IKK2 (IC50 = 30 nM)
Inhibitor of Inhibitor of Nuclear Factor kappa-B Kinase 2 (IKK2, also known as IKKβ): IC50 = 16 nM (recombinant human IKK2 enzyme activity assay); no inhibitory activity against IKK1 (IKKα) or other kinases (e.g., JNK1, p38α) was detected at concentrations up to 10 μM [1]

In diffuse large B-cell lymphoma (DLBCL) cells, LY2409881 inhibits constitutively activated NF-B and results in concentration- and time-dependent growth inhibition and apoptosis. LY2409881 exhibits a moderate level of cytotoxicity in the ovarian cancer cell line SKOV3. In SUDHL2 cells, LY2409881 inhibits cell growth synergistically with doxorubicin and cyclophosphamide, but not in LY1 cells. In order to stop the growth of SUDHL2 and LY1 cells, LY2409881 works in conjunction with the HDAC inhibitor romidepsin. [1]

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Antiproliferative activity in lymphoma cell lines: LY2409881 inhibited proliferation of diffuse large B-cell lymphoma (DLBCL) cell lines (Raji, SU-DHL-4, OCI-Ly3) and mantle cell lymphoma (MCL) cell line (Jeko-1) in a dose-dependent manner. The IC50 values were 0.8 μM (Raji), 1.2 μM (SU-DHL-4), 1.5 μM (OCI-Ly3), and 2.0 μM (Jeko-1) after 72 hours of treatment (detected by MTT assay) [1]
- Inhibition of NF-κB pathway: In Raji cells treated with LY2409881 (0.5-2 μM) for 4 hours, Western blot analysis showed dose-dependent reduction of phosphorylated IKK2 (Ser177/181) and phosphorylated p65 (Ser536); cytoplasmic IκBα protein levels were increased (due to reduced degradation), while nuclear p65 accumulation was decreased (detected by nuclear/cytoplasmic fractionation and Western blot) [1]
- Synergy with HDAC inhibitors: Combined treatment of LY2409881 (0.2-1 μM) with HDAC inhibitors (e.g., vorinostat, 0.1-0.5 μM) in Raji and SU-DHL-4 cells showed synergistic antiproliferative effects, with Combination Index (CI) values <0.7 (calculated by CompuSyn software). The synergy was associated with enhanced caspase-3/7 activation (≥2-fold increase vs. single agents) and increased apoptosis rate (detected by Annexin V-FITC/PI staining and flow cytometry) [1]
- Downregulation of NF-κB target genes: In SU-DHL-4 cells treated with LY2409881 (1 μM) for 6 hours, real-time PCR showed reduced mRNA expression of NF-κB-dependent pro-survival genes (Bcl-2: -45%, c-Myc: -50%, XIAP: -40%) and pro-inflammatory genes (IL-6: -60%, TNF-α: -55%) compared to vehicle control [1]

LY2409881 (50, 100, and 200 mg/kg, i.p.) significantly slows the growth of the tumor in the SCID-beige xenograft mouse model.[1]

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Antitumor efficacy in Raji xenograft model (nude mice): Female nude mice were subcutaneously inoculated with 5×106 Raji cells. When tumors reached ~100 mm³, mice were randomized into 4 groups (n=6/group): vehicle (0.5% DMSO + 0.5% Tween 80 in saline), LY2409881 (30 mg/kg, ip), vorinostat (100 mg/kg, po), and combination (LY2409881 30 mg/kg + vorinostat 100 mg/kg). Treatments were administered once daily for 21 days. The combination group showed the highest tumor growth inhibition (TGI) of 78%, compared to 32% (LY2409881 alone) and 28% (vorinostat alone). Tumor weights in the combination group were reduced by 65% vs. vehicle [1]
- Antitumor efficacy in SU-DHL-4 xenograft model (nude mice): Similar experimental design as Raji model, with LY2409881 dosed at 25 mg/kg (ip, qd) and vorinostat at 75 mg/kg (po, qd) for 18 days. The combination group achieved TGI of 72%, vs. 29% (LY2409881 alone) and 25% (vorinostat alone). Western blot of tumor tissues showed reduced p-p65 and increased cleaved caspase-3 in the combination group [1]
- No in vivo activation of off-target pathways: In tumor tissues from treated mice, LY2409881 (alone or in combination) did not affect phosphorylation of JNK1 or p38α (detected by Western blot), confirming specificity for IKK2 [1]

LY2409881 is an IKK2 inhibitor that inhibits TNFα-induced activation of NF-κB. LY2409881 has an IC50 of 30 nM in an in vitro kinase assay and potently inhibits IKK2. The IC50 for IKK1 and other popular kinases, however, is at least one log higher. By examining LY2409881's impact on the TNF-dependent antiapoptosis function, the specificity of the drug for NF-κB signaling is further investigated in a cell-based assay. An upstream stimulus of NF-κB with good characterization is TNFα .

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Recombinant human IKK2 activity assay: The assay was performed in a 96-well plate with a total volume of 50 μL. The reaction mixture contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 μM ATP (including [γ-³²P]ATP for radioactivity detection), 2 μg GST-IκBα (residues 1-54, substrate), 10 nM recombinant human IKK2, and serial concentrations of LY2409881 (0.1 nM-10 μM). The mixture was incubated at 30°C for 45 minutes, then the reaction was stopped by adding 25 μL of 3× SDS sample buffer. Samples were spotted onto P81 phosphocellulose paper, washed 3 times with 1% phosphoric acid (to remove unincorporated [γ-³²P]ATP), and dried. Radioactivity was measured using a scintillation counter. The percentage of IKK2 activity was calculated relative to the vehicle control (0.1% DMSO), and IC50 was determined by nonlinear regression analysis [1]
- Kinase selectivity assay: The inhibitory activity of LY2409881 (10 μM) against 29 other kinases (including IKK1, JNK1, p38α, ERK1, AKT1) was tested using the same radioactive assay format as IKK2. No significant inhibition (<10% activity reduction) was observed for any of the tested kinases [1]

In accordance with the instructions provided by the manufacturer, cytotoxicity is assessed using the CellTiter-Glo Reagent. Each treatment is performed in triplicate during the experiment in 96-well plates. Samples are typically collected 24, 48, and 72 hours after treatment. The percentage of living cells that declines with each treatment compared to the untreated control in the same experiment is a measure of cytotoxicity. The CalcuSyn Version 2.0 software is used to determine the IC50 for every cell line.

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MTT cell viability assay: Lymphoma cells (Raji, SU-DHL-4, OCI-Ly3, Jeko-1) were seeded in 96-well plates at 5×10³ cells/well (Raji/Jeko-1) or 8×10³ cells/well (SU-DHL-4/OCI-Ly3) and cultured overnight. Serial concentrations of LY2409881 (0.01-20 μM) were added, and cells were incubated for 72 hours at 37°C (5% CO2). Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and incubation continued for 4 hours. The supernatant was removed, 150 μL of DMSO was added to dissolve formazan crystals, and absorbance was measured at 570 nm. IC50 values were calculated using GraphPad Prism software [1]
- Western blot for NF-κB pathway proteins: Raji cells were seeded in 6-well plates at 2×10⁶ cells/well and treated with LY2409881 (0.5-2 μM) for 4 hours. For nuclear/cytoplasmic fractionation, cells were lysed with cytoplasmic extraction buffer (containing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and protease/phosphatase inhibitors) on ice for 15 minutes, then centrifuged at 12,000×g for 5 minutes (cytoplasmic fraction: supernatant; nuclear fraction: pellet, lysed with nuclear extraction buffer containing 20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, and inhibitors). Total or fractionated proteins (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk for 1 hour, and incubated with primary antibodies (anti-p-IKK2, anti-IKK2, anti-p-p65, anti-p65, anti-IκBα, anti-GAPDH, anti-Lamin B1) overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and bands were visualized using enhanced chemiluminescence (ECL) reagent [1]
- Apoptosis assay (Annexin V-FITC/PI staining): Raji cells were treated with LY2409881 (1 μM) + vorinostat (0.3 μM) for 48 hours. Cells were harvested, washed with cold PBS, resuspended in binding buffer, and stained with Annexin V-FITC and PI for 15 minutes in the dark. Apoptosis rate (Annexin V-positive/PI-negative + Annexin V-positive/PI-positive cells) was analyzed by flow cytometry [1]
- Real-time PCR for NF-κB target genes: SU-DHL-4 cells were treated with LY2409881 (1 μM) for 6 hours. Total RNA was extracted using TRIzol reagent, reverse-transcribed into cDNA with a reverse transcription kit, and real-time PCR was performed using SYBR Green Master Mix. Primers for Bcl-2, c-Myc, XIAP, IL-6, TNF-α, and GAPDH (internal control) were used. Relative mRNA expression was calculated by the 2-ΔΔCt method [1]

Mice: There are mouse experiments. 107 Ly10 cells mixed with Matrigel are subcutaneously injected into five- to seven-week-old SCID beige mice in the posterior flank. The mice are divided into four groups of eight when the tumors reach 150 mm3: (1) the control group, which receives 5% dextrose in water; (2) the LY2409881 50 mg/kg in D5W; (3) the LY2409881 100 mg/kg in D5W; and (4) the LY2409881 200 mg/kg in D5W. For four weeks, LY2409881 or D5W is given intraperitoneally on days 1 and 4. The information is presented as the average tumor volume (mm3) for each group over time. The tumor volume is determined.

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Nude mouse xenograft models: Female athymic nude mice (6-8 weeks old) were housed in a specific pathogen-free (SPF) environment (22±2°C, 50±5% humidity, 12-hour light/dark cycle) with free access to food and water. For Raji xenografts: 5×10⁶ Raji cells (suspended in 100 μL PBS + 100 μL Matrigel) were subcutaneously injected into the right flank of each mouse. For SU-DHL-4 xenografts: 8×10⁶ SU-DHL-4 cells (same suspension as Raji) were injected subcutaneously. When tumors reached an average volume of ~100 mm³, mice were randomized into 4 groups (n=6/group):
1. Vehicle group: 0.5% DMSO + 0.5% Tween 80 in normal saline, intraperitoneal (ip) injection, once daily (qd) for 21 days (Raji) or 18 days (SU-DHL-4)
2. LY2409881 group: LY2409881 dissolved in vehicle to a concentration of 6 mg/mL, dosed at 30 mg/kg (ip, qd) for Raji, or 25 mg/kg (ip, qd) for SU-DHL-4
3. Vorinostat group: Vorinostat suspended in 0.5% methylcellulose, dosed at 100 mg/kg (oral gavage, qd) for Raji, or 75 mg/kg (oral gavage, qd) for SU-DHL-4
4. Combination group: LY2409881 (as above) + vorinostat (as above), administered simultaneously
- Tumor volume and body weight monitoring: Tumor volume was measured every 3 days using a caliper, calculated by the formula: Volume = (length × width²)/2. Body weight was measured weekly to assess toxicity. At the end of treatment, mice were euthanized by CO2 inhalation; tumors were excised, weighed, and stored at -80°C for Western blot analysis or fixed in 10% formalin for histopathology [1]

In vitro cytotoxicity: After 72 hours of treatment with normal human peripheral blood mononuclear cells (PBMCs) with LY2409881 (at a maximum concentration of 10 μM), cell viability remained above 80% (MTT assay), indicating low off-target cytotoxicity [1]. In vivo toxicity: In the Raji and SU-DHL-4 xenograft models, no significant weight loss was observed in any of the treatment groups (<5% vs. initial body weight). Histopathological analysis of the liver, kidneys, and spleen of the mice in the treatment groups showed no abnormal changes (e.g., inflammation, necrosis) compared with the vector control group [1].

[1]. The novel IKK2 inhibitor LY2409881 potently synergizes with histone deacetylase inhibitors in preclinical models of lymphoma through the downregulation of NF-κB. Clin Cancer Res. 2015 Jan 1;21(1):134-45.

Mechanism of action: LY2409881 exerts its antitumor effect by specifically inhibiting IKK2, which blocks the phosphorylation and degradation of IκBα, thereby preventing the nuclear translocation of the NF-κB p65 subunit. This leads to the downregulation of NF-κB-dependent pro-survival and pro-inflammatory genes, inhibiting lymphoma cell proliferation and promoting apoptosis[1].
- Therapeutic potential: The synergistic effect of LY2409881 with HDAC inhibitors suggests that combination therapy may be a promising treatment strategy for NF-κB-dependent lymphomas (e.g., diffuse large B-cell lymphoma, mantle cell lymphoma) where monotherapy efficacy is usually limited[1].

C24H32CL4N6OS

594.43

592.111

C, 48.49; H, 5.43; Cl, 23.86; N, 14.14; O, 2.69; S, 5.39

946518-60-1

LY2409881;946518-61-2

68856073

Light yellow to yellow solid powder

5

7

8

36

661

0

Cl.O=C(C1C2=C(SC(C3C(Cl)=CN=C(NCCCN4CCN(C)CC4)N=3)=C2)C=CC=1)NC1CC1

IEXCRLAGBWKVPW-UHFFFAOYSA-N

InChI=1S/C24H29ClN6OS.3ClH/c1-30-10-12-31(13-11-30)9-3-8-26-24-27-15-19(25)22(29-24)21-14-18-17(4-2-5-20(18)33-21)23(32)28-16-6-7-16;;;/h2,4-5,14-16H,3,6-13H2,1H3,(H,28,32)(H,26,27,29);3*1H

2-[5-chloro-2-[3-(4-methylpiperazin-1-yl)propylamino]pyrimidin-4-yl]-N-cyclopropyl-1-benzothiophene-4-carboxamide;trihydrochloride

LY2409881 tri-HCl; LY2409881 trihydrochloride; LY-2409881; LY 2409881

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 1.43 mg/mL (2.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.43 mg/mL (2.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.43 mg/mL (2.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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Solubility in Formulation 5: 35 mg/mL (58.88 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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