Discovery, Synthesis, and Evaluation of 2,4-Diaminoquinazolines as a Novel Class of Pancreatic β-Cell-Protective Agents against Endoplasmic Reticulum (ER) Stress

Article abstract of DOI:10.1021/acs.jmedchem.6b00041

Pancreatic insulin-producing β-cell dysfunction and death plays central roles in the onset and progression of both type 1 and type 2 diabetes. Current antidiabetic drugs cannot halt the ongoing progression of β-cell dysfunction and death. In diabetes, a major cause for the decline in β-cell function and survival is endoplasmic reticulum (ER) stress. Here, we identified quinazoline derivatives as a novel class of β-cell protective agents against ER stress-induced dysfunction and death. A series of quinazoline derivatives were synthesized from dichloroquiazoline utilizing a sequence of nucleophilic reactions. Through SAR optimization, 2,4-diaminoquinazoline compound 9c markedly protects β-cells against ER stress-induced dysfunction and death with 80% maximum rescue activity and an EC₅₀ value of 0.56 μM. Importantly, 9c restores the ER stress-impaired glucose-stimulated insulin secretion response and survival in primary human islet β-cells. We showed that 9c protects β-cells by alleviating ER stress through the suppression of the induction of key genes of the unfolded protein response and apoptosis.

Full text of DOI:10.1021/acs.jmedchem.6b00041

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Article
Discovery, Synthesis and Evaluation of 2,4-diaminoquinazolines
as a Novel Class of Pancreatic # Cell-Protective
Agents against Endoplasmic Reticulum (ER) Stress
hongliang duan, Jae Wook Lee, Sung Won Moon, Daleep Arora, Yu Li, Hui-ying Lim, and Weidong wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00041 • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 9, 2016
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Discovery, Synthesis and Evaluation of 2,4-diaminoquinazolines as a Novel Class of Pancreatic β
Cell-Protective Agents against Endoplasmic Reticulum (ER) Stress
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†
¶
§∫¶*
§
†
£
#
Hongliang Duan, Jae Wook Lee, Sung Won Moon, Daleep Arora, Yu Li, Hui-Ying Lim, and
Weidong Wang†£*
†
th
Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13 Street,
Oklahoma City, Oklahoma 73104, United States
§Natural Product Research Center, Korea Institute of Science and Technology, 679 Saimdang-ro,
Gangneung, Gangwon-do, 210-340, Republic of Korea
∫Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon,
3
05-333, Republic of Korea
#Department of Physiology, The University of Oklahoma Health Science Center, 941 Stanton L. Young
Blvd., Oklahoma City, Oklahoma 73104, United States
£Department of Medicine, Division of Endocrinology, Department of Physiology, Harold Hamm
Diabetes Center, The University of Oklahoma Health Science Center, 941 Stanton L. Young Blvd.,
Oklahoma 73104, United States
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ABSTRACT
Pancreatic insulin-producing β-cell dysfunction and death plays central roles in the onset and
progression of both type 1 and type 2 diabetes. Current antidiabetic drugs cannot halt the ongoing
progression of β-cell dysfunction and death. In diabetes, a major cause for the decline in β cell function
and survival is endoplasmic reticulum (ER) stress. Here, we identified quinazoline derivatives as a
novel class of β cell protective agents against ER stress-induced dysfunction and death. A series of
quinazoline derivatives were synthesized from dichloroquiazoline utilizing a sequence of nucleophilic
reactions. Through SAR optimization, a 2,4-diaminoquinazoline compound 9c markedly protects β
cells against ER stress-induced dysfunction and death with 80% maximum rescue activity and an EC50
value of 0.56 M. Importantly, 9c restores the ER stress-impaired glucose-stimulated insulin secretion
response and survival in primary human islet β cells. We showed that 9c protects β cells by alleviating
ER stress through the suppression of the induction of key genes of the unfolded protein response and
apoptosis.
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INTRODUCTION
Diabetes has become a serious public health problem with tremendous social and economic burden on
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society; currently, it is estimated that 380 million people are suffering from diabetes worldwide. Loss
of pancreatic insulin-producing β cell mass or function is a critical event in the pathogenesis of both
type 1 diabetes (T1D) and type 2 diabetes (T2D).2-5 Thus, preservation and expansion of β cells is a
promising therapeutic approach for patients with diabetes. Unfortunately, currently the most commonly
prescribed antidiabetic drugs cannot halt the progression of β cell dysfunction and death.
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Figure 1. Structures of small molecules protective against β cell ER stress.
In diabetes, a major cause for the decline of pancreatic β cell function and mass is ER stress, a
condition in which misfolded or unfolded proteins accumulate in the ER.6-8 In T2D, insulin resistance
forces β cells to synthesize more insulin that exceeds the cellular capacity of ER for protein folding,
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thus causing ER stress. If this threshold is crossed, β cell dysfunction and apoptosis will ensue. In
T1D in which β cells are known to be destroyed by auto-immune reaction, ER stress has also been
implicated, and ER stress-reducing chemical chaperon has been shown to protect β cells and prevent
the onset of T1D in mouse models.9-11
ER stress is mediated by three ER membrane-associated proteins, inositol-requiring protein 1
(
IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6), which act as
unfolded protein sensors.6-8, 12 In unstressed cells, these sensors are maintained in an inactivate 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
1
3
and activating the sensors. IRE1, PERK, and ATF6 each activates a series of events aimed at
restoring ER homeostasis by altering the translation, folding, and post-translational modification of
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secreted and membrane proteins, a process termed the unfolded protein response (UPR). Failure to
re-establish ER homeostasis eventually triggers cell death under chronic or severe ER stress.
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As β cells must produce and rapidly secrete insulin in response to postprandial increases in blood
glucose levels, they maintain a very large pool of proinsulin mRNA (~20% of the total cellular mRNA)
and can increase proinsulin protein synthesis 25-fold upon glucose stimulation.14, 15 This surge in
proinsulin synthesis places a heavy burden on the protein-folding capacity of the ER; β cells are
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therefore particularly susceptible to ER stress. Thus, compounds that prevent β cells from ER
stress-induced damage hold promise as potential therapeutic agents for diabetes.
Small molecules have been identified that can regulate ER stress and/or the UPR. However, some of
these small molecules are tissue- and/or cell type-specific as they inhibit ER stress in one cell type but
not in others.16-25 For instance, salubrinal, which inhibits dephosphorylation of eIF2 (a PERK target),
protects neuronal cells and PC12 cells from ER stress but triggers apoptosis in β cells.26-28 The
aforementioned unique features of β cells may in part explain why compounds that protect other cell
types from ER stress fail to protect β cells27, 28 and contribute to a scarcity of β cell-protective small
molecules.
To date, only a few chemotypes have been reported to exhibit β cell-protective activities against ER
2
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stress through a high throughput β cell survival screen. Examples include
1
-((3s,5s,7s)-adamantan-1-yl)-3-(2-(methylthio)benzyl)urea (1) (Figure 1),
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5-((4-chloro-2-nitro-5-(1H-pyrrol-1-yl)phenyl)thio)-4H-1,2,4-triazol-3-amine (2) (Figure 1), and
several bacteriostatic antibiotics. There are also several other β cell-protective small molecules that do
not target ER stress and are not effective against ER stress, such as the STAT1 signaling inhibitor
BRD0476
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(N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tet
rahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfona
mide).30, 31 In a further extended β cell survival screen, a 2,4-diaminoquinazoline compound (5a) was
identified as β cell-protective agent against ER stress. Therefore, a series of 2,4-diaminoquinazoline
analogs were synthesized for their capacity to protect β cells from ER stress. SAR studies identified 9c
as a potent compound that suppresses ER stress-induced death and dysfunction of INS-1 β cells with
8
0% maximum rescue activity and an EC50 of 0.56 M. Importantly, compound 9c is also effective in
primary human islet β cells. In addition, we show that 9c inhibits ER stress-induced expression of UPR
and apoptotic genes in β cells. Compound 9c represents a new β cell-protective chemotype with a
2
,4-diaminoquinazoline skeleton.
CHEMISTRY
To identify potent β cell-protective quinazoline analogs, we initiated structure activity guided
optimization of 2,4-diaminoquinazoline. We planned modification of chemical structure at C-4 position
and following to C-2 of 2,4-diaminoquinazoline. The synthetic route employed for the synthesis of
2
,4-diaminoquinazolines is summarized in Scheme 1. The key intermediate, dichloroquinazoline 3,
was synthesized by the chlorination of benzoyleneurea with phosphorus oxychloride and catalytic
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amount of N,N-dimethylaniline. After a regioselective nucleophilic substitution at C-4 was performed
with various benzyl amines and phenethyl amine in the presence of DIEA, intermediate 3 was
converted into a series of monoamine derivatives, 4a-n, bearing methoxy, bromo, chloro, and fluoro in
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R .
Scheme 1. Synthesis of 2,4-diaminoquinazoline derivatives.a
aReagents and conditions: (i) dimethylaniline, POCl
, 120°C, 24 hrs, 69%; (ii) aryl amine, DIEA,
3
n-BuOH, 40°C, 2 hrs, 65-85%; (iii) L-prolinol or 2-piperidinemethanol, DIEA, n-BuOH, 120°C,
overnight, 50-85%.
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A second nucleophilic substitution at the C-2 of 4a-n with L-prolinol in the presence of DIEA yield
compounds, 5a-m. The β cells protection activities of compounds (5a-m) were summarized in Table 1.
To investigate the structure-activity relationship between five member ring and six membered ring, we
introduced 2-piperidinylmethanol at C-2 position of quinazoline. Compounds (6a-n) were synthesized
by nucleophilic substitution at the C-2 of 4a-n with 2-piperidinylmethanol in the presence of DIEA.
Bioactivities of compounds (6a-n) were summarized in Table 2. We next investigated the bioactivities
related with length of amine linker at C-4 substitution. Benzyl amines were selected to compare with
phenethylamines. The synthesis of intermediate (7a-d) was performed with various benzyl amines in
the presence of DIEA. A nucleophilic substitution at C-2 position with L-prolinol or
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-piperidinylmethanol in the presence of DIEA yielded compounds, 8c-d and 9a-d.33
Table 1. Activity of L-prolinol derivatives on the survival of INS-1 cells treated with Tm.
Maximum activitya
EC50 (μM)b
Compd
R
H
5a
54.4%
0.48 ± 0.19
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5b
4-F
4-Cl
4-Br
4-CH
3-F
56.2%
57.3%
44.1%
34.2%
51.3%
51.5%
53.2%
54.9%
52.5%
50.6%
39.0%
56.6%
2.13 ± 0.41
0.51 ± 0.09
0.65 ± 0.16
0.66 ± 0.21
1.15 ± 0.19
1.05 ± 0.18
0.78 ± 0.24
1.51 ± 0.13
1.70 ± 0.23
1.44 ± 0.27
1.88 ± 0.32
3.56 ± 0.15
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5e
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f
5
g
3-Cl
3-Br
5h
5
i
3-OMe
2-F
5
j
5k
2-Cl
5
l
2-Br
5
m
2-OMe
aMaximum activity value is reported as % rescue from Tm (0.1 μg/mL) -induced reduction of cell
viability; the values for Tm treatment alone and control (DMSO, without Tm) treatment are designated
b
as 0% and 100%, respectively in all tables. EC50 values (the concentrations that reach half-maximal
activity) for INS-1 cell viability are calculated with GraphPad Prism from the data of ten 2-fold serial
titration points in all tables. All experiments were performed in triplicate.
RESULTS AND DISCUSSION
A series of 2,4-diaminoquinazoline derivatives incorporating a 2-(hydroxymethyl)pyrrolidin-1-yl
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group at the C-2 position of a quinazoline ring were first synthesized and tested for their β
cell-protective activity against ER stress using a CellTiter-Glo cell viability assay in rat INS-1 β cells.
Treatment of INS-1 β cells with tunicamycin (Tm), a potent ER stress inducer that causes the
accumulation of misfolded proteins due to inhibition of N-linked glycosylation, drastically reduced cell
viability at 72 h compared with DMSO-treated cells, as measured by intracellular ATP levels with a
CellTiter-glo kit (Figure 2). The maximum activities and the concentrations that reach half-maximal
activity (EC50) of the compounds were evaluated by the degree of increase in viability of INS-1 cells
co-treated with the compounds in the presence of Tm compared with Tm treatment alone. As shown in
Table 1, derivatives of the phenethylamine moieties substituted at para, meta, and ortho positions
exhibited maximum activities ranging from ~34.2% to 57.3% as opposed to that as 100% in the
absence of Tm, with no position being conferred to be more potent. However, more derivatives
substituted at para position, including 5a, 5c, 5d and 5e, appeared to exhibit lower EC50s at
approcimately 0.5 μM than derivatives substituted at meta and ortho positions (Table 1).
We then changed the 2-(hydroxymethyl)pyrrolidin-1-yl group at the C-2 position of the quinazoline
ring to 2-(hydroxymethyl)piperidin-1-yl as racemic mixtures to explore the SAR (Table 2). Compared
to the 2-(hydroxymethyl)pyrrolidin-1-yl derivatives, the maximum activities of the
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-(hydroxymethyl)piperidin-1-yl derivatives remained in a similar range except 6j (Table 2). However,
the EC50s of these compounds improved over their 2-(hydroxymethyl)pyrrolidin-1-yl counterparts by
2-3 fold (6a, 6c, 6d, 6e, 6h, 6l, and 6n versus 5a, 5c, 5d, 5e, 5g, 5k, and 5m respectively), with 6b
~
improving by ~7 fold (6b versus 5b). 6j showed significantly improved maximum activity at 69.2% vs.
its 2-(hydroxymethyl)pyrrolidin-1-yl counterpart 5i (54.9%), but its EC50 also increased. Notably,
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among the 2-(hydroxymethyl)piperidin-1-yl derivatives, except bromo substitution which showed
similar EC50, the derivatives with para substitution, including 6a-f, exhibited relatively low EC50
s
compared to ortho- or meta- substituted derivatives (6g-n) (Table 2) . Taken together, this series of
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SAR studies indicates the 2-(hydroxymethyl)piperidin-1-yl moiety to be more favorable than its
2
-(hydroxymethyl)pyrrolidin-1-yl counterpart in β cell protection against ER stress.
Table 2. Activity of 2-piperidinemethanol derivatives on the survival of INS-1 cells treated with Tm.
Compd
R
Maximum activity
55.7%
EC50 (μM)
0.36 ± 0.21
0.31 ± 0.26
0.17 ± 0.05
0.34 ± 0.09
0.14 ± 0.04
0.31 ± 0.12
1.88 ± 0.18
0.79 ± 0.30
0.77 ± 0.40
6
a
H
6
b
4-F
4-Cl
4-Br
4-CH
47.5%
6c
57.6%
6
d
52.8%
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3
33.6%
6f
4-OMe
3-F
58.6%
6
g
57.3%
6
h
3-Cl
46.2%
6i
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6j
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69.2%
60.3%
51.4%
44.2%
46.9%
2.59 ± 0.31
1.46 ± 0.46
0.47 ± 0.45
1.41 ± 0.31
1.60 ± 0.15
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6m
2-Br
6
n
2-OMe
Next, we shifted the SAR study to the phenethylamine moiety. When the amine linker length was
shortened from two carbons to one, most resulting benzylamine compounds exhibited similar potency
and maximum activity to their phenethylamine counterparts (6a versus 9a, and 6c versus 9b) (Tables 2
and 3). However, for the 4-OCH derivatives, a benzyl group (9c) significantly improved the maximum
3
activity over its phenethylamine counterpart 6f (80.4% versus 58.6%), with an EC50 in the
sub-micromolar range (0.56 μM) (Tables 2 and 3 and Figure 2A). However, a derivative substituted at
both meta and para positions with OCH
3
moieties (9d) exhibited less favorable activity than 9c (Table
3
). We also synthesized the 2-(hydroxymethyl)pyrrolidine counterparts of both 9c and 9d, and
observed that 9c and 9d showed more favorable activities than their 2-(hydroxymethyl)pyrrolidine
derivatives in maximum activity (80.4% vs 57.2% and 69.4% vs 44.9%, respectively) (Table 3).
Overall, all the SAR studies established that 9c is more potent β cell-protective compound against ER
stress in this series. Compared to RH01687 (a compound known to be β cell-protective against ER
stress, as shown in Figure 2B, 9c exhibited similar maximum activity to that of RH01687 but at
significantly lower concentration (5 M for 9c vs. 30 M for RH01687).
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To explore whether the chirality of 9c influenced its potency, the R (R-9c) and S (S-9c) isomers of
compound 9c were synthesized. As shown in Figure 2C, both the R-9c, and S-9c compounds exhibited
similar EC50s and maximum activities in increasing the viability of INS-1 cells after Tm treatment. We
therefore chose racemic 9c for further characterization of its mechanism of action in promoting β cell
survival and function against ER stress.
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Table 3. Activity of benzylamine derivatives on the survival of INS-1 cells treated with Tm.
Maximum
Compd
R
n
EC50 (μM)
activity
57.2%
44.9%
56.4%
54.7%
80.4%
69.4%
8
c
4-OMe
1
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2
4.05 ± 0.13
1.85 ± 0.39
0.23 ± 0.07
0.21 ± 0.08
0.56 ± 0.18
3.64 ± 0.38
8d
3,4-diOMe
H
9
a
9
b
4-Cl
9c
4-OMe
3,4-diOMe
9
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Figure 2. Protective effects of 9c and its chiral forms on the viability of INS-1 cells after Tm treatment.
(A) INS-1 cells were treated with or without Tm (0.1 μg/mL) in the presence of 9c at the indicated
concentrations or DMSO for 72 h. (B) INS-1 cells were treated with 9c (5 μM), RH01687 (30 μM), or
DMSO in the presence of Tm (0.1 μg/mL), or with DMSO in the absence of Tm, for 72 h. 5 μM of 9c
and 30 μM of RH01687 were used as each at its chosen concentration respectively exhibited the
maximum protective activity in INS-1 cells based on dose-dependent curves. (C) INS-1 cells were
treated with or without Tm (0.1 μg/mL) in the presence of compounds (5 μM) or DMSO for 72 h. The
cell viability was determined using the CellTiter-Glo assay, and was normalized as 1 for DMSO alone
(in all figures unless specified). The results are the means of 3 replicate wells and are representative of
3
independent experiments. Bars indicate SD. ** P < 0.01 compared with Tm alone. DMSO
concentration in all conditions (in all figures unless specified) is 0.1%.
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Figure 3. Compound 9c increases the viability of β cells against ER stress. (A) INS-1 cells were
treated with or without Tm (0.1 μg/mL) in the presence of 9c or DMSO for 72 h. The cell viability was
determined using MTT. (B, C) βTC6 cells were treated with or without Tm (0.25 μg/mL) in the
presence of 9c or DMSO for 72 h. The cell viability was determined using CellTiter-Glo assay (B) or
MTT (C). (D) INS-1 cells were treated with or without BFA (0.2 μg/mL) in the presence of 9c (20 μM)
or DMSO for 72 h. The cell viability was determined by CellTiter-Glo. The results in all panels are the
means of 3 replicate wells and are representative of 3 independent experiments. * P < 0.05 and ** P <
0
.01 compared with Tm or BFA treated alone. Bars indicate SD.
In the above SAR studies, we used the intracellular ATP level measured by the CellTiter-glo assay as a
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surrogate for cell viability. To rule out the possibility of an ATP-specific (rather than viability) effect of
the compounds, we used the MTT assay, which measures the activity of NAD(P)H-dependent cellular
oxidoreductase enzymes, as an orthogonal method to measure cell viability. As shown in Figure 3A,
Tm treatment reduced the MTT reading in INS-1 cells compared to that of DMSO treatment, and
co-treatment with 9c resulted in an increase in viability of INS-1 cells. To confirm that the protective
effect of 9c on  cells is not INS-1 cell-specific, another β cell line, TC6, was used. As expected, Tm
induced a reduction in viability in βTC6 cells, and co-treatment with 9c rescued the viability of βTC6
cells in a dose-dependent manner, as assessed by both intracellular ATP level and activity of
NAD(P)H-dependent cellular oxidoreductase enzymes (Figures 3B and 3C). To determine whether the
protective effect of 9c on  cells is specific to Tm-induced stress, we used another ER stressor, BFA,
which inhibits a key guanine nucleotide exchange factor essential for the transport of proteins from the
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ER to the Golgi, to treat INS-1 cells. Indeed, compound 9c also protected INS-1 cells against BFA
3
4
(Figure 3D). All these results indicate that compound 9c protects β cell survival against ER stress.
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Figure 4. Compound 9c protects INS-1 cells against Tm-induced apoptosis. (A) INS-1 cells were
treated with or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for the indicated
times. Cleaved caspase-3 and PARP were determined by Western blotting. α-Tubulin was used as a
loading control. The data shown are representative of 3 independent experiments. (B) INS-1 cells were
treated with or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for 24 h, and live-cell
phase-contrast images were acquired (magnification 10×)
To determine whether the increase in cell viability following treatment with 9c was caused by a
suppression of apoptotic cell death, levels of cleaved caspase-3 and cleaved PARP were assessed by
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Western blotting. Caspase-3, a member of executioner caspases, plays essential roles in initiating
apoptotic signaling and executing the final stages of cell death as it is responsible for the proteolytic
cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP)
which is a family of proteins involved 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 caspase-3 and
PARP is an indication of apoptosis.35, 36 Tm treatment for 24 h significantly induced both cleaved
caspase-3 and cleaved PARP protein levels in INS-1 cells (Figure 4A). However, 9c co-treatment
significantly reversed Tm-induced cleavage of both caspase-3 and PARP (Figure 4A). These results
demonstrate that 9c inhibits Tm-induced activation of caspase 3 and apoptosis in INS-1 cells.
Consistent with this, significantly more viable cells were observed with Tm and 9c co-treatment than
with Tm alone (Figure 4B).
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ER stress also impairs the most important function of β cells: the biosynthesis and secretion of insulin.
Multiple steps of insulin synthesis and secretion are impaired under ER stress; they include insulin
gene transcription, insulin mRNA stability, protein translation, and the insulin protein secretory
process.6, 7, 37, 38 First, we examined whether compound 9c could rescue Tm-suppressed mRNA levels
of insulin genes. As expected, Tm treatment of INS-1 cells decreased the mRNA levels of both insulin
genes, INS1 and INS2, but this reduction was completely rescued by 9c (Figure 5A and B). Second,
we examined whether compound 9c affects the expression of β cell transcription factors PDX1 and
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MafA, which control β cell identity and the expression of insulin genes.39, 40 Chronic exposure to
supraphysiologic concentrations of glucose and its associated ER stress cause the down-regulation of
expression of PDX1 and MafA.41-45 Consistent with this notion, Tm decreased the levels of PDX1 and
MafA mRNA expression levels in INS-1 cells. Co-treatment with 9c almost completely reversed this
decrease to normal levels (Figure 5C and D). Next, we explored whether compound 9c re-establishes
Tm-impaired glucose-stimulated insulin secretion (GSIS). As shown in Figure 5E, Tm treatment
abolished the insulin secretion caused by high concentration of glucose treatment (25 mM) in INS-1
cells. Addition of 9c significantly rescued the GSIS in Tm-treated cells. Taken together, these data
demonstrate that 9c restores ER stress-impaired  cell survival and function.
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Figure 5. Compound 9c reverses Tm-suppressed  cell function. (A-D) INS-1 cells were treated with
or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for 24 h. The mRNA levels of
INS1 (A), INS2 (B), PDX1 (C), and MafA (D), were analyzed by qRT-PCR. The results are the means
of 3 replicate wells and are representative of 3 independent experiments. * P < 0.05 and ** P < 0.01.
Bars indicate SD. (E) Insulin secretion by INS-1 cells incubated with 2.5 mM and 25 mM glucose in
the presence of Tm (0.1 μg/mL) and 9c. Secreted insulin was measured by ELISA after 24 h treatment.
* P < 0.05. The amount of insulin secreted in response to 2.5 mM glucose in the absence of Tm was set
to 1.0.
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Figure 6. Compound 9c decreases XBP1s mRNA levels induced by Tm. INS-1 cells were treated with
or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for the indicated times. (A)
XBP1s mRNA levels were analyzed by qRT-PCR. The results are expressed as the fold-increase over
mRNA levels in untreated control cells and are the means of 3 replicate wells and representative of 3
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independent experiments. * P < 0.05 and ** P < 0.01. Bars indicate SD. (B) XBP1 mRNA levels were
analyzed by RT-PCR and the products were resolved by agarose gel electrophoresis. The full-length
(unspliced, XBP1u) and spliced (XBP1s) forms of XBP1 mRNA are indicated. Cyclophilin A mRNA
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was used as an internal control. The data shown are representative of 3 independent experiments.
Next, we investigated the mechanism by which 9c protects β cells against ER stress. In response to ER
stress, all three branches of the UPR, IRE1, PERK and ATF6, are activated to either restore cellular
homeostasis/survival or lead to cell death, depending on the severity of ER stress. First, we asked
whether 9c affects the activation of IRE1α in β cells under ER stress. Activated IRE1α cleaves X-box
binding protein-1 (XBP1) mRNA to generate a spliced form (XBP1s) that is translated into a potent
transcription factor which controls expression of UPR genes encoding factors involved in ER protein
folding and degradation.7, 12, 46 We therefore determined the effect of 9c on IRE1α-mediated XBP1
splicing in INS-1 cells in the presence of Tm. As shown in Figure 6A, INS-1 cells treated with Tm
exhibited an increase in XBP1s mRNA, and this increase was suppressed by 9c co-treatment, as
measured by qRT-PCT using XBP1 splicing-specific primers. Likewise, electrophoretic separation of
spliced and unspliced forms of XBP1 after RT-PCR amplification of total XBP1 mRNA revealed that
9
c inhibits the Tm-induced generation of XBP1s mRNA (Figure 6B). These results indicate that 9c
inhibits the activation of the IRE1–XBP1 pathway of the UPR.
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Figure 7. Compound 9c inhibits Tm-induced Bip and GRP94 expression in INS-1 cells. INS-1 cells
were treated with or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for the indicated
times. Bip (A) and GRP94 (B) mRNA levels were analyzed by qRT-PCR. The results are expressed as
the fold-increase over mRNA levels in untreated control cells and are the means of 3 replicate wells
and representative of 3 independent experiments. * P < 0.05 and ** P < 0.01.
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Second, we asked whether 9c affects the activation of ATF6 in β cells under ER stress. Activated ATF6
acts as a homodimer or as an ATF6–XBP1s heterodimer to control the up-regulation of select UPR
target genes including the chaperone proteins BiP and GRP94.12, 46 We evaluated the effect of 9c on the
mRNA levels of chaperones Bip and GRP94 in β cells in the presence of Tm. As expected, we found
that both Bip and GRP94 mRNAs were up-regulated in INS-1 cells treated with Tm. These increases
were almost completely suppressed by 9c co-treatment (Figure 7A and B).
We then determined the effect of 9c on the activation of the PERK pathway in β cells under ER stress.
Activated PERK phosphorylates eukaryotic translation initiator factor 2α (eIF2α), which in turn
attenuates general protein synthesis to relieve ER load. EIF2α phosphorylation also allows the
selective translation of ATF4 mRNA, which encodes a transcription factor that induces the expression
of the pro-apoptotic gene C/EBP-homologous protein (CHOP).6, 8, 9, 46 Thus, we used ATF4 and CHOP
expression levels as markers of PERK pathway activation. Tm treatment of INS-1 cells significantly
increased the mRNA levels of both ATF4 and CHOP, whereas co-treatment with 9c resulted in a
decrease in both their levels (Figure 8A and B). Tm treatment also increased the ATF4 protein level
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with the peak time at 8h and CHOP protein level starting at 8 h (Figure 8C). Consistent with its effects
on ATF4 and CHOP mRNA transcription, 9c co-treatment also decreased ATF4 and CHOP protein
levels in Tm-treated INS-1 cells (Figure 8C). Together, our results that 9c inhibited the ER
stress-mediated activation of all three UPR pathways indicate that 9c protects β cell survival by
alleviating ER stress.
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Figure 8. Compound 9c inhibits Tm-induced ATF4 and CHOP up-regulation in INS-1 cells. (A, B)
INS-1 cells were treated with or without Tm (0.1 μg/mL) in the presence of 9c (20 μM) or DMSO for
the indicated times. ATF4 (A) and CHOP (B) mRNA levels were analyzed by qRT-PCR. The results
are expressed as the fold-increase over mRNA levels in untreated control cells and are the means of 3
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replicate wells and representative of 3 independent experiments. * P < 0.05 and ** P < 0.01. Bar
indicates SD. (C) INS-1 cells were treated with or without Tm (0.1 μg/mL) in the presence of 9c (20
μM) or DMSO for the indicated times. ATF4 and CHOP protein levels were determined by Western
bloting. α-Tubulin was used as a loading control. The data shown are representative of 3 independent
experiments.
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Finally, we investigated whether 9c exhibits similar protective effects on primary human islet β cells to
exclude the possibility that our findings are merely unique to rodent β cells. Rodent models of
mammalian β cell biology have been invaluable tools for the understanding of β cell physiology and
diabetes pathogenesis; however, human and rodent β cells differ in fundamental ways. For example,
the pancreatic islet architecture is markedly different between humans and rodents. In rodents, the
islets are more organized, comprising a large core of β cells (representing ~80% of islet cells)
enveloped by a layer of  cells and other endocrine cells. However, in humans, β cells account for only
4
7
~
50% of islet cells and are scattered throughout the islet. Rodent β cells also show significant
47
regenerative capacity that human β cells lack. Moreover, among hundreds of manipulations reported
to prevent or cure T1D in the NOD diabetic mouse model, very few demonstrate a limited efficacy in
T1D patients.48, 49 Therefore, agents that function well in rodent systems need to be confirmed in
human systems. We utilized primary human islets to evaluate whether 9c protects  cell survival and
function impaired by ER stress. Another advantage of human islets is that they are primary cells and,
therefore, share more authentic properties with β cells than immortalized cell lines.
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First, we determined whether 9c suppresses the apoptosis of primary human β cells induced by Tm
treatment as assessed by TUNEL, which detects fragmentation of DNA, a marker of apoptotic cell
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death,. As expected, Tm treatment markedly increased TUNEL staining in insulin cells (15% TUNEL
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insulin cells compared to 2.5% with DMSO) (Figure 9A and B). Compound 9c co-treatment
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significantly decreased the percentage of TUNEL insulin cells to 3.5% (Figure 9A and B). Next, we
investigated whether 9c restored the Tm-induced insulin secretion defect. Tm treatment markedly
diminished the high glucose-stimulated increase in insulin secretion, but the addition of 9c
significantly reversed this effect and restored the Tm-impaired GSIS (Figure 9C). These data indicate
that 9c is equally effective for the protection of human  cells as for rodent  cells.
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Figure 9. Compound 9c protects human β cells against Tm-induced ER stress. (A) TUNEL staining of
human β cells. Primary human islets were treated with 1 μg/mL of Tm with or without compound 9c
(10 μM) for 48 h before TUNEL staining. Anti-insulin antibody was used to mark insulin-positive β
cells, and DAPI was used as a nuclear marker. Magnification is 40×. (B) Quantification of TUNEL
staining from 10 fields of images. ** P < 0.01 compared with Tm treatment alone. (C) Insulin
secretion by human islets (50 of equal size) incubated with 2.5 mM or 20 mM glucose in the presence
of Tm (1 μg/mL) and 9c. Secreted insulin was measured by ELISA after 48 h treatment. For A and B,
the values have been normalized to total cellular protein. The baseline insulin secretion at 2.5 mM
glucose was normalized as 1. ** P < 0.01.
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Figure 10. Proposed model of signaling pathways involved in 9c-mediated β cell-protective effects
against ER stress. ER stress induces activation of three branches of UPR (PERK, IRE1, and ATF6),
leading to up-regulation of ATF4, XBP1s, Bip, GRP4, CHOP and eventual activation caspase 3 and
cell apoptosis, while diminishing the expression of PDX1, MAFA and insulin genes, leading to β cell
dysfunction. Compound 9c protects β cell against ER stress-mediated dysfunction and death by
down-regulating ATF4, XBP1s, Bip, GRP94, CHOP, and caspase 3 and up-regulating PDX1, MAFA
and insulin genes.
Conclusion
In summary, we discovered and optimized 2,4-diaminoquinazoline analogs as a novel class of agents
that suppress ER stress-induced β cell death and dysfunction. The 2-(hydroxymethyl)piperidin-1-yl-
containing analogues showed improved potency and maximum activity over their
2
-(hydroxymethyl)pyrrolidin-1-yl counterparts, of which the 4-OCH - and benzylamine-containing 9c
3
exhibited optimal β cell-protective activity. In addition, we have demonstrated that 9c alleviates ER
stress/UPR response by inhibiting Tm-induced up-regulation of key genes involved in the unfolded
protein response and apoptosis. Figure 10 proposes a model of signaling events leading to the
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protective effect of 9c in β cells against ER stress-induced dysfunction and cell death. Importantly, the
c effects on rodent β cells are translatable to human cells as 9c also shows potent protective activities
9
on primary human islet β cells, a prerequisite for potential drug development for human diabetes.
Studies to improve the pharmacokinetic properties of 9c are ongoing as part of our program to develop
novel chemotypes as a β cell-protective leads for further anti-diabetic drug development.
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EXPERIMENTAL SECTION
1
. Cell culture
INS-1 cells were cultured in RPMI 1640 (Corning, NY, USA) supplemented with 10% FBS (Atlanta
Biologicals, Norcross, GA), HEPES (10 mM, Life Technologies, CA, USA), sodium pyruvate (1 mM,
Corning), 2-mercaptoethanol (50 μM, Sigma, St Louis, MO, USA) and antibiotics (100 UI/mL
penicillin and 100 μg/mL streptomycin, Corning). βTC6 cells were cultured in DMEM (Corning) with
15% FBS, sodium pyruvate (1 mM, Corning), non-essential amino acids (1 mM, Thermo, IL, USA),
GlutaMAX (1 mM, Life Technologies) and antibiotics (100 UI/mL penicillin and 100 μg/mL
streptomycin). Human islets were obtained from the Integrated Islet Distribution Program (Duarte, CA)
in accordance with Oklahoma Medical Research Foundation’s internal review board (IRB) and ethical
guidelines for the use of human tissue. Standard viability was 80-90% and purity was >80%. Islets
were maintained in CMRL medium (Life Technologies) supplemented with 10% FBS. All cells were
grown at 37 °C in a humidified 5% CO atmosphere.
2
2
. Cell survival assay
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INS-1 cells or βTC6 cells were seeded at 3 × 10 cells/well in a 384-well plate and treated with
compounds at the indicated concentrations. After 3 d treatment, the medium was aspirated and 20
μL/well of CellTiter-Glo reagent (Promega, WI, USA) was added. Cell viability was measured with an
EnVision multilabel plate reader (PerkinElmer, MA, USA).
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. RNA isolation and qRT-PCR
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INS-1 cells were seeded at 4 × 10 cells/well in 6-well plates and treated with compounds for the
indicated times. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according
to the manufacturer’s protocol, and 2 μg of total RNA was reverse transcribed using a Superscript kit
(Invitrogen). Real-time PCR was performed in 96-well format using SYBR Select Master Mix
(
Applied Biosystems, Foster City, CA) with an ABI 7500 PCR system (Applied Biosystems). The
primer sequences used were: Rat CHOP: F, 5’-GAAATCGAGCGCCTGACCAG-3’ and R,
5’-GGAGGTGATGCCAACAGTTCA-3’. Rat ATF4: F, 5’- TCCTGAACAGCGAAGTGTTG-3’ and R,
5
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’- GTGTCTGAGGCACTGACCAA-3’. Rat Bip: F, 5’-CTATTCCTGCGTCGGTGTATT-3’ and R,
’-GGTTGGACGTGAGTTGGTTCT-3’. Rat GRP94: F, 5’- TCCCCCTTAATGTTTCCCGTG-3’ and
R, 5’-TAGCCCTTCTTCAGAAGCCTC-3’. Rat XBP1s: F, 5’- CTGAGTCCGAATCAGGTGCAG-3’
and R, 5’- ATCCATGGGAAGATGTTCTGG-3’. Rat XBP1 for regular PCR (XBP1u and XBP1s): F,
5
5
’-GCTTGTGATTGAGAACCAGG-3’, R, 5’-GAAAGGGAGGCTGGTAAGGAAC-3’. Rat Ins1: F,
’- GTCCTCTGGGAGCCCAAG-3’ and R, 5’- ACAGAGCCTCCACCAGG-3’. Rat Ins2: F, 5’-
ATCCTCTGGGAGCCCCGC-3’ and R, 5’- AGAGAGCTTCCACCAAG-3’. Rat PDX1: F,
5
’-GAGGACCCGTACAGCCTACA-3’ and R, 5’- CGTTGTCCCGCTACTACGTT-3’. Rat MafA: F,
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5’- AGCGGTCATATTTTCGCAAC-3’ and R, 5’- CTCTACAGGGAGCAGCGAAC-3’. Rat
Cyclophilin
A:
F,
5’-GGTGACTTCACACGCCATAA-3’
and
R,
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’-CTTCCCAAAGACCACATGCT-3’.
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. Western blotting
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INS-1 cells were seeded in 60-mm dishes at 8 × 10 cells/dish and treated for the indicated times. Cells
were then washed with PBS and lysed with lysis buffer (Cell Signaling Technology, Danvers, MA)
containing EDTA (Thermo, IL) and phosphatase inhibitors (Thermo). Aliquots of 20 μg total protein
were separated on 7% SDS-PAGE gels (Life Technologies) and transferred to PVDF membranes (Life
Technologies). The membranes were probed with primary antibodies followed by the appropriate
HRP-conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG, 1:3000; Santa
Cruz Biotechnology, CA, USA). Blots were then developed. The primary antibodies and dilutions used
were: CHOP (1:1000, MA1-250, Thermo), ATF4 (1:1000, 10835-1-AP, ProteinTech Group, IL, USA),
cleaved caspase 3 (1:1000, 9661, Cell Signaling Technology, MA, USA), PARP (1:1000, 9542L, Cell
Signaling Technology), and α-tubulin (1:3000, SC-8035, Santa Cruz Biotechnology).
5
. MTT assay
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INS-1 cells or βTC6 cells were seeded at 3 × 10 cells/well in a 384-well plate and treated with
compounds at the indicated concentrations. After 3 d treatment, the medium was aspirated and 10 μl of
MTT reagent (Cayman Chemical, MI, USA, prepared according to manufacturer’s instruction) to each
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