Novel SIRT1 activator MHY2233 improves glucose tolerance and reduces hepatic lipid accumulation in db/db mice

Article abstract of DOI:10.1016/j.bmcl.2018.01.021

The NAD⁺-dependent deacetylase SIRT1, which is associated with the improvement of metabolic syndromes, such as type 2 diabetes, is a well-known longevity-related gene. Several in vitro and in vivo studies have shown the known protective effects of SIRT1 activators, such as resveratrol and SRT1720, on diabetes- or obesity-induced fatty liver and insulin resistance. Here, we newly synthesized 18 benzoxazole hydrochloride derivatives based on the structure of resveratrol and SRT1720. We performed an in vitro SIRT1 activity assay to identify the strongest SIRT1 activator. The assay confirmed MHY2233 to be the strongest SIRT1 activator (1.5-fold more potent than resveratrol), and docking simulation showed that the binding affinity of MHY2233 was higher than that of resveratrol and SRT1720. To investigate its beneficial effects, db/db mice were orally administered MHY2233 for 1 month, and various metabolic parameters were assessed in the serum and liver tissues. MHY2233 markedly ameliorated insulin signaling without affecting body weight in db/db mice. In particular, the mRNA expression of lipogenic genes, such as acetyl CoA carboxylase, fatty acid synthase, and sterol regulatory element-binding protein, which increased in db/db mice, decreased following oral treatment with MHY2233. In conclusion, the novel SIRT1 activator MHY2233 reduced lipid accumulation and improved insulin resistance. This finding may contribute toward therapeutic approaches for fatty liver disease and glucose tolerance.

Full text of DOI:10.1016/j.bmcl.2018.01.021

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel SIRT1 activator MHY2233 improves glucose tolerance and reduces
hepatic lipid accumulation in db/db mice
a
a
a
a,b
a
a
a
Min Jo Kim , Hye Jin An , Dae Hyun Kim , Bonggi Lee , Hye Jin Lee , Sultan Ullah , Su Jeong Kim ,
a
a,b
a
a
a
c
Hyoung Oh Jeong , Kyoung Mi Moon , Eun Kyeong Lee , Jungho Yang , Jinia Akter , Pusoon Chun ,
Hyung Ryong Moon , Hae Young Chung
Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Daegu 41062, Republic of Korea
College of Pharmacy, Inje University, Gyeongsangnam-do 50834, Republic of Korea
a,
⇑
a,
⇑
a
b
c
a r t i c l e i n f o
a b s t r a c t
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Article history:
The NAD -dependent deacetylase SIRT1, which is associated with the improvement of metabolic syn-
Received 13 October 2017
Revised 4 January 2018
Accepted 12 January 2018
Available online xxxx
dromes, such as type 2 diabetes, is a well-known longevity-related gene. Several in vitro and in vivo stud-
ies have shown the known protective effects of SIRT1 activators, such as resveratrol and SRT1720, on
diabetes- or obesity-induced fatty liver and insulin resistance. Here, we newly synthesized 18 benzoxa-
zole hydrochloride derivatives based on the structure of resveratrol and SRT1720. We performed an
in vitro SIRT1 activity assay to identify the strongest SIRT1 activator. The assay confirmed MHY2233 to
be the strongest SIRT1 activator (1.5-fold more potent than resveratrol), and docking simulation showed
that the binding affinity of MHY2233 was higher than that of resveratrol and SRT1720. To investigate its
beneficial effects, db/db mice were orally administered MHY2233 for 1 month, and various metabolic
parameters were assessed in the serum and liver tissues. MHY2233 markedly ameliorated insulin signal-
ing without affecting body weight in db/db mice. In particular, the mRNA expression of lipogenic genes,
such as acetyl CoA carboxylase, fatty acid synthase, and sterol regulatory element-binding protein, which
increased in db/db mice, decreased following oral treatment with MHY2233.
Keywords:
SIRT1
MHY2233
Lipogenesis
Glucose tolerance
In conclusion, the novel SIRT1 activator MHY2233 reduced lipid accumulation and improved insulin
resistance. This finding may contribute toward therapeutic approaches for fatty liver disease and glucose
tolerance.
Ó 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
The NAD -dependent deacetylase (SIRT1) is a NAD+-dependent
lin secretion, lipid mobilization, and fatty acid oxidation in various
organs.
8
–10
histone deacetylase, and its activity is closely related to the bene-
ficial effect of calorie restriction (CR).1 Moreover, recent evidence
indicates that SIRT1 regulates glucose–lipid metabolism through
its deacetylase activity toward many known substrates such as
peroxisome proliferator-activated receptor gamma coactivator 1-
alpha (PGC-1
,2
Insulin resistance is a pathological condition in which cells fail
to respond to normal signaling, and it is commonly associated with
metabolic syndromes, such as type 2 diabetes, hypertension, and
fatty liver disease. Furthermore, it is an important characteristic
of metabolic syndrome, and it has been shown that to obtain the
adequate biological effects of insulin, more insulin is required in
such conditions. A defect in insulin signaling can cause insulin
3
a
) and liver kinase B1 (LKB1). SIRT1 activators, such
as resveratrol and SRT1720, and SIRT1 overexpression have benefi-
cial effects on glucose and lipid homeostasis in diabetic condition.
4
1
1
SIRT 1 improved insulin sensitivity, especially under insulin-resis-
tant conditions by repressing PTP1B, a major negative regulator of
insulin action and protected diet-induced-obesity and insulin
resistance. Under insulin-resistant conditions, the supply of free
fatty acid from other tissues increases because of the impaired sup-
pression of lipid oxidation, which leads to hepatic lipid accumula-
5
–7
12,13
resistance.
Furthermore, SIRT1 is involved in the control of glu-
tion.
Therefore, SIRT1 may be a new therapeutic target for the
cose homeostasis through the regulation of gluconeogenesis, insu-
prevention of metabolic diseases such as insulin resistance and
subsequent type 2 diabetes.
Sterol regulatory element-binding proteins (SREBPs) regulate
lipid synthesis as transcription factors. SREBP-1c, a lipogenic
⇑
Corresponding authors.
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr (H.Y.
1
4
Chung).
activator, is an important in vivo target of SIRT1, and SIRT1
https://doi.org/10.1016/j.bmcl.2018.01.021
0
960-894X/Ó 2018 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Kim M.J., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.021

2
M.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
deacetylates and inhibits SREBP-1c activity by decreasing its stabil-
in vitro studies, SRT1720 was shown to increase insulin sensitivity
1
5
ity and occupancy at the lipogenic target gene promoters. Fur-
thermore, SIRT1 improves insulin resistance and hepatic steatosis
through the inhibition of ER stress and mTORC1, and acts as a neg-
and ameliorate mitochondrial and oxidative metabolism in an
1
7,18
obese model.
Furthermore, SRT1720 treatment of MSG mice
with obesity and insulin resistance reduced liver lipid accumula-
1
6
19
ative regulator of UPR signaling in type 2 diabetes models. The
SIRT1 activator SRT1720 is being studied for the treatment of
metabolic and chronic diseases related to aging. In previous
tion and lipogenic gene expression. Thus, we investigated the
effects of oral treatment with MHY2233, a novel SIRT1 activator,
on lipid synthesis and insulin signaling in db/db mice.
in vitro SIRT1 activity assay
2
2
1
1
50
00
50
00
*
**
###
2
.0
.5
.0
.5
.0
#
1
1
5
0
**
0
0
n
Co
s
Re
3
3
22
0
T
3
W
/db
db
23
2
Name
233
Autodock
-14.77
Dock
-206.61
-36.67
2
Resveratrol
SRT1720
-8.07
-13.28
-239.57
Fig. 1. MHY2233 is a potent SIRT1 activator. (A) Measurement of SIRT1 activity in vitro. (B) Effect of MHY2233 on p53 deacetylation. p53 deacetylation was quantified by
western blotting. In the western blot analysis, MHY2233 decreased the nuclear fraction protein levels of acetyl p53. TF IIB was used as nuclear internal control. One
representative blot of each protein is shown from 3 independent experimental groups that yielded similar results. (C) Docking simulation was performed to identify
interaction between MHY2233 and the ligand binding affinity. (D) Structure of MHY2233. MHY2233 has strong binding affinity compared with the known SIRT1 activators
resveratrol and SRT1720.
Body weight
Food intake
6
0
30
20
10
0
WT
db/db
MHY2233
WT
db/db
MHY2233
40
20
0
0
5
0
5
0
0
2
1
0
15
20
25
30
1
15
25
30
days
days
TG (serum)
##
TC (serum)
NEFA (nonesterified fatty acids)
1500
#
1
500
5
00
00
00
00
00
#
#
##
4
1000
*
1000
*
3
2
***
5
00
500
0
1
0
0
b
3
/db
db
2
3
3
WT
/
d
db
3
/db
db
3
3
WT
2
22
WT
2
2
Fig. 2. Effect of MHY2233 on metabolic parameters. (A) Body weight (B) Food intake (C) Triglyceride (D) Total cholesterol (E) NEFA concentration in serum was quantified
using the colorimetric assay kit, respectively after administrating MHY2233 to db/db mice. Data represents the mean ± S.E.M. One-way analysis of variance (ANOVA) was
used to determine the statistical result: ##p < 0.01, and
###
p < 0.001 versus the WT and the db/db mice, p < 0.05, p < 0.001 versus the db/db and MHY2233-treated db/db
*
***
mice. 2233, MHY2233 5 mg/kg/day; WT, misty mice; db/db, non-treated db/db mice.
Please cite this article in press as: Kim M.J., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.021

M.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
To screen SIRT1 activators, we newly synthesized 18 benzoxa-
zole hydrochloride derivatives based on the structure of the well-
known SIRT1 activators resveratrol and SRT1720. The in vitro SIRT1
activity assays were performed to identify the strongest SIRT1 acti-
vator. MHY2233 increased SIRT1 activity, and the effect was higher
ties in obese and diabetic subjects. In db/db mice, MHY2233 signif-
icantly reduced serum TG, TC, and NEFA levels (Fig. 2C and E).
These data suggest that MHY2233 ameliorates serum TG, TC, and
NEFA levels in diabetes.
To investigate the beneficial effects of MHY2233, we performed
the glucose tolerance test (GTT). GTT revealed that MHY2233
improved obesity-induced glucose intolerance after 3 weeks of
treatment (Fig. 3A). We also evaluated fasting glucose and insulin
levels. As expected, fasting glucose and insulin levels increased in
db/db mice, which confirmed the development of insulin resis-
tance in db/db mice and amelioration by MHY2233 treatment
(Fig. 3B and C). We also investigated the insulin signaling pathway
in the liver tissue of db/db mice. The administration of MHY2233
restored the insulin signaling pathway, including tyrosine phos-
phorylation of IRS-1 and serine phosphorylation of Akt (Fig. 3D
and E). These results suggest that MHY2233 improves insulin resis-
tance in db/db mice.
To determine whether MHY2233 ameliorates lipid accumula-
tion via observed Histological analysis in db/db mice liver. Lipid
droplets accumulated in db/db mice liver, but that MHY2233
reduced this accumulation (Fig. 4A). To confirm this effect, hepatic
triglyceride levels were measured in db/db mice. MHY2233
improved hepatic triglycerides concentration (Fig. 4B). These
than that of resveratrol (Fig. 1A). Studies have suggested that SIRT1
associates with and deacetylates p53.2
0,21
Western blotting
revealed that MHY2233 suppressed acetylation of p53 in db/db
mice (Fig. 1B). The predicted binding affinity of resveratrol to SIRT1
was À8.07 kcal/mol in AutoDock 4.2 program and À36.07 kcal/mol
in Dock6 program, whereas the binding affinity of MHY2233 to
SIRT1 was À14.77 kcal/mol in AutoDock 4.2 program and
À206.61 kcal in Dock6 program (Fig. 1C). These results suggest that
the binding affinity of MHY2233 to SIRT1 may be higher than that
of the positive control, resveratrol, and SRT1720.
To investigate the beneficial effects of MHY2233 in vivo,
MHY2233 was orally administered at a concentration of 5 mg/kg/-
day for 30 days in db/db mice. Body weight and food intake were
not change between untreated and MHY2233 treated db/db mice
(
Fig. 2A and B). To investigate the physiological effects of
MHY2233 in db/db mice, serum triglyceride (TG), total cholesterol
TC), and non-esterified fatty acids (NEFA) profiles were analyzed
to determine whether MHY2233 attenuates metabolic abnormali-
(
Fig. 3. MHY2233 improves insulin resistance in db/db mice. (A) Glucose concentrations were determined using an intraperitoneal glucose tolerance test (GTT) by
#
#
###
administration of 2 g/kg glucose after overnight fasting. (B) Fasting serum glucose and (C) insulin levels. The data are presented as mean ± SEM (n = 5). p < 0.01,
p < 0.001
*
**
vs. WT; p < 0.05, p < 0.01 vs. db/db. (D and E) Expression of genes involved in insulin signaling was analyzed by western blotting using liver samples of db/db mice. b-actin
was used as cytosolic internal control. In the western blot analysis, the protein levels of insulin resistance-related genes were decreased by MHY2233. One representative blot
of each protein is shown from 3 independent experimental groups that yielded similar results. 2233, MHY2233 5 mg/kg/day; WT, misty mice; non-treated db/db mice; Akt,
Protein kinase B; IRS, Insulin receptor substrate.
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4
M.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Hepatic Triglyceide
300
#
##
*
200
100
0
3
3
WT
/db
b
22
d
Fig. 4. MHY2233 improved hepatic steatosis in db/db mice liver. (A) A histological examination based on hematoxylin-eosin staining showed lipid accumulation in the livers.
B) Hepatic triglycerides were extracted and measured. The data are shown as the mean ± SEM (n = 5). ###p < 0.001 versus the WT and the db/db mice, p < 0.05 versus the db/
*
(
db and MHY2233-treated db/db mice. 2233, MHY2233 5 mg/kg/day; WT, misty mice; db/db, non-treated db/db mice.
related genes, including SREBP isoforms (1c, 1a), FASN, and ACC.
The expression of these genes was found to be higher in the db/
db mice groups than that of the control group, whereas the expres-
sion decreased in MHY2233-treated db/db mice (Fig. 5A). Further-
more, we investigated the mRNA expression of genes involved in b-
oxidation, including carnitine palmitoyltransferase-1 (CPT-1) and
peroxisomal acyl-coenzyme A oxidase 1 (ACOX-1) the expression
did not change in db/db mice (Fig. 5B). Therefore, MHY2233 regu-
lated lipogenic genes but not b-oxidation genes in the obese model.
Previously study, SIRT activators and SIRT1 overexpression have
various beneficial effects on glucose and lipid homeostasis in dia-
2
3
betes. Furthermore, SIRT1 is generally localized to the nucleus
and deacetylates p53, FOXOs, histone, and non-histone proteins.24
Our study indicated that MHY2233 decreased acetyl p53, and the
binding affinity of MHY2233 was higher than that of resveratrol,
SRT1720, SIRT1 activator (Fig. 1). Also, MHY2233 ameliorated glu-
cose intolerance and insulin signaling pathway in the obese model
(
Fig. 3). Moreover, MHY2233 decreased lipid synthesis-related
gene expression but did not change b-oxidation gene expression
Fig. 5). In summary, SIRT1 activators have various beneficial
(
effects that mitigate insulin resistance. MHY2233 improves insulin
resistance by decreasing fatty acid synthase gene expression and
improving insulin signaling in diabetes. In conclusion, MHY2233
is a potential pharmaceutical agent that can ameliorate metabolic
syndrome associated with diabetes.
Fig. 5. Effect of MHY 2233 on lipid metabolism genes in db/db mice liver. mRNA
levels of (A) SREBP-1c, SREBP-1a, FASN, and ACC (B) CPT1 and ACOX1 in the liver
tissue were analyzed by qRT–PCR. Results were normalized to the expression of a
Acknowledgments
#
#
reference gene GAPDH. The data are shown as the mean ± SEM (n = 5). p < 0.01,
and # p < 0.001 versus the WT and the db/db mice, p < 0.05, p < 0.01, p < 0.001
##
*
**
***
This research was supported by the National Research Founda-
tion of Korea (NRF) funded by the Korea government (MSIP) (2009-
0083538, NRF-2015R1A2A201004137, NRF-2013M3A9B6076413).
We thank Aging Tissue Bank for providing aged tissue.
versus the db/db and MHY2233-treated db/db mice. 2233, MHY2233 5 mg/kg/day;
WT, misty mice; db/db, non-treated db/db mice.
results indicated that MHY2233 alleviated hepatic lipid
accumulation.
A. Supplementary data
The transcription factor SREBP plays a major role in the effect of
insulin on the transcription of hepatic lipogenic genes such as fatty
acid synthase. We evaluated the expression of lipid synthesis-
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.01.021.
2
2
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M.J. Kim et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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