Small-molecule inducer of β cell proliferation identified by high-throughput screening

Article abstract of DOI:10.1021/ja309304m

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to ident

Full text of DOI:10.1021/ja309304m

Communication
pubs.acs.org/JACS
Small-Molecule Inducer of β Cell Proliferation Identified by High-
Throughput Screening
Weijun Shen,†,‡,§,⊥ Matthew S. Tremblay,†,‡,§,⊥ Vishal A. Deshmukh,† Weidong Wang,†
Christophe M. Filippi,‡ George Harb,‡ You-qing Zhang,‡ Anwesh Kamireddy,‡ Janine E Baaten,‡
Qihui Jin,‡ Tom Wu,‡ Jonathan G. Swoboda,† Charles Y. Cho,‡ Jing Li,‡ Bryan A. Laffitte,‡
Peter McNamara,‡ Richard Glynne,*,‡ Xu Wu,‡,∥ Ann E. Herman,‡,# and Peter G. Schultz*,†,§
†Department of Chemistry and the Skaggs Institute for Molecular Biology, 10550 North Torrey Pines Road, The Scripps Research
Institute, La Jolla, California 92037, United States
‡The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United
States
§California Institute for Biomedical Research, 11119 North Torrey Pines Road, Suite 100, La Jolla, California 92037, United States
S
* Supporting Information
growth-arrested β cell line. Further screening efforts have
ABSTRACT: The identification of factors that promote β
cell proliferation could ultimately move type 1 diabetes
treatment away from insulin injection therapy and toward
a cure. We have performed high-throughput, cell-based
screens using rodent β cell lines to identify molecules that
induce proliferation of β cells. Herein we report the
discovery and characterization of WS6, a novel small
molecule that promotes β cell proliferation in rodent and
human primary islets. In the RIP-DTA mouse model of β
cell ablation, WS6 normalized blood glucose and induced
concomitant increases in β cell proliferation and β cell
number. Affinity pulldown and kinase profiling studies
implicate Erb3 binding protein-1 and the IκB kinase
pathway in the mechanism of action of WS6.
identified a number of additional chemotypes capable of
inducing proliferation of R7T1 cells, including the diarylurea
WS111,12 (Figure 1A). Preliminary analogue synthesis (see
β cells are a subset of cells in the pancreatic islets, which
themselves comprise only a few percent by mass of the
pancreas. Despite their small numbers, β cells play a dominant
role in metabolism by regulating glucose homeostasis through
the production of insulin. In type 1 diabetes (T1D), a
combination of genetic and environmental factors results in
the selective targeting of β cells by the immune system,
ultimately leading to loss of functional β cell mass.1,2 The
current standard of care for T1D involves stringent patient self-
monitoring to maintain glucose homeostasis via insulin
injection.3 A more robust therapy can be envisioned that
addresses β cell dysfunction directly by promoting regeneration
of β cell mass. Small molecules,4−6 growth factors,7 and ectopic
expression of transcription factors8 and cell cycle genes9,10 have
been described that are capable of inducing β cell proliferation,
but robust, pharmacological proliferation of human islets
remains elusive. Herein we report the identification of WS6, a
small molecule capable of inducing proliferation of primary
rodent and human islets in vitro and mouse β cells in vivo, and
correcting hyperglycemia in the RIP-DTA mouse model of β
cell ablation.
Figure 1. Diarylurea compounds induce proliferation of the mouse β
cell line R7T1. (A) Chemical structures of diarylurea compounds
WS1, WS3, WS6, and WS9. (B) Dose-dependent effects of WS6 and
analogues on R7T1 β cell proliferation. (C) Expanded R7T1 cell
clusters after 5 days of WS6 treatment (bright field image).
detailed synthetic scheme and procedures in the Supporting
Information) yielded diarylurea WS3 and diarylamide WS6,
which induced R7T1 proliferation in dose response, with EC50
values of 0.074 μM and 0.28 μM, respectively (Figure 1B).
Proliferation of R7T1 cells, which are cultured in suspension
and grow as clusters, was apparent by visible inspection (Figure
1C).
Unlike the previously reported molecule, BAY K8644, which
displayed moderate activity in primary islets, initial experiments
suggested that WS6 was robustly active in primary rodent and
In previous studies, we described a high-throughput screen
for the identification of proliferative small molecules using a
Received: September 19, 2012
Published: January 19, 2013
© 2013 American Chemical Society
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Communication
human islets. In order to quantitatively characterize WS6’s
activity in primary cells, we sought to develop a high-
throughput islet proliferation assay. However, intact islets
clusters are not amenable to traditional automated manipu-
lation because of their large and heterogeneous size distribution
(50−300 μm). To overcome this, we developed a dispersed
islet proliferation assay, wherein intact islets are dissociated
with trypsin to yield a single-cell suspension, plated in
monolayer on poly-D-lysine-coated 384-well plates in standard
islet medium (10% FBS, 5.5 mM glucose RPMI), and then
assayed by automated high-content imaging for EdU
incorporation into insulin-positive cells. The addition of the
antibiotic Geneticin helps to suppress fibroblast outgrowth of
the dispersed human islet cultures, whereas this is not required
for the analogous rat islet culture.13 In this assay format,
proliferation of both rat and human islets is induced by
overexpression of cell cycle activators CDK7, cyclin D1, and
cyclin H9 (Figure S1). Encouragingly, WS6 induced up to 4%
of rat β cells to proliferate, with an EC50 of 0.4 μM (Figure
2A). In the same format, WS6 also induced 3% of human β
Images representative of those used for quantification are
shown in Figure 3B (rat) and Figure 3D (human).
Figure 3. WS6 causes proliferation of primary rat and human β cells in
intact islet format. (A) WS6 induces primary rat β cell proliferation in
dose response after 4 day treatment in standard islet media. (B)
Representative images of proliferating rat β cells with WS6 treatment.
(C) WS6 proliferates human primary β cells in dose response after 4
day treatment. (D) Representative images of proliferating human β
cells treated with WS6.
Next, we set out to evaluate the effect of the compound on β
cell proliferation and hyperglycemia in a rodent model of T1D.
RIP-DTA mice are double transgenic mice that express both
the reverse tetracycline transactivator (rtTA) under control of
the rat insulin promoter (RIP) and diphtheria toxin A (DTA)
under control of a tetracycline-sensitive operon.14 Treatment of
these mice with doxycycline (Dox) causes selective expression
of DTA in their pancreatic β cells, which leads to β cell ablation
and onset of hyperglycemia. RIP-DTA mice were fed Dox in
the drinking water until the onset of overt diabetes (blood
glucose reading >300 mg/dL, typically 4−10 days), at which
point Dox treatment was discontinued and treatment with WS6
was initiated (5 mg/kg every other day via intraperitoneal
injection). Pharmacokinetic studies with WS6 at 50 mg/kg
revealed a CMAX of ∼5 μM and T1/2 of ∼2 h (Figure S4); the
dose used in the RIP-DTA study was lowered to 5 mg/kg for
optimal tolerability during the 45 day chronic treatment.
Treatment with WS6 caused a progressive reduction of blood
glucose over time, starting around 2 weeks (Figure 4A). The
reduction in glucose occurred with a concomitant increase in β
cell proliferation from 3.8% to 6.4%, as assessed by Ki-67/
insulin costaining of pancreatic sections in mice sacrificed at the
end of week 1, when we hypothesize the proliferation rate
increase relevant to blood glucose lowering could be optimally
measured (Figure 4B, left panel). At the termination of the
study, we observed ∼50% increase in β cell mass in WS6-
treated mice (Figure 4B, right panel).
Figure 2. WS6 induces primary β cell proliferation in dissociated
format. (A) WS6 causes proliferation of rat dissociated islets in dose
response. (B) Representative images of proliferating rat β cells. (C)
WS6 induces human β cell proliferation in dose response. (D)
Representative images of proliferating human β cells in dissociated
format.
cells to proliferate, with a similar potency to the rat β cells
(Figure 2C). For illustration purposes, high magnification
confocal microscope images show the proliferating rat (Figure
2B) and human (Figure 2D) β cells in dissociated format.
We also tested the activity of WS6 in intact islet cultures.10
Islets were cultured with WS6 for 4 days in standard islet
media, fixed and embedded in paraffin, and serial sections were
cut and stained for insulin and Ki67, a marker of proliferation
present during the G1, S, G2, and M phases of cell cycle. β cell
proliferation was quantified by counting the percentage of
insulin-positive cells that colocalize with Ki67. Encouragingly,
WS6 induced significant proliferation compared to controls in
both rat (Figure 3A) and human (Figure 3C) intact islets.
In order to understand the mechanism of action of WS6, a
biotin-conjugated analogue (WS9) was synthesized for affinity-
based target identification experiments. WS9 proliferates R7T1
β cells 3-fold with an EC50 of 0.66 μM, comparable to WS6 but
with somewhat reduced maximal efficacy (Figure 1B). To
identify putative binding partners of WS6, affinity chromatog-
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IKKε plays a role in regulating the NFκB pathway, which is
involved in the proinflammatory stress response. To confirm a
role for these proteins in the β cell proliferation activity of WS6,
we analyzed the effects of blocking their activity either
pharmacologically or with shRNAs. Multiple candidate shRNAs
(Sigma) for knockdown of EBP1 in R7T1 cells were generated
(Figure 5B); the best construct, shRNA-92, induced significant
proliferation of R7T1 cells (Figure 5C). In contrast, the IKK
inhibitor IKK-16 (Tocris) alone had little effect on R7T1
proliferation. To evaluate a possible synergistic effect of EBP1
and IKK, we assayed IKK-16 in combination with shRNA-92
and saw that this combination fully recapitulated the effect of
WS6 on R7T1 proliferation (Figure 5C), suggesting both
pathways contribute to the activity of WS6. In the presence of
EBP1 shRNA, WS6 and IKK-16 cotreatment had similar effects
on proliferation. Overexpression of EBP1 in R7T1 cells reduced
the ability of WS6 to induce proliferation (Figure 5D),
implicating a direct involvement of EBP1 in the mechanism
of action of WS6. Given EBP1’s antiproliferative role in other
cell types via suppression of E2F-mediated cell cycle gene
expression,16 we examined expression of E2F, cyclin-D1 and
-D2, and EBP1 in R7T1 cells treated with WS6 (1 μM) for
various times (Figure 5E). E2F mRNA was induced 4.5-fold at
12 h, while cyclin-D1 was induced 3.5-fold at 8 h. Cyclin-D2
was modestly induced (1.8-fold) at 12 h, while changes in EBP1
expression were not statistically significant. We also observed a
2-fold increase in an E2F promoter-driven luciferase reporter
gene assay carried out in R7T1 cells (Figure S3). We tested the
possibility that WS6 invoked the GLP1R pathway, a known
regulator of cyclin-D expression and β cell proliferation,20 but
no cAMP response element activation was observed (Figure
S5).
Figure 4. WS6 ameliorates diabetes in RIP-DTA mice. (A) Fed blood
glucose levels (mean SEM) over time in mice treated with either 5
mg/kg i.p. WS6 (black squares) or water (gray circles). (B) Percentage
of Ki-67-expressing β cells (left) and β mass (right) (mean SEM) in
the pancreas of RIP-DTA mice after 1 and 6 weeks of treatment,
respectively, with either WS6 (black bars) or water (gray bars).
raphy was performed by incubating WS9 with R7T1 cell lysate
followed by streptavidin pulldown. Two-dimensional poly-
acrylamide gel electrophoresis and silver staining (Figure S5)
revealed several spots that were pulled down by WS9 (5 μM)
but were competed off by incubation with WS6 (500 μM).
Tandem mass spectrometry identified IKKε and EBP1 among
the proteins that were differentially bound to WS9. The identity
of these two proteins was further supported by Western
blotting of WS9-enriched lysate with anti-EBP1 and anti-IKKε
antibodies (Figure 5A).
Erb3 binding protein-1 (EBP1, also called PA2G4) is
classified as a tumor suppressor on the basis of its ability to
repress cell cycle regulated genes through binding to histone
deacetylase and retinoblastoma protein.15,16 The IκB kinase
In summary, we have identified a novel small molecule
capable of inducing proliferation of pancreatic β cells. WS6 is
among a few agents reported to cause proliferation of β cells in
vitro or in vivo.6,17,18 While the extensive medicinal chemistry
that would be required to improve the selectivity, efficacy, and
tolerability of WS6 is beyond the scope of this work, further
optimization of WS6 may lead to an agent capable of
promoting β cell regeneration that could ultimately be a key
component of combinatorial therapy for this complex disease.19
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed protocols for cell culture, in vitro β cell proliferation
assays in cell line and primary rat and human islets, affinity
pulldown, shRNA knockdown, and organic synthesis of WS3
and WS6. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Figure 5. WS6 target identification and validation. (A) Affinity pull-
down and WB confirmation of IKKε and EBP1 after enrichment of
R7T1 lysate with WS9 (5 μM); both were competed off in the
presence of excess WS6 (100×). (B) Western blot of EBP1
knockdown with shRNA 89−93 (EBP1 knockdown constructs) and
scrambled control lentivirus, and lentiviral EBP1 overexpression in
R7T1 cells. (C) EBP1 knockdown with shRNA 92 in R7T1 causes
proliferation, which is enhanced with either WS6 (1 μM) or IKK
inhibitor IKK-16 (1 μM) cotreatment. (D) EBP1 overexpression shifts
the potency of WS6 in R7T1 proliferation. (E) Transcriptional
changes induced by WS6 treatment (1 μM); for each target gene,
change relative to GAPDH was calculated and then normalized to
change induced by DMSO at each time point.
Corresponding Author
schultz@scripps.edu; rglynne@gnf.org
Present Addresses
∥Cutaneous Biology Research Center, Massachusetts General
Hospital, Cambridge, MA 02129
#Genentech, 1 DNA Way, San Francisco, CA 94080
Author Contributions
⊥W.S. and M.S.T. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors acknowledge the efforts of John Walker and Eric
Peters for experimental support. This work was supported by
the JDRF and the Novartis Research Foundation. M.S.T. and
J.G.S. were supported by postdoctoral fellowships from the
JDRF. The authors also thank Patricia Kilian and Richard Insel
of JDRF for their support and insight.
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