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Supplementary Information
The discovery and characterization of K-756, a novel Wnt/β-catenin pathway inhibitor
targeting tankyrase
Ryoko Okada-Iwasaki12, Yuichi Takahashi1, Yasuo Watanabe1, Hiroshi Ishida1, Jun-ichi Saito1,
Ryuichiro Nakai1, Akira Asai2
1
R&D Division, Kyowa Hakko Kirin Co., Ltd., Shizuoka, 411-8731, Japan, 2Center for Drug
Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka,
411-8526, Japan
Corresponding Author: Ryuichiro Nakai ([email protected]), R&D
Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Sunto-gun, Shizuoka, 411-8731,
Japan
Phone: +81-55-989-3289, FAX: +81-55-986-7430
Supplementary Figures and Legends 1-5
Supplementary Tables 1-2
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Supplementary Figure S1. Target gene knockdown and downstream gene inhibition by
CTNNB1 siRNAs. Cells were transfected with siRNA. After 48 hours, the cells were collected
for an RT-PCR, Western blotting and reporter assay. A, Target gene knockdown by CTNNB1
siRNA#1 and #2. B, Active β-catenin and whole β-catenin protein suppression by CTNNB1
siRNAs. C, CTNNB1 siRNAs inhibited the reporter activity in DLD-1/TCF-Luc cells but not
in DLD-1/mtTCF-Luc cells. D, The mRNA expression of the genes downstream of the
Wnt/β-catenin pathway after CTNNB1 siRNA transfection.
Supplementary Figure S2. A, K-756 did not inhibit the cell growth of DLD-1/TCF-Luc cells.
K-756 was added to the cells in 96-well plates. After 144 hours, cell growth inhibitory activity
was measured by an XTT assay. B, CTNNB1 knockdown by siRNAs in COLO 320DM and
SW403 cells. Cells were transfected with 5 nmol/L of siRNA. After 72 hours, the cells were
collected for an RT-PCR. CTNNB1 expression was normalized by GAPDH. C, The inactive
analogue K-050 did not inhibit the cell growth of COLO 320DM cells. K-050 was added to
the cells in 96-well plates. After 144 hours, the antiproliferative activity was measured by an
XTT assay.
Supplementary Figure S3. A, XAV939 and B, IWR-1 stabilized Axin 1 and 2 and decreased
active β-catenin in COLO 320DM and SW403 cells. COLO 320DM and SW403 cells were
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treated with XAV939 or IWR-1 in 10 cm-dishes. After 24 hours, the cells were collected for a
Western blot analysis. C, K-756 showed higher extent of Axin stabilization and active
β-catenin suppression than XAV939 in long term cultured COLO 320DM and SW403 cells.
The cells were treated with K-756 and XAV939 in 6-well plates. After 0, 72, 120 and 144
hours, cells were collected for a Western blot analysis. D, K-756 and IWR-1 tended to
increase G2/M phase population in COLO 320DM cells. The cells were treated with indicated
concentrations of K-756 and IWR-1 in 12-well plates. After 120 hours, the cells treated with
K-756 and IWR-1 were collected and fixed with 70% ethanol. As a positive control for G2/M
phase arrest, 1 μmol/L of CDK1/2 inhibitor, BMS-265246 (Selleckchem, Houston, TX) was
added to the cells and collected after 72 hours treatment. The cells were stained with PI by
PI/RNase staining buffer (BD bioscience, Franklin Lakes, NJ) and cell cycle was analyzed by
Tali Image-Based Cytometer (Invitrogen).
Supplementary Figure S4. A, PK data of K-756 in Balb/c mice. K-756 dissolved in 0.5%
MC400 was administered orally to Balb/c mice
at doses of 1,10 or 100 mg/kg. At 0.5, 1, 2,
4, 7, 10 (1 and 10 mg/kg p.o. mice) or 24 (100 mg/kg p.o. mice) hours after administration,
plasma was collected. Each point represents average plasma concentration of the compound
detected in two mice. The black line indicates the plasma concentration converted reporter
inhibition IC50 nmol/L of K-756 in DLD-1/TCF-Luc cells in vitro. B, K-756 inhibited the
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expression of LGR5 in DLD-1/TCF-Luc cells. The cells were treated with K-756 and
XAV939 in 12-well plates. After 24 hours, cells were collected for RT-PCR. K-756 inhibited
C, the FGF20, D, the LGR5 and E, the reporter activity in DLD-1/TCF-Luc cell xenograft
tumors after 1 or 2 days administration. Mice were subcutaneously implanted with
DLD-1/TCF-Luc cells. After 14 days, 0.5% MC 400 or K-756 was orally administered to the
mice once a day for 1 or 2 days. Twenty-four hours after the last administration, the tumors
were collected from the mice. RNA was extracted from the tumor; after mRNA extraction, an
RT-PCR was performed. After protein lysis, a reporter assay was performed. Each column
represents the mean + S.E. (n = 5); the asterisks indicate a statistically significant difference
in comparison to the vehicle treated group (*, p < 0.05; **, p < 0.005) in a one-way ANOVA
followed by a Dunnett’s test.
Supplementary Figure S5. A, K-756 stabilized Axin1 in PC-9 and NCI-H322 cells and
suppressed active β-catenin in PC-9 cells. The cells were treated with K-756 in 10-cm dishes.
After 24 hours, the cells were collected for a Western blot analysis. B, K-756 changed the
expression of Wnt/β-catenin downstream genes in NCI-H322 cells. Cells were seeded in
12-well plates. The next day, K-756 was added to the cells. After 24 hours, cells were
collected for RT-PCR.
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