition, partial deletion of the SIRT6 gene decreased myocardial functional recovery following I/R in a Langendorff perfusion model. Similarly, the protective effects of SIRT6 were also observed in cultured cardiomyocytes following hypoxia/reoxygenation. Intriguingly, SIRT6 was noticed to up-regulate AMP/ATP and then activate the adenosine 5′-monophosphate-activated protein kinase (AMPK)-forkhead box O3α (FoxO3α) axis and further initiated the downstream antioxidant-encoding gene expression (manganese superoxide dismutase and catalase), thereby decreasing cellular levels of oxidative stress and mediating cardioprotection in the ischemic heart. These results suggest that SIRT6 protects the heart from I/R injury through FoxO3α activation in the ischemic heart in an AMP/ATP-induced AMPK-dependent way, thus upregulating antioxidants and suppressing oxidative stress.
Article
Anthocyanins are a class of water-soluble flavonoids, which show a range of pharmacological effects, such as prevention of cardiovascular disease, obesity control and antitumour activity. Their potential antitumour effects are reported to be based on a wide variety of biological activities including antioxidant; anti-inflammation; anti-mutagenesis; induction of differentiation; inhibiting proliferation by modulating signal transduction pathways, inducing cell cycle arrest and stimulating apoptosis or autophagy of cancer cells; anti-invasion; anti-metastasis; reversing drug resistance of cancer cells and increasing their sensitivity to chemotherapy. In this review, the latest progress on the anticancer activities of anthocyanins and the underlying molecular mechanisms is summarized using data from basic research in vitro and in vivo, from clinical trials and taking into account theory and practice. Linked articles: This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.
Chapter
SIRT6 has been shown to possess weak deacetylation, mono-ADP-ribosyltransferase activity, and deacylation activity in vitro. SIRT6 selectively deacetylates H3K9Ac and H3K56Ac. Several SIRT6 assays have been developed including HPLC assays, fluorogenic assays, FRET, magnetic beads, in silico, and bioaffinity chromatography assays. Herein, we describe detailed protocols for the HPLC based activity/inhibition assays, magnetic beads deacetylation assays, bioaffinity chromatographic assays as well as fluorogenic and in silico assays.
Article
We discovered in the current study that six side chain-to-side chain cyclic pentapeptides harboring a central N(ε)-dodecyl (or tetradecyl)-thiocarbamoyl-lysine residue all behaved as highly potent (nM level) inhibitors against human SIRT6-catalyzed deacylation reaction. Moreover, one compound was also found to be selective for SIRT6 versus SIRT2/3/5 (∼20-, ∼11-, and >940-fold, respectively), despite its modest (∼2.3-fold) SIRT6 inhibitory selectivity versus SIRT1. These compounds could be valuable leads for the identification of further potent and selective human SIRT6 inhibitors.
Article
Anthocyanins are naturally occurring molecules belonging to the flavonoid class characterized by the presence of chromophores. Apart from their well-known antioxidant activity, they show a wide variety of health-promoting properties for human health, ranging from cytoprotective, antimicrobial and antitumour activities to neuroprotective, anti-obesity and lipidomic potential, properties for which anthocyanins have been prescribed as medicines in several countries for thousands of years. Despite this, these phytochemicals have received less attention than other flavonoids, and there is still a gap in the literature, particularly regarding pharmacological and toxicological aspects. Moreover, epidemiological evidence suggests a direct correlation between anthocyanin intake and a lower incidence of chronic and degenerative diseases. In light of this, the aim of this review is to cover the current literature on anthocyanins, their biological in vitro and in vivo effects and their potential therapeutic applications, as well as their bioavailability and pharmacokinetics, all of which are essential to gain a better understanding of their biological effectiveness and potential toxicity.
Article
Sirtuin 6 (SIRT6) is an NAD+-dependent histone deacetylase enzyme that is involved in multiple molecular pathways related to aging. Initially, it was reported that SIRT6 selectively deacetylated H3K9Ac and H3K56Ac; however, it has more recently been shown to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups in vitro. Subsequently, fatty acids were demonstrated to increase the catalytic activity of SIRT6. In this study, we investigated whether a series of N-acylethanolamines (NAEs), quercetin, and luteolin could regulate SIRT6 activity. NAEs increased SIRT6 activity, with oleoylethanolamide having the strongest activity (EC50 value of 3.1 μM). Quercetin and luteolin were demonstrated to have dual functionality with respect to SIRT6 activity; namely, they inhibited SIRT6 activity with IC50 values of 24 and 2 μM, respectively, and stimulated SIRT6 activity more than sixfold (EC50 values of 990 and 270 μM, respectively).