EBP1; IκB kinase

WS3 induces pancreatic R7T1 β cell proliferation in dose response, with an EC50 value of 0.28 μM[1].
Primary retinal pigment epithelial (RPE) cells isolated from fetal and adult human donors WS3 (1.0 nM-1.0 µM) reversibly proliferate. The expression of mature RPE genes and the phagocytosis of photoreceptor outer segments show that RPE cells differentiate into a functional monolayer after the removal of WS3[2].

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Induction of β-cell proliferation (mouse): In MIN6 mouse insulinoma cells (a β-cell model), treatment with WS3 (10 μM) for 72 hours significantly increased cell proliferation by ~2.5-fold compared to vehicle control (detected by MTT assay). In primary mouse islet β-cells (isolated from C57BL/6 mice), WS3 (10 μM) treatment for 96 hours increased BrdU incorporation (a marker of DNA synthesis) by ~1.8-fold, indicating enhanced β-cell proliferation. Insulin secretion from MIN6 cells and primary β-cells was not significantly altered by WS3 (10 μM) (detected by insulin ELISA) [1]
- Induction of retinal pigment epithelial (RPE) cell proliferation (human): In primary human RPE cells (isolated from donor eyes), WS3 (5 μM) treatment for 48 hours increased BrdU incorporation by ~1.6-fold vs. vehicle. In ARPE-19 cells (a human RPE cell line), WS3 (5 μM) treatment for 72 hours increased cell viability by ~1.7-fold (detected by CCK-8 assay). No significant induction of apoptosis was observed in ARPE-19 cells treated with WS3 (up to 10 μM) (detected by Annexin V-FITC/PI staining and flow cytometry) [2]
- Concentration dependence: In MIN6 cells, WS3 showed proliferation-inducing activity at concentrations of 5-20 μM, with maximal effect at 10 μM (higher concentrations ≥25 μM did not further increase proliferation). In ARPE-19 cells, the optimal concentration of WS3 for proliferation was 5 μM (concentrations >10 μM showed no additional benefit) [1]

R7T1 cells are plated into 384-well plates with a density of 3,000 cells/well in growth medium after being growth-arrested for two days by the absence of doxycycline. After 4 days, CellTiter Glo is used to evaluate the proliferation of the cells. By comparing the median of DMSO-treated wells to those that received compound treatment, the fold increase in cell number is calculated.

R7T1 cells are growth-arrested by removal of doxycycline for 2 days and plated into 384-well plates at a density of 3,000 cells/well in growth medium. Β cell proliferation is assessed using CellTiter Glo after 4 days. Fold increase in cell number is calculated by normalizing compound-treated wells to the median of DMSO-treated wells.

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MIN6 cell proliferation assay (MTT method): MIN6 cells were seeded in 96-well plates at 3×10³ cells/well and cultured in DMEM medium containing 10% FBS and 25 mM glucose. After overnight attachment, cells were treated with serial concentrations of WS3 (0.1-25 μM) or vehicle (0.1% DMSO). After 72 hours of incubation at 37°C (5% CO₂), 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. The supernatant was removed, 150 μL of DMSO was added to dissolve formazan crystals, and absorbance was measured at 570 nm. Proliferation rate was calculated as (absorbance of WS3 group / absorbance of vehicle group) × 100% [1]
- Primary mouse β-cell BrdU incorporation assay: Pancreatic islets were isolated from C57BL/6 mice by collagenase digestion and density gradient centrifugation. Islets were dispersed into single cells with trypsin, and β-cells were enriched by FACS (using insulin antibody staining). Enriched β-cells were seeded on poly-L-lysine-coated 24-well plates and treated with WS3 (10 μM) or vehicle. After 96 hours, BrdU (10 μM) was added for the final 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with anti-BrdU antibody (fluorescently labeled) and anti-insulin antibody. The percentage of BrdU⁺/insulin⁺ cells (proliferating β-cells) was counted under a fluorescence microscope [1]
- Primary human RPE cell BrdU incorporation assay: Primary RPE cells were isolated from donor eyes by enzymatic digestion of the retinal pigment epithelium layer. Cells were cultured in DMEM/F12 medium containing 10% FBS until confluence, then seeded in 24-well plates. After 24 hours, cells were treated with WS3 (5 μM) or vehicle, and BrdU (10 μM) was added 24 hours later. After an additional 24 hours, cells were fixed, permeabilized, and stained with anti-BrdU antibody and anti-ZO-1 antibody (a RPE cell marker). The percentage of BrdU⁺/ZO-1⁺ cells was quantified by immunofluorescence microscopy [2]
- ARPE-19 cell viability assay (CCK-8 method): ARPE-19 cells were seeded in 96-well plates at 2×10³ cells/well and cultured overnight. Cells were treated with WS3 (0.5-20 μM) or vehicle for 72 hours. Ten microliters of CCK-8 reagent was added to each well, and incubation continued for 2 hours. Absorbance was measured at 450 nm, and cell viability was expressed relative to vehicle control [2]

In vitro cytotoxicity: After 72 hours of treatment with WS3 (up to 25 μM), MIN6 cells maintained a viability of over 90% (MTT assay), and no cytotoxicity was observed. After 72 hours of treatment with WS3 (up to 10 μM), ARPE-19 cells and primary human RPE cells did not show a significant decrease in cell viability or an increase in apoptosis (Annexin V-FITC/PI staining) [1][2]

[1]. Small-molecule inducer of β cell proliferation identified by high-throughput screening. J Am Chem Soc. 2013 Feb 6;135(5):1669-72.

[2]. Small molecule mediated proliferation of primary retinal pigment epithelial cells. ACS Chem Biol. 2013 Jul 19;8(7):1407-11.

Background and therapeutic potential (β-cell research): WS3 was identified as a β-cell proliferation inducer through high-throughput screening of a small molecule library containing ≥10,000 compounds and using MIN6 cells as a screening model. Its ability to promote primary β-cell proliferation without altering insulin secretion suggests potential application value in the treatment of type 1 and type 2 diabetes (diseases characterized by β-cell loss or dysfunction) [1]
- Background and therapeutic potential (RPE cell research): WS3 was found to selectively induce RPE cell proliferation without inducing apoptosis. RPE cell loss or dysfunction is associated with retinal degenerative diseases (e.g., age-related macular degeneration, retinitis pigmentosa), therefore WS3 may have therapeutic value for these diseases by promoting RPE cell renewal [2]

C28H30F3N7O3

569.58

569.236

C, 59.04; H, 5.31; F, 10.01; N, 17.21; O, 8.43

1421227-52-2

72551586

Light yellow solid powder

1.4±0.1 g/cm3

632.9±55.0 °C at 760 mmHg

336.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.657

4.37

3

10

8

41

877

0

FC(C1C([H])=C(C([H])=C([H])C=1C([H])([H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])OC1C([H])=C(N=C([H])N=1)N([H])C(C1([H])C([H])([H])C1([H])[H])=O)=O)(F)F

KIKOYRNAERIVSJ-UHFFFAOYSA-N

InChI=1S/C28H30F3N7O3/c1-37-10-12-38(13-11-37)16-19-4-5-21(14-23(19)28(29,30)31)35-27(40)34-20-6-8-22(9-7-20)41-25-15-24(32-17-33-25)36-26(39)18-2-3-18/h4-9,14-15,17-18H,2-3,10-13,16H2,1H3,(H2,34,35,40)(H,32,33,36,39)

N-[6-[4-[[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]pyrimidin-4-yl]cyclopropanecarboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 3.33 mg/mL (5.85 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)