Discovery of a Benzamide Derivative That Protects Pancreatic β-Cells against Endoplasmic Reticulum Stress

Article abstract of DOI:10.1021/acs.jmedchem.7b00435

Endoplasmic reticulum (ER) stress-mediated pancreatic insulin-producing β-cell dysfunction and death are critical elements in the onset and progression of both type 1 and type 2 diabetes. Here, through cell-based high throughput screening we identified be

Full text of DOI:10.1021/acs.jmedchem.7b00435

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Article
Discovery of a benzamide derivative that protects
pancreatic #-cells against endoplasmic reticulum stress
Hongliang Duan, Yu Li, Daleep K Arora, Depeng Xu, Hui-Ying Lim, and Weidong Wang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Jun 2017
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Discovery of a benzamide derivative that protects pancreatic βꢀcells against endoplasmic
reticulum stress
Hongliang Duan,† Yu Li£, Daleep Arora£, Depeng Xu, † HuiꢀYing Lim#, and Weidong Wang†£*
£Department of Medicine, Division of Endocrinology, The University of Oklahoma Health Science Center, 941
Stanton L. Young Blvd., Oklahoma City, Oklahoma 73104, United States
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†Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma
City, Oklahoma 73104, United States
#Department of Physiology, The University of Oklahoma Health Science Center, 941 Stanton L. Young Blvd.,
Oklahoma City, Oklahoma 73104, United States
ABSTRACT
Endoplasmic reticulum (ER) stressꢀmediated pancreatic insulinꢀproducing βꢀcell dysfunction and death
are critical elements in the onset and progression of both type 1 and type 2 diabetes. Here, through
cellꢀbased high throughput screening we identified benzamide derivatives as a novel class of βꢀcell
protective agents against ER stressꢀinduced dysfunction and death. Through structureꢀactivity
relationship optimization, a 3ꢀ(Nꢀpiperidinyl)methyl benzamide derivative 13d markedly protects
βꢀcells against ER stressꢀinduced dysfunction and death with near 100% maximum rescue activity and
an EC50 value of 0.032 ꢀM. Compound 13d alleviates ER stress in βꢀcells by suppressing ER
stressꢀmediated activation of all three branches of unfolded protein response (UPR) and apoptotic
genes. Finally, we show that 13d significantly lowers blood glucose levels and increases concomitant
βꢀcell survival and number in a streptozotocinꢀinduced diabetic mouse model. Identification of
βꢀcellꢀprotective small molecules against ER stress provides a new promising modality for the
treatment of diabetes.
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Introduction
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Diabetes, a group of metabolic diseases in which blood sugar levels are abnormally high over a
prolonged period, has become a serious public health problem with tremendous social and economic
burden on society; as of 2015, 415 million people were estimated to have diabetes worldwide.1 The
dysfunction and death of insulinꢀproducing pancreatic βꢀcells are critical elements in the pathogenesis
of type 1 (T1D) and type 2 (T2D) diabetes.2, 3 Increasing evidence indicates that endoplasmic
reticulum (ER) stress, a condition in which misfolded proteins accumulate in the ER, plays an
important role in the decline in pancreatic βꢀcell function and mass in diabetes.4, 5 Thus, prevention of
functional βꢀcell death by targeting ER stress is a promising therapeutic approach for patients with
diabetes. Unfortunately, no existing antiꢀdiabetic drugs are capable of halting the progression of βꢀcell
dysfunction and death.
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In T2D, βꢀcells are forced to synthesize more insulin due to higher metabolic demands of obesity and
insulin resistance, which typically exceeds the cellular capacity of the ER for protein folding, and
eventually leads to ER stress and βꢀcell dysfunction and death.3 In addition, the common causes for
βꢀcell dysfunction and death in T2D, including lipotoxicity, glucotoxicity, oxidative stress, amyloid
deposition, and insulin mutations, have been known to be associated with unresolvable chronic ER
stress.4, 6, 7 In T1D in which βꢀcells are destroyed by an autoꢀimmune reaction, ER stress has also been
implicated, and an ER stressꢀreducing chemical chaperone has been reported to prevent the onset of
T1D in mouse models by protecting βꢀcell survival.8ꢀ10
ER stress induces activation of the unfolded protein response (UPR) through three ER membrane
proteins, inositolꢀrequiring protein 1α (IRE1α), PKRꢀlike ER kinase (PERK), and activating
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transcription factor 6 (ATF6), which act as unfolded protein sensors. 4, 6, 11, 12 In unstressed cells, these
sensors are maintained in an inactive state through interaction with the protein chaperone binding
immunoglobulin protein (BiP). Under ER stress, unfolded and misfolded proteins accumulate in the ER
and bind to and sequester BiP, thereby releasing and activating the sensors.13 Upon initial or mild ER
stress, IRE1α, PERK, and ATF6 each activate a series of events aimed at restoring ER homeostasis by
altering the translation, folding, and postꢀtranslational modification of secreted and membrane
proteins.11, 12, 14, 15 However, failure to adequately reꢀestablish ER homeostasis eventually triggers cell
death, as in the case of chronic or severe ER stress.11, 12, 14ꢀ16
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Despite of the importance of ER stress in mediating βꢀcell dysfunction and death in the pathogenesis of
diabetes, only a handful of small molecules have so far been reported to exhibit βꢀcellꢀprotective
activities against ER stress.17, 18 A major reason for the scarcity of βꢀcellꢀprotective small molecules
could lie in the unique property of βꢀcells. βꢀcells normally produce and rapidly secrete insulin in
response to increases in blood glucose levels after food intake. To achieve this, they maintain a very
large pool of proinsulin mRNA (~20% of the total cellular mRNA) and increase proinsulin protein
synthesis 25ꢀfold upon glucose stimulation.19, 20 This surge in proinsulin synthesis places a heavy
burden on the proteinꢀfolding capacity of the ER; βꢀcells are therefore particularly susceptible to ER
stress.6 This βꢀcell property may also in part explain why compounds that protect other cell types from
ER stress fail to protect βꢀcells.21, 22 Furthermore, the existing βꢀcellꢀprotective small molecules17, 18 yet
still suffer from the issue of low potency, often with EC50 values ranging from singleꢀ to doubleꢀdigit
ꢀM.
In this study, we used a βꢀcell survivalꢀbased high throughput screening approach to identify small
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molecules that protect βꢀcells against ER stressꢀinduced apoptosis, and discovered
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Nꢀ(3ꢀchlorophenethyl)ꢀ2,3ꢀdihydrobenzo[b][1,4]dioxineꢀ6ꢀ carboxamide (1, Figure 1), a benzamide
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derivative, as a βꢀcellꢀprotective small molecule against ER stress, from ~50,000 compounds screened.
Although 1 is structurally novel, its weak activity on βꢀcellꢀprotection (the maximum activity is 42%
with an EC50 value at 21 µM) prompted us to initiate a lead optimization campaign and a mechanistic
study on the βꢀcellꢀprotection of this novel class of compounds.
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Compound 1,
EC50 = 21 µM, Maximum activity = 42%
Figure 1. Structure and cell protective effect on INSꢀ1 cells of compound 1.
CHEMISTRY
Synthetic routes for the preparation of the target compounds are summarized in Schemes 1ꢀ3. The
2ꢀ/3ꢀ/4ꢀ(Nꢀpiperidinyl)methyl benzoic acid methyl esters 6, 4, and 2, were synthesized from the
corresponding bromomethylbenzoic acid methyl ester and piperidine. After a subsequent hydrolysis
step with sodium hydroxide, the (Nꢀpiperidinyl)methyl substituted esters were converted to the key
acid intermediates 7, 5, and 3. Finally, condensation of these carboxylic acids with a series of
substituted benzylamines or phenylethylamines resulted in the desired compounds, 11aꢀd, 12aꢀh, and
13aꢀd.
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Scheme 1. Synthesis of 4ꢀ(Nꢀpiperidinyl)methyl benzamide derivatives.a
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aReagents and conditions: (i) piperidine, CH2Cl2, reflux, 75%; (ii) NaOH, MeOH/H2O, rt, 87%; (iii)
benzylamines or phenylethylamines, HATU, DIEA, CH2Cl2, rt, 70ꢀ80%.
Scheme 2. Synthesis of 3ꢀ(Nꢀpiperidinyl)methyl benzamide derivatives.a
aReagents and conditions: (i) piperidine, CH2Cl2, reflux, 71%; (ii) NaOH, MeOH/H2O, rt, 83%; (iii)
benzylamines or phenylethylamines, HATU, DIEA, CH2Cl2, rt, 75ꢀ85%.
Scheme 3. Synthesis of 2ꢀ(Nꢀpiperidinyl)methyl benzamide derivatives.a
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aReagents and conditions: (i) piperidine, K2CO3, THF, rt, 76%; (ii) NaOH, MeOH/H2O, rt, 89%; (iii)
4ꢀchlorobenzylamine, HATU, DIEA, CH2Cl2, rt, 73%.
Table 1. Activity of Nꢀbenzylbenzamide derivatives 8aꢀ8h on the survival of INSꢀ1 cells treated with
Tm.a
Maximum activity
(% rescue)a
EC50
Compd
R1
R2
(µM)b
3ꢀCl
H
42% ± 12%
47% ± 9%
29% ± 6%
50% ± 11%
51% ± 17%
21 ± 5.7
19 ± 6.9
>30
1
3,4ꢀ
8a
8b
8c
8d
3,4ꢀ
3,4ꢀ
3,4ꢀ
3,4ꢀ
2ꢀOMe
4ꢀF
21 ± 3.2
15 ± 1.6
4ꢀCl
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4ꢀOMe
26% ± 8%
>30
>30
8e
8f
3,4ꢀ
3,4ꢀ
3,4ꢀDiꢀOMe
5% ± 7%
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3ꢀCl
3ꢀCl
41% ± 14%
46% ± 17%
25 ± 5.2
24 ± 2.1
8g
8h
3,4ꢀDiꢀOMe
aMaximum activity value is reported as % rescue from Tm (0.1 ꢁg/mL) ꢀinduced reduction of cell
viability; the values for Tm treatment alone and control (DMSO, without Tm) treatment are designated
as 0% and 100%, respectively in all tables. bEC50 values (the concentrations that reach halfꢀmaximal
activity) for INSꢀ1 cell viability are calculated with GraphPad Prism from the data of ten 2ꢀfold serial
titration points in all tables. All experiments were performed in triplicate in all tables.
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Results and Discussion
A structureꢀactivityꢀrelationship (SAR) study was conducted to improve the weak potency of lead
compound 1. Newly synthesized or commercially available compounds were tested for their
βꢀcellꢀprotective activity against ER stress in rat INSꢀ1 βꢀcells. Treatment of INSꢀ1 βꢀcells with
tunicamycin (Tm), a potent ER stress inducer that inhibits Nꢀlinked glycosylation of proteins and
causes the accumulation of misfolded proteins, significantly reduced cell viability at 72 h compared
with DMSOꢀtreated cells, as measured by an intracellular ATP levelꢀbased cell viability assay (Figure
1A). The maximum activities and the concentrations that reach halfꢀmaximal activity (EC50) of the
compounds were evaluated by the degree of increase in viability of INSꢀ1 cells coꢀtreated with the
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compounds in the presence of Tm compared with Tm treatment alone. Our initial focus was to explore
the right phenyl ring of 1 while keeping the left dihydrobenzodioxine ring intact (Table 1). While
removal of the 3ꢀchloro group in the right phenyl ring of 1 (8a) did not markedly affect the EC50 values
and maximum activity levels of the resultant compound, the introduction of a methoxy group, such as
2ꢀmethoxy (8b), 4ꢀmethoxy (8e), and 3,4ꢀdimethoxy (8f), led to less potency in that an increase in the
EC50 values and reduction in the maximum activity were observed compared to the parent compound 1.
However, replacement with 4ꢀfluorine (8c) and 4ꢀchlorine (8d) on the position 4 in the right phenyl
ring appeared to improve the maximum activity levels of these compounds (42% for 1 to 50% for 8c
and 51% for 8d) with a similar EC50 values.
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Table 2. Activity of Nꢀbenzylbenzamide derivatives 9aꢀ9f on the survival of INSꢀ1 cells treated with
Tm.
Maximum activity
Compd
R
EC50 (µM)
(% rescue)
43% ± 12%
38% ± 14%
57% ± 11%
69% ± 9%
69% ± 12%
68% ± 10%
2ꢀCl
2ꢀBr
25 ± 6.9
25 ± 2.1
9.5 ± 1.2
5.5 ± 0.7
2.8 ± 0.5
3.6 ± 0.9
9a
9b
9c
9d
9e
9f
3ꢀCl
3ꢀBr
4ꢀCl
3,4ꢀDiꢀCl
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We then investigated the consequence of modifying the middle linker region, with the ethylene linker
shortened to a methylene linker. We found that the methylene derivatives with modifications at
position 2 (2ꢀchlorine (9a) and 2ꢀbromo (9b)) exhibited similar activity to that of hit compound 1.
However, the methylene derivatives with modifications at positions 3 and 4, such as 3ꢀchlorine (9c),
3ꢀbromo (9d), 4ꢀchlorine (9e), and 3,4ꢀdiꢀchlorine (9f), exhibited significantly improved EC50 values
and maximum activity compared to the ethylene linker derivatives (Table 2 vs. Table 1). Of note,
compound 9e improved its EC50 value over its ethylene counterpart 8d by almost 6ꢀfold (EC50 2.8 µM
for 9e vs. 15 µM for 8d) and the maximum activity (69% for 9e vs. 51% for 8d). Thus, the methylene
group appeared to be a preferable linker.
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Table 3. Activity of Nꢀphenethylbenzamide derivatives 10aꢀ10p on the survival of INSꢀ1 cells treated
with Tm.
Maximum activity
Compd
R
EC50 (µM)
(% rescue)
6% ± 5%
30% ± 8%
<0
2ꢀMe
2ꢀSꢀiPr
3ꢀF
ꢀ
10a
10b
10c
10d
10e
>30
ꢀ
ꢀ
ꢀ
3ꢀNO2
3ꢀNH2
<0
<0
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3ꢀOEt
3ꢀNHSO2Me
49% ± 11%
26 ± 4.5
25 ± 2.1
20 ± 5.4
>30
10f
10g
10h
10i
24% ± 8%
25% ± 5%
54% ± 8%
37% ± 6%
31% ± 7%
87% ± 9%
42% ± 11%
56% ± 8%
23% ± 6%
<0
4ꢀMe
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4ꢀOꢀpropargyl
4SO2ꢀNMe2
19 ± 2.1
11 ± 1.9
21 ± 3.5
17 ± 6.1
15 ± 3.2
9.9 ± 2.1
ꢀ
10j
4ꢀ(Nꢀpyrrolidinyl)methyl
4ꢀ(Nꢀpiperidinyl)methyl
3,4ꢀDiꢀCl
10k
10l
10m
10n
10o
10p
3ꢀMeꢀ4ꢀOMe
2,3,4ꢀtrifluoro
2,3,4,5ꢀtetrafluoro
Next, we shifted our SAR study to the left phenyl ring. Since removal of the 3ꢀchloro group on the
right ring did not affect the potency (8a vs. 1), we kept the right phenyl ring unsubstituted. The
resulting compounds with various substituents at the left phenyl ring are listed in Table 3. Most
derivatives exhibited similar or reduced maximum activity or EC50 values compared with the
dihydrobenzodioxine derivative 8a. However, the 4ꢀ(Nꢀpiperidinyl)methyl group derivative (10l)
exhibited significantly improved maximum activity (87% for 10l vs. 47% for 8a) and a similar EC50
value (21 µM for 10l vs. 19 µM for 8a). In contrast, introduction of a 4ꢀ(Nꢀpyrrolidinyl)methyl group
(10k) resulted in a reduced maximum activity (31%). Taken together, these results demonstrate that the
4ꢀ(Nꢀpiperidinyl)methyl group rendered favorable activity to the scaffold.
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Table 4. Activity of 4ꢀ(Nꢀpiperidinyl)methyl benzamide derivatives 10l, 11aꢀ11d on the survival of
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INSꢀ1 cells treated with Tm.
O
N
n
R
H
N
Maximum activity
(% rescue)
EC50
Compd
n
R
(µM)
2
2
2
1
1
H
87% ± 6%
21 ± 3.5
21 ± 4.2
24 ± 2.7
9.5 ± 1.1
1.8 ± 0.2
10l
11a
11b
11c
11d
4ꢀCl
82% ± 10%
71% ± 11%
77% ± 7%
3,4ꢀDiꢀCl
4ꢀCl
3,4ꢀDiꢀCl
78% ± 10%
As we observed more favorable activity of the methylene middle linker than ethylene derivatives in
compounds with dihydrobenzodioxine ring for the left phenyl moiety, we investigated whether
methylene middle linker exhibits similar advantage in the 4ꢀ(Nꢀpiperidinyl)methyl background. As
summarized in Table 4, all the derivatives (11aꢀ11d) exhibited significant maximum activity levels (all
greater than 70%) regardless of the middle linker region. However, the derivatives with methylene
middle linker exhibited significant improvements on the EC50 values over the ethylene derivatives (11c
vs. 11a and 11d vs. 11b). In particular, the EC50 value of methylene derivative with 3,4ꢀdiꢀchlorine
substitution in the right phenyl ring (11d) improved by 13ꢀfold compared to that of the corresponding
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ethylene derivative 11b (EC50 1.8 µM for 11d vs. 24 µM for 11b). These results indicate that the
combination of the 4ꢀ(Nꢀpiperidinyl)methyl moiety in the left phenyl ring and the methylene middle
linker confers the compound favorable activity on βꢀcell protection.
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Table 5. Activity of (Nꢀpiperidinyl)methyl benzamide derivatives 12aꢀ12h, 13aꢀd on the survival of
INSꢀ1 cells treated with Tm.
Substitution position of
Maximum activity
(% rescue)
EC50
Compd
R
(Nꢀpiperidinyl)methyl
(µM)
2
3
3
3
3
4
4
4
4ꢀCl
H
73% ± 13%
95% ± 8%
6.9 ± 1.2
0.35 ± 0.06
2.04 ± 0.18
0.41 ± 0.06
0.24 ± 0.06
13 ± 1.6
12a
12b
12c
12d
12e
12f
2ꢀCl
3ꢀCl
4ꢀCl
2ꢀCl
3ꢀCl
3ꢀBr
93% ± 7%
90% ± 11%
93% ± 13%
87% ± 15%
80% ± 9%
2.2 ± 0.3
4.9 ± 0.7
12g
12h
81% ± 12%
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3ꢀOMe
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3ꢀCF3
92% ± 10%
0.96 ± 0.16
1.12 ± 0.21
0.25 ± 0.04
0.03 ± 0.01
13a
13b
13c
13d
93% ± 5%
91% ± 14%
97% ± 8%
4ꢀCF3
We next investigated whether the position of (Nꢀpiperidinyl)methyl group in the left phenyl ring affects
βꢀcellꢀprotective activity while keeping the methylene middle linker. A series of derivatives (12aꢀ12h,
13aꢀ13d) were subsequently synthesized and these are summarized in Table 5. Clearly, substitution of
(Nꢀpiperidinyl)methyl group at position 3 (12bꢀ12e) showed significant improvements over positions 2
and 4 (12a, and 12fꢀ12h) in both maximum activity and EC50 values. Furthermore, we noticed that
among the 3ꢀ(Nꢀpiperidinyl)methyl derivatives (12cꢀ12e), substitutions with chlorine at meta and para
positions in the rightꢀhand phenyl ring (12d and 12e) improved the EC50s over the orthoꢀsubstituted
derivative 12c. We finally examined whether the electron status in the right phenyl ring influences the
potency on the 3ꢀ(Nꢀpiperidinyl)methyl derivatives. We therefore substituted the compounds with the
electronꢀdonating group ꢀOMe (13a and 13b) and the electronꢀwithdrawing group ꢀCF3 (13c and 13d)
at the metaꢀ and paraꢀ positions on the rightꢀhand phenyl ring. The electronꢀwithdrawing groups, ꢀCF3
(13c and 13d) and ꢀCl (12d and 12e) at the metaꢀ and paraꢀ positions significantly exhibited more
favorable EC50s compared to their electronꢀdonating group (ꢀOMe) counterparts (13a and 13b). In
particular, the compound with the substitution of the strong electronꢀwithdrawing CF3 in the paraꢀ
position (13d) exhibited the most potent property with a near 100% maximum activity and an EC50 at
0.032 µM. We therefore chose 13d for further characterization of its mechanism of action in promoting
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βꢀcell survival and function against ER stress.
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Figure 1. Protective effects of 13d on the viability of INSꢀ1 cells after Tm treatment. (A, B) INSꢀ1
cells were treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d or DMSO for 72 h. Cell
viability was determined by intracellular ATP assay (A) or MTT assay (B). (C) βTC6 cells were treated
with or without Tm (0.3 ꢁg/mL) in the presence of 13d or DMSO for 72 h. The cell viability was
determined by the intracellular ATP level. (D) INSꢀ1 cells were treated with 13d or DMSO for 72 h.
The cell viability was determined by the intracellular ATP level. Results in AꢀC are expressed as the
fold change compared with that of no Tm treatment (set to 1.0). The result in D is expressed as the
foldꢀchange compared to no 13d treatment (set to 1.0). Results in all panels are the means of 3 replicate
wells and are representative of 3 independent experiments. Error bars indicate standard deviation (SD).
* P < 0.05, ** P < 0.01, and *** P < 0.001 compared with Tm treated alone.
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In the above SAR studies, we used the measurement of the intracellular ATP level as a surrogate assay
for cell viability. We observed that compound 13d rescued the viability of INSꢀ1 βꢀcells under Tm
treatment in a doseꢀdependent manner and that 13d retained maximum βꢀcellꢀprotective activity within
a wide range of concentrations from 0.16~20 ꢁM (Figure 1A). To confirm the authenticity of 13d’s
protective effect on βꢀcell viability (not simply an increase in ATPꢀspecific measurement), we used the
MTT assay, which measures the activity of NAD(P)Hꢀdependent cellular oxidoreductase enzymes, as
an orthogonal method to measure cell viability. As shown in Figure 1B, Tm treatment reduced MTT
reading in INSꢀ1 cells compared to that of DMSO treatment, and coꢀtreatment with 13d restored the
viability of INSꢀ1 cells. To determine whether the protective effect of 13d on βꢀcells is specific to
INSꢀ1 cells, another βꢀcell line, βTC6, was used. As expected, Tm induced a reduction in viability in
βTC6 cells, and coꢀtreatment with 13d rescued the viability of βTC6 cells in a doseꢀdependent manner,
as assessed by the intracellular ATP level (Figure 1C). In addition, compound 13d on its own had no
effect on βꢀcell viability in both cell lines (Figure 1C, 1D). Taken together, these results indicate that
compound 13d protects βꢀcells against ER stress.
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Caspase 3
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α-Tubulin
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Figure 2. Compound 13d protects INSꢀ1 cells against Tmꢀinduced apoptosis. (A) INSꢀ1 cells were
treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d at indicated concentrations or DMSO
for 24 h. Cleaved caspaseꢀ3 and PARP were determined by Western blotting. αꢀTubulin was used as a
loading control. The data shown are representative of 3 independent experiments. (B) INSꢀ1 cells were
treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d (0.1 ꢁM) or DMSO for 48 h, and
liveꢀcell phaseꢀcontrast images were acquired (magnification 10×). The images shown are
representative of 3 independent experiments. (C) TUNEL staining of INSꢀ1 cells. INSꢀ1 cells were
treated with 0.1 ꢁg/mL of Tm with or without compound 13d for 24 h before TUNEL staining. DAPI
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was used as a nuclear marker. Magnification is 20×. The images shown are representative of 3
independent experiments. (D) Quantification of TUNEL staining from 20 fields of images. ** P < 0.01
compared with Tm treatment alone.
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To determine whether the increase in βꢀcell viability following treatment with 13d is caused by a
suppression of apoptotic cell death, several apoptosisꢀrelated makers were measured. First, levels of
cleaved caspaseꢀ3 and cleaved PARP were assayed by Western blotting. Caspaseꢀ3, a member of
executioner caspases activated by cleavage, plays essential roles in initiating apoptotic signaling and
executing the final stages of cell death, whereas the nuclear enzyme poly (ADPꢀribose) polymerase
(PARP) plays an important role in a number of cellular processes involving DNA repair and cell death.
Under normal condition, caspase 3 exists as inactive proenzyme. However, upon severe ER stress,
caspase 3 undergoes proteolytic cleavage to produce two subunits that dimerize to form the active
enzyme, which in turns cleaves PARP. Hence, appearance of the cleaved forms of both caspaseꢀ3 and
PARP is an indication of apoptosis.23, 24 Tm treatment for 24 h significantly induced both cleaved
caspaseꢀ3 and cleaved PARP protein levels in INSꢀ1 cells (Figure 2A). However, 13d coꢀtreatment
completely or significantly diminished Tmꢀinduced cleavage of both caspaseꢀ3 and PARP (Figure 2A).
Consistent with this, significantly more viable INSꢀ1 cells were observed with Tm and 13d
coꢀtreatment than with Tm alone (Figure 2B). A further assessment of apoptotic INSꢀ1 cell death was
performed using TUNEL assays to detect nuclear DNA fragmentation (a consequence of cell apoptosis)
in situ. Tm treatment resulted in a significant increase in TUNELꢀpositive cells (7% TUNELꢀpositive
cells compared to less than 0.2% in control group), while coꢀtreatment with Tm and 13d remarkably
reduced the TUNELꢀpositive cells (0.5%) (Figures 2C and 2D). Taken together, these results
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demonstrate that 13d can suppress Tmꢀinduced βꢀcell apoptosis.
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E
1.7 mM 20 mM
**
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DMSO
13d
Tm
Tm + 13d
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Figure 3. Compound 13d reversed Tmꢀsuppressed βꢀcell dysfunction. (AꢀD) INSꢀ1 cells were treated
with or without Tm (0.1 ꢁg/mL) in the presence of 13d at indicated concentrations or DMSO for 24 h.
The mRNA levels of INS1 (A), INS2 (B), PDX1 (C), and MafA (D), were analyzed by qRTꢀPCR.
Relative mRNA levels were normalized against the housekeeping gene Cyclophilin A using the
comparative CT method. The results are the means of 3 replicate wells and are representative of 3
independent experiments. * P < 0.05 and ** P < 0.01 compared with Tm treatment alone. Bars indicate
SD. (E) Insulin secretion by INSꢀ1 cells incubated with 1.7 mM and 20 mM glucose in the presence or
absence of Tm (0.1 ꢁg/mL) and 13d. Secreted insulin was measured by ELISA after 24 h treatment. *
P < 0.05. The amount of insulin secreted in response to 1.7 mM glucose in the absence of Tm was set
to 1.0 and was normalized with total protein concentration.
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In addition to βꢀcell apoptosis, ER stress also leads to βꢀcell dysfunction, namely, the impairment of
biosynthesis and secretion of insulin. First, we examined whether compound 13d reverses the
Tmꢀsuppressed mRNA levels of insulin genes. As expected, Tm treatment of INSꢀ1 cells decreased the
mRNA levels of both insulin genes, INS1 and INS2, but this reduction was completely rescued by 13d
(Figures 3A and 3B). Second, we examined whether compound 13d affects the expression of βꢀcell
transcription factors PDX1 and MafA, which control βꢀcell identity and the expression of insulin
genes.25, 26 Tm treatment decreased the levels of PDX1 and MafA mRNA expression levels in INSꢀ1
cells, however, these decreases were completely rescued by 13d coꢀtreatment (Figures 3C and 3D).
Next, we investigated whether compound 13d reꢀestablishes Tmꢀimpaired glucoseꢀstimulated insulin
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secretion (GSIS). As shown in Figure 3E, Tm treatment abolished the insulin secretion caused by high
concentration of glucose treatment (20 mM) in INSꢀ1 cells. Addition of 13d significantly rescued the
GSIS in Tmꢀtreated cells. Taken together, these data demonstrate that 13d restores ER stressꢀimpaired
βꢀcell survival and function.
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Figure 4. Compound 13d inhibits Tmꢀinduced ATF4 and CHOP upꢀregulation in INSꢀ1 cells. (A, B)
INSꢀ1 cells were treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d or DMSO for 8 h.
ATF4 (A) and CHOP (B) mRNA levels were analyzed by qRTꢀPCR. Relative mRNA levels were
normalized against the housekeeping gene Cyclophilin A using the comparative CT method. The
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results were expressed as the foldꢀincrease over mRNA levels in untreated control cells and are the
means of 3 replicate wells and representative of 3 independent experiments. ** P < 0.01 compared
with Tm treatment alone. Bar indicates SD. (CꢀE) INSꢀ1 cells were treated with or without Tm (0.1
ꢁg/mL) in the presence of 13d or DMSO for 8 hATF4 and CHOP protein levels were determined by
Western bloting (C). αꢀTubulin was used as an internal control. Quantification of ATF4 (D) and CHOP
(E) protein levels was analyzed by ImageJ program, which was normalized by αꢀTubulin protein level.
The data shown are representative of 3 independent experiments.
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Having established that compound 13d restores ER stressꢀimpaired βꢀcell survival and function, we
next investigated the mechanism of action by which 13d protects βꢀcells against ER stress. Chronic or
severe ER stress activates all three branches of the UPR, PERK, IRE1α, and ATF6, leading to eventual
cell death. First, we determined the effect of 13d on the activation of the PERK pathway in βꢀcells
under ER stress. PERK activation phosphorylates eukaryotic translation initiator factor 2α (eIF2α),
which in turn allows for the upꢀregulation of activating transcription factor 4 (ATF4) and of the
proꢀapoptotic gene C/EBPꢀhomologous protein (CHOP).4, 8, 11, 27 Thus, we used ATF4 and CHOP
expression levels as markers of PERK pathway activation. Tm treatment of INSꢀ1 cells significantly
increased the mRNA levels of both ATF4 and CHOP, whereas coꢀtreatment with 13d almost
completely abolished the Tmꢀinduced increase in both mRNA levels (Figures 4A and 4B). In addition,
after treatment with Tm for 8 h, CHOP mRNA level increased 7ꢀfold compared with the control group,
and coꢀtreatment with compound 13d inhibited Tmꢀinduced CHOP expression with an IC50 value of
0.037 µM (Figure 4C). Moreover, this IC50 value was almost equal to the EC50 value of 13d for cell
survival (Table 5). In addition, 13d had no effect on the mRNA levels of ATF4 and CHOP on its own
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(Figure 4A and 4B). These results indicate that 13d inhibits the activation of PERKꢀATF4ꢀCHOP
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XBP1s
Cyclophilin A
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Figure 5. Compound 13d inhibits Tmꢀinduced XBP1s mRNA expression in INSꢀ1 cells. INSꢀ1 cells
were treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d or DMSO for 8 h. (A) XBP1s
mRNA levels were analyzed by qRTꢀPCR. Relative mRNA levels were normalized against the
housekeeping gene Cyclophilin A using the comparative CT method. ** P < 0.01 compared with Tm
treatment alone. Bars indicate SD. (B) XBP1 mRNA levels were analyzed by RTꢀPCR and the products
were resolved by agarose gel electrophoresis. The fullꢀlength (unspliced, XBP1u) and spliced (XBP1s)
forms of XBP1 mRNA are indicated. Cyclophilin A mRNA was used as a loading control.
Quantification was analyzed by ImageJ program shown at the bottom. The data shown are
representative of 3 independent experiments.
Next, we determined the effect of 13d on the activation of IRE1α in βꢀcells under ER stress. Activation
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of the IRE1α leads to cleavage of Xꢀbox binding proteinꢀ1 (XBP1) mRNA and this generates a spliced
form (XBP1s) that is translated into a potent transcription factor that controls the expression of UPR
genes involved in ER protein folding and degradation.6, 12, 27 We therefore used XBP1 splicing as a
marker for the activation of the IRE1α pathway and determined the effect of 13d on IRE1αꢀmediated
XBP1 splicing in INSꢀ1 cells in the presence of Tm. As shown in Figure 5A, Tm markedly increased
the level of XBP1s mRNA in INSꢀ1 cells, but this increase was abolished by 13d coꢀtreatment, as
measured by qRTꢀPCT using XBP1 splicingꢀspecific primers. Similarly, an electrophoretic separation
of spliced and unspliced forms of XBP1 after RTꢀPCR amplification of total XBP1 mRNA revealed
that 13d inhibits the Tmꢀinduced generation of XBP1s mRNA (Figure 5B). These results indicate that
13d inhibits Tmꢀinduced activation of IRE1αꢀXBP1 pathway.
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Figure 6. Compound 13d decreases Bip and GRP94 mRNA levels induced by Tm. INSꢀ1 cells were
treated with or without Tm (0.1 ꢁg/mL) in the presence of 13d or DMSO for 8 h. Bip (A) and GRP94
(B) mRNA levels were analyzed by qRTꢀPCR. Relative mRNA levels were normalized against the
housekeeping gene Cyclophilin A using the comparative CT method. The results are the means of 3
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replicate wells and representative of 3 independent experiments. ** P < 0.01 compared with Tm
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We then investigated the effects of 13d on the activation of ATF6 in βꢀcells under ER stress. Activated
ATF6 acts as a homodimer or as an ATF6–XBP1s heterodimer to control the upꢀregulation of select
UPR target genes including the chaperone proteins BiP and GRP94.12, 27 Therefore, these two
chaperone proteins were evaluated in the INSꢀ1 cells in the presence of Tm. Both Bip and GRP94
mRNAs were upꢀregulated by treatment with Tm, while coꢀtreatment with Tm and compound 13d
almost completely reversed these increases (Figure 6A and 6B).
Thus far, our observation that 13d protected βꢀcells against ER stressꢀinduced cell death and
dysfunction and that 13d inhibited ER stressꢀmediated activation of all three UPR pathways revealed a
strong correlation between the βꢀcell protective effect of 13d and its suppression of ER stress response.
If 13d protects βꢀcells through the inhibition of ER stress response, a structurally similar but
βꢀcellꢀinactive analog of 13d would be expected to possess no inhibitory effect on ER stressꢀinduced
UPR response. Indeed, we observed that as a negative control, the βꢀcellꢀinactive analog 8f had no
effect on the UPR pathways. As shown in Figure 7, 8f coꢀtreatment had no obvious effect on the
mRNA levels of ATF4, CHOP, Bip, GRP94 or XBP1s. These results together support the notion that
13d protects βꢀcell survival by alleviating ER stress.
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Figure 7. Inactive compound 8f does not decrease ER stress related mRNA levels induced by Tm.
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INSꢀ1 cells were treated with or without Tm (0.1 ꢁg/mL) in the presence of 8f or DMSO for 8 h. ATF4
(A), CHOP (B), Bip (C), GRP94 (D), and XBP1s (E) mRNA levels were analyzed by qRTꢀPCR.
Relative mRNA levels were normalized against the housekeeping gene Cyclophilin A using the
comparative CT method. The results are the means of 3 replicate wells and representative of 3
independent experiments. Error bars indicate SD.
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We next evaluated the in vivo efficacy of 13d using a streptozotocin (STZ)ꢀinduced diabetic mouse
model. STZ induces ER stress in βꢀcells in addition to the increase in reactive oxygen species (ROS)
production and DNA alkylation.28, 29 C57BL/6 mice injected intraperitoneally with low dose STZ (50
mg/kg) once daily for 5 consecutive days develop diabetes; blood glucose levels increased gradually
within a week of STZ treatment. Coꢀtreatment with 13d (5 mg/kg) lowered the glucose levels
significantly in a timeꢀdependent manner (314 mg/dL (13d) vs. 422 mg/dL (vehicle) after 3 weeks of
treatment) (Figure 8A). The intraperitoneal glucose tolerance testing on overnight fasted animals
showed lower blood glucose levels at 15, and 30 min in mice coꢀtreated with STZ and 13d, compared
to vehicleꢀtreated STZ group (Figure 8B).
To evaluate whether 13d treatment influences the survival of βꢀcells in STZ animals, histology on
pancreatic sections was analyzed. We observed that 13dꢀtreated mice had more than double the total
pancreatic islet βꢀcell area compared to vehicleꢀtreated mice (67.4 ± 5.9% for STZ + 13d vs. 30.2 ± 7.9%
for STZ + saline, Figures 8C and 8D). In addition, 13d had no obvious effect on the average number of
αꢀcells between vehicle and 13d groups (Figure 8C). These results indicate that 13d ameliorates
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STZꢀinduced diabetes in mice by preserving βꢀcell survival.
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Figure 8. Antiꢀdiabetic effect of 13d in the STZꢀinduced diabetic mouse model. (A) Fasting blood
glucose levels were measured in STZꢀinjected mice treated with vehicle control (n = 6) or 13d (n = 7).
C57BL/6J mice were injected intraperitoneally once daily for 5 days with STZ (50 mg/kg body weight)
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and with either vehicle (n = 6 mice) or 13d (5 mg/kg body weight; n = 7 mice). Injections of vehicle or
13d alone were then continued for 2 more weeks. (B) Glucose tolerance test. Blood glucose levels
measured at indicated time points after intraperitoneal injection of glucose (2g/kg body weight). (C)
Pancreases were sectioned and slides were stained with antiꢀinsulin antibody (green, βꢀcell marker),
antiꢀglucagon antibody (red, αꢀcell marker), and DAPI (blue). Slides were imaged with an Olympus
FV1000 confocal microscope. Representative images are shown in (C). (D) Quantification of total islet
area per section and βꢀcell number per islet. Total area of all islets per section calculated for a total of
six sections using insulinꢀpositive cells to demarcate islets. Data are the means ± SEM of six sections
from three mice. *p < 0.05 **p < 0.01, and ***p < 0.001 by Student’s tꢀtest.
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Conclusion
We discovered and optimized 3ꢀ(Nꢀpiperidinyl)methyl benzamide derivatives as potent agents that
suppress ER stressꢀinduced βꢀcell death and dysfunction. Our lead optimization has led to the
identification of the 3ꢀ(Nꢀpiperidinyl)methyl benzamide analog 13d with near 100% βꢀcellꢀprotective
activity and an EC50 of 0.032 ꢀM against ER stress. Importantly, we showed that 13d significantly
lowers blood glucose levels and increases βꢀcell survival in an STZꢀinduced diabetic mouse model. In
addition, we demonstrated that 13d alleviates ER stress/UPR response by inhibiting Tmꢀinduced
upꢀregulation of all three branches of unfolded protein response (UPR) and apoptosis. Figure 9
proposes a model of signaling events leading to the protective effect of 13d in βꢀcells against ER
stressꢀinduced dysfunction and cell death.
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Figure 9. Proposed model of signaling pathways involved in 13dꢀmediated βꢀcellꢀprotective effects
against ER stress. ER stress induces activation of three branches of UPR (ATF6, IRE1α, and PERK),
leading to upꢀregulation of Bip, GRP4, XBP1s, ATF4, and CHOP. These events result in the reduction
in the expression of PDX1, MAFA and insulin genes, leading to βꢀcell dysfunction, and the eventual
activation of caspase 3 and cell apoptosis. Compound 13d protects βꢀcells against ER stressꢀmediated
dysfunction and death by attenuating ER stress through upꢀregulating PDX1, MAFA and insulin genes
and downꢀregulating ATF4, XBP1s, Bip, GRP94, CHOP, and caspase 3.
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Our current work has unraveled compound 13d as a representative of a new βꢀcellꢀprotective
chemotype containing a 3ꢀ(Nꢀpiperidinyl)methyl benzamide skeleton with a near 100% maximum
protection activity and an EC50 value of ~30 nM. Studies on the pharmacokinetic properties of 13d are
ongoing as part of our program to develop novel chemotypes as βꢀcellꢀprotective leads for further
antiꢀdiabetic drug development.
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