A potent free fatty acid receptor 1 agonist with a glucose-dependent antihyperglycemic effect

Article abstract of DOI:10.1039/c9cc04040d

We present a novel free fatty acid receptor 1 (FFA1) agonist, named PAFA, with a strong agonist potency (EC₅₀ = 6.04 nM) and good serum stability. PAFA significantly stimulates insulin secretion in INS-1 cells in a glucose-dependent manner and exhibits high antihyperglycemic effects in db/db mice without adverse effects.

Full text of DOI:10.1039/c9cc04040d

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Conformational control of nonplanar free base porphyrins:
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DOI: 10.1039/C9CC04040D
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A potent free fatty acid receptor 1 agonist with a glucose-
dependent antihyperglycemic effect †
Xuekun Wang,‡a,b Yurui Xu,‡a Shujun Feng,a Xinyu Huang,a Xia Meng,a Jiao Chen,c Leilei Guo,a
Junliang Ge,a Jikang Zhang,a Jianmei Chen,a Li Cheng,a Kai Gu,a,d Yu Zhang,*a Qing Jiang*e and
Xinghai Ning*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We present a novel free fatty acid receptor 1 (FFA1) agonist, named activities of phospholipase C (PLC), which can promote inositol
PAFA, with a strong agonist potency (EC50=6.04 nM) and good 1,4,5-triphosphate (IP3)-mediated intracellular calcium influx
serum stability. PAFA significantly stimulates insulin secretion in and the activation of protein kinase C (PKC), leading to insulin
11
INS-1 cells in a glucose-dependent manner, and exhibits high secretion from pancreatic β-cells.10,
In addition, FFA1
antihyperglycemic effects in db/db mice without adverse effects.
mediates glucose-stimulated insulin secretion (GSIS),8 and
regulates the secretion of glucose-dependent insulinotropic
peptide (GIP) and glucagon-like peptide-1 (GLP-1).12 Therefore,
Type 2 diabetes mellitus (T2DM) is a metabolic disease,
associated with insulin resistance induced hyperglycemia.
T2DM has a serious impact on people's lives, and causes a
number of disabling and life-threatening health problems,
FFA1 represents
a
potential target for safe insulin
secretagogues with a limited risk of hypoglycemia.
In the last two decades, several FFA1 agonists have been
developed, which stimulated insulin secretion and exhibited
antidiabetic potential. Among all the agonists available,
phenylpropanoic acid was known as the first FFA1 agonist, and
a range of derivatives had been developed accordingly (Fig. S1A,
ESI†).13, 14 However, phenylpropanoic acid derivatives exhibited
poor pharmacokinetic properties with low oral bioavailability,
due to susceptibility to β-oxidation.15 For example, compound
2, a biaryl analogue, could be rapidly oxidized and decomposed
by β-oxidase, leading to ablation of the agonist activity.16 Many
strategies have been exploited to improve metabolic stability of
phenylpropanoic acid derivatives. Particularly, the β-position of
the carboxylic acid was modified to generate a fused-ring (TAK-
875 and AM-5262), which can greatly improve biostability.17
However, the β-position modification commonly increases the
steric hindrance, resulting in reduced agonist potency or
increased cytotoxicity.16, 18, 19 For instance, TAK-875, the most
advanced FFA1 agonist in clinical trials, has good
pharmacokinetic profiles, but it was terminated in the phase III
clinical trial due to unexpected hepatotoxicity.20, 21 Thus, there
is a great interest to develop new FFA1 agonists with low
leading to a huge burden to global healthcare budgets.1,
2
Although a broad range of oral antidiabetic agents have been
launched in the clinic, such as sulfonylurea, metformin, α-
glucosidase inhibitors, thiazolidinediketone derivatives, DPP-4
4
enzyme inhibitors, etc.3, Their clinical impacts have been
limited due to poor pharmacokinetics, unsatisfactory glycemic
control, serious adverse effects, or particularly frequent
emergence of drug resistance.5, 6 Therefore, novel antidiabetic
drugs with good oral bioavailability, high efficacy and sustained
safety are greatly desirable.
Free fatty acid receptor 1 (FFA1, also known as GPR40) is
highly expressed in pancreatic β-cells, and can be activated by
free fatty acids (FFAs), which are an important signaling
molecules in numerous physiological processes.7 FFA1 is mainly
coupled with the G protein α-subunit of the Gq family (Gαq).8, 9
The activation of Gαq protein-coupled receptors increases
aDepartment of Biomedical Engineering, Nanjing National Laboratory of
Microstructures, College of Engineering and Applied Sciences, Nanjing University,
Nanjing 210093, P.R. China.
bDepartment of Pharmaceutical Engineering, College of pharmacy; Liaocheng
University, Liaocheng 252059, P.R. China.
cytotoxicity
and
improved
pharmacokinetic
and
pharmacodynamic profiles.14
cJiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, P.R.
China.
In this study, we utilized the pharmacophore based drug
design approach to develop a novel FFA1 agonist, named
phenylthioacetic acid-based FFA1 agonist (PAFA). PAFA is
c o m p o s e d o f b i p h e n y l s u l p h o n a t e f r a g m e n t a n d
phenylthioacetic acid, which are responsive for the improved
dColleges of Medicine, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P.R. China.
eThe Center of Diagnosis and Treatment for Joint Disease, Drum Tower Hospital
Affiliated with the Medical School of Nanjing University, Nanjing 210008, P.R.
China.
†Electronic Supplementary Information (ESI) available: Experimental section and
supplymentary figures. See DOI: 10.1039/x0xx00000x
‡ These authors contributed equally to this work.
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hydroxides, 27 affording PAFA with a quantitative yield. Agonist
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activity of PAFA was then evaluated by tDhOeI:F1L0I.P10R3c9a/Clc9iCuCm04a0s4s0aDy
in CHO cells stably expressing FFA1. TAK-875 was used as the
positive control. As shown in Fig. 2A and Fig. S6 (ESI†), PAFA
showed a strong agonist activity towards FFA1 with EC50 of 6.04
nM, which is 3-fold higher than TAK-875, indicating that PAFA is
a potent FFA1 agonist.
To investigate the insulinotropic effects of PAFA, glucose-
stimulated insulin secretion assay (GSIS) was performed in INS-
1 cells in the presence of 11 mM glucose. Fig. 2B shows that
PAFA stimulated an increase in GSIS in a concentration
dependent manner, and was able to significantly improve
insulin secretion at doses of more than 1.0 μM. Particularly,
PAFA could only promote insulin secretion at high glucose
concentration (11 mM), indicating that PAFA has a glucose-
dependent mechanism (Fig. 2C). In addition, PAFA rapidly
prompted insulin secretion, and an increase in insulin levels was
observed with increasing incubation period, demonstrating that
Fig. 1 (A) Pharmacophore analyses of FFA1 agonists and the design of PAFA using
the pharmacophore based drug design approach. (B) The binding model between
PAFA and FAA1. The phenylthioacetic acid moiety is buried within hydrophilic
region, and has strong ionic/polar interactions with Arg 1835.39, Arg 2587.35, Tyr
913.37and Tyr 2406.51. The biphenyl sulphonate moiety is exposed to exterior
hydrophobic region. (C) The docking simulation studies of the PAFA-FAA1
complex. The carboxylate moiety of PAFA is highly coordinated with several key
residues on the charge cluster of FAA1.
the pharmacokinetics and pharmacodynamics, respectively.
PAFA is an orally bioavailable agonist, and can effectively
stimulate glucose dependent insulin secretion. In addition,
PAFA exhibits good oral bioavailability, desirable
biocompatibility, favorable antihyperglycemic activity and low
risk of hypoglycemia, indicating that it is
antidiabetic drug candidate for treating T2DM.
a promising
Molecular modeling studies were performed to reveal the
binding mode between TAK-875 and hFFA1. Fig. S1B(ESI†)
shows that the phenyl carboxylate moiety of TAK-875 binds to
the surface non-canonical site,22 and forms hydrogen bonds
with Arg 1835.39, Arg 2587.35, Tyr 913.37 and Tyr 2406.51, indicating
that it plays key role in pharmacodynamics. In addition,
biphenyl sulphonate moiety only serves to improve the
properties of absorption, distribution, metabolism and
excretion (ADME),23 suggesting its pharmacokinetic function.
However, TAK-875 has relatively weaker binding affinity (126%,
total binding scores), compared to compound 2 (140%, total
binding scores) with a liability of β-oxidation, indicating that the
carboxylate moiety increases metabolic stability but decreases
agonist potency (Fig. S1C, ESI†). Therefore, we chose the
metabolically stable phenylthioacetic acid moiety and biphenyl
sulphonate fragment of TAK-875 to construct the new FFA1
agonist, which are responsive for improved agonist activities
and pharmacokinetic properties, respectively (Fig. 1A). The
Docking simulation studies verified that PAFA is a potent FAA1
agonist, and has the strong interaction with hFFA1 (143%, total
binding scores, Fig. 1B and 1C, Table. S1, ESI†), which is identical
with compound 2.
Fig. 2 (A) EC50 values of FFA1 agonists, which represent the mean of six
determinations. (B) PAFA stimulated insulin secretion. INS-1 cells were incubated
with 11mM glucose in the presence of different concentrations of PAFA and TAK-
875 for 2 h, and the levels of insulin were measured. (C) The glucose dependent
insulin secretion. INS-1 cells were incubated with different concentrations of
glucose in the presence of 3 μM PAFA, TAK-875 or glibenclamide for 2 h, and the
levels of insulin were measured. (D) Time dependent effects of PAFA on insulin
secretion. INS-1 cells were incubated with 11mM glucose in the presence of either
3 μM PAFA or TAK-875, and the levels of insulin were measured. Data are
expressed as the mean fold-increase over the control group ± SD (n = 6). P values
were determined by Student's t-test (**p <0.01, *** p <0.001).
PAFA regulates insulin secretion in a time-dependent manner
(Fig. 2D). Furthermore, PAFA displayed a more potency for
stimulating insulin secretion than TAK-875, generating a two-
fold higher insulin levels. More importantly, PAFA-triggered
insulin secretion could be inhibited by FFA1 antagonist
DC260126, indicating that PAFA is a specific FFA1 agonist (Fig.
S7, ESI†). Therefore, PAFA is a promising anti-diabetic drug
candidate carrying a low risk of hypoglycemia.
Biostability of FFA1 agonists is a key factor determining
their application scopes. PAFA showed good serum stability,
and no decomposition was observed after 24h incubation,
indicating that sulfur substitution can prevent β-oxidation with
minimal effects on its agonist activity (Fig. S8, ESI†). We
further investigated the antihyperglycemic effects of PAFA in
ICR mice. Overnight fasted ICR mice were orally administered
with the mixture of either different dose of PAFA or TAK-875
(20 mg/kg) with methylcellulose, and 30 min later, mice
PAFA was synthesized as outlined in Fig. S2 (ESI†). Anilines 6
was firstly synthesized by reducing the nitro group of compound
5 with the mixture of iron and ammonium chloride,24 followed
by converting amine to latent thiol using the method reported
by Hori et al.25, 26 The thiol group of compound 7 was then
reacted with methyl chloroacetate to generate the
intermediate 8. Biphenyl fragment 925, 26 was coupled directly
to intermediate 8, followed by hydrolyzing with aqueous
2 | J. Name., 2012, 00, 1-3
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received an oral administration of glucose (2 g/kg).28 The time- glucose excursion in db/db mice, and its antihVyipeweArgrtilcyleceOmnlinice
DOI: 10.1039/C9CC04040D
dependent changes in blood glucose levels were measured effects were successfully persisted for
4 weeks. Most
importantly, long-term exposure did not impair antidiabetic
efficacy of PAFA, and indicating low risk of pancreatic β-cell
exhaustion. Moreover, the PAFA-treated mice had similar body
weight as the control group without reducing pancreatic insulin
contents, suggesting that PAFA is an effective antidiabetic drug
candidate without adverse effects.
Patients with type
2 diabetes are susceptible to
hypoglycaemia, which should be considered for antidiabetic
treatments. The preliminary data indicate that FFA1 agonists
may be associated with a low risk of hypoglycaemia.8, 12 We
therefore investigated the risk of PAFA to induce hypoglycemia.
As shown in Fig. 4B, orally administered PAFA did not cause low
glucose levels in the fasted mice even at the concentration of
60 mg/kg, and maintained glucose levels in a safe range. In
contrast, glibenclamide, a classical antidiabetic drug, induced
serious hypoglycemia in the fasted mice, leading to a high risk
of morbidity and mortality. These results confirm that PAFA
stimulates plasma insulin secretion with a glucose-dependent
manner, and is not associated with unexpected hypoglycemia.
Fig. 3 (A) Dose-dependent effects of PAFA on blood glucose. ICR mice were treated
with PAFA or TAK-875, and the blood glucose levels were measured. (B) Time-
dependent insulin levels in ICR mice treated with either TAK-875 or PAFA. (C) The
change in blood glucose levels during an OGTT in fasted db/db mice, treated with
PAFA or TAK-875. (D) The change in insulin secretion during an OGTT in fasted
db/db mice, treated with PAFA or TAK-875. Values are mean ± SD (n = 6).
and compared. Fig. 3A shows that PAFA could rapidly reduce
blood glucose levels, and exhibited stronger hypoglycemic
a
c
t
i
v
i
t
y
t
h
a
n
T
A
K
-
875. In addition, the changes in area under the curve (AUC) of
glucose from 0 to 120 min (AUC0–120 min) were calculated and
compared. Fig. S9 (ESI†) shows that PAFA significantly
diminished AUC0–120
of glucose after a single oral
min
administration. For example, PAFA induced a significant
reduction in AUC (34.9%), which is 1.5-fold higher than TAK-875
(23.0%), indicating that PAFA is a potent FFA1 agonist.
Importantly, PAFA had antihyperglycemic effects in a dose-
dependent manner, and could effectively decrease blood
glucose levels with high dose of PAFA. In addition, a single oral
administration of PAFA significantly enhanced plasma insulin
secretion 15 min after glucose challenge (Fig. 3B), suggesting
that PAFA can rapidly regulate blood glucose levels.
Although there are currently no FFA1 agonists approved for
clinical use, Phase II clinical trials of TAK-875 demonstrated that
TAK-875 could reduce blood glucose levels, increase insulin
levels, and result in a 1.2% reduction in hemoglobin A1C
levels.29 Therefore, the antihyperglycemic and insulinotropic
effects of PAFA and TAK-875 in db/db mice were systemically
measured and compared. A single oral administration of PAFA
significantly reduced glucose excursion during oral glucose
tolerance tests (OGTT), and showed better antihyperglycemic
effects than TAK-875 (Fig. 3C and Fig. S10, ESI†). In addition,
PAFA was found to be more potent at stimulating insulin
secretion, compared to TAK-875 (Fig. 3D), indicating high
antidiabetic activity of PAFA.
Fig. 4 (A) The long-term treatment of PAFA and TAK-875 in db/db mice. A 4-week
treatment of PAFA on fasting blood glucose levels in db/db mice. (B) The risk of
PAFA induced hypoglycaemia. (C) Hepatotoxicity assessment of PAFA. Cytotoxicity
of PAFA and TAK-875 against L02 cells. (D) The H&E stained histopathological
images (×200) of liver tissues. Values are mean ± SD (n = 6). P values were
determined by Student's t-test (*p <0.05, **p <0.01, *** p <0.001).
Hepatotoxicity is one of key problems leading to clinical
failure of FFA1 agonists. For example, TAK-875, the most
clinically advanced drug candidate, was terminated in phase Ⅲ
clinical trials due to serious hepatotoxicity.21 Therefore, MTT
assay was used to evaluate the cytotoxicity of PAFA against
human liver L02 cells. Fig. 4C shows that TAK-875 exhibited
obvious cytotoxicity at 100 μM, whereas, PAFA did not affect
cell viability even at a high concentration of 200 μM. In addition,
the risk of chronic hepatotoxicity induced by PAFA was
subsequently investigated in db/db mice. The mice were orally
administered PBS, TAK-875 (20 mg/kg), or PAFA (20 mg/kg)
daily for 4 weeks, and toxicological parameters, including body
weight, food consumption, biochemical parameters and
histopathology, were monitored. There were no significant
differences on the body weight and food consumption between
the PAFA administrated group and control group (Fig. S11A,
Furthermore,
we
also
investigated
long-term
antihyperglycemic effects of PAFA and TAK-875 in db/db mice
for 4 weeks. An OGTT was performed during the treatment. Fig.
4A shows that, in comparison of TAK-875, PAFA could
effectively maintain normal glucose homeostasis during 4-week
treatment. Particularly, PAFA could effectively reduce the blood
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J. Name., 2013, 00, 1-3 | 3
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Komatsu, F. Matsumura, Y. Noguchi, T. Shinohara, S.
ESI†). In addition, Fig. S11B (ESI†) shows that PAFA did not
elevate plasma levels of enzymes associated with
hepatotoxicity, such as alanine aminotransferase (ALT),
aspartate transaminase (AST) and total bilirubin (TBIL).
Moreover, histological studies show that PAFA did not induce
liver injury, and only mild fatty degeneration of hepatocytes,
hepatic fibrosis and inflammation were found in the portal area
(Fig. 4D). All these data indicate good biocompatibility of PAFA.
In conclusion, we utilize the pharmacophore based drug
design approach to develop a novel phenylthioacetic acid-based
FFA1 agonist (PAFA), possessing high antihyperglycemic activity
and oral bioavailability. We identify that PAFA has a stronger
agonist potency (EC50=6.04 nM) than TAK-875 (EC50=19.26 nM),
the most advanced FFA1 agonist in clinical trials. In addition,
PAFA significantly stimulates insulin secretion in INS-1 cells in a
glucose-dependent manner. More importantly, oral
administration of PAFA exhibits high antihyperglycemic effects
in db/db mice during treatment without adverse effects.
Therefore, PAFA is a promising antidiabetic drug candidate for
treating type 2 diabetes mellitus, and has a high potential for
clinical applications.
X.N. and Q.J. designed and developed the research. X.W.,
L.C., K.G., and J.G. performed the synthesis. X.H., X.M., J.C., L.G.,
J.Z., and J.C. contributed to evaluate the profiles. Y.X., S.F., and
Y.Z. wrote the manuscript. The works were supported by the
National Key Research and Development Program of China
(Grant No. 2018YFB1105400), the National Natural Science
Foundation of China (Grant No. 21708019), the Thousand
Talents Program for Young Researchers, the Natural Science
Foundation of Jiangsu Province (Grant No. BK20180334), the
Shuangchuang Program of Jiangsu Province, the Fundamental
Research Funds for Central Universities Nanjing University and
the Scientific Research Foundation of Graduate School of
Nanjing University (Grant No. 2017ZDL04).
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Conflicts of interest
There are no conflicts to declare.
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