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| 1mg |
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| ln Vitro |
The initial hit compound 1 inhibited IL-15-dependent proliferation of 32Dβ cells with an IC50 of 17.3 µM.
Optimization of the linker length led to compound 43 (n=7), which showed significantly improved anti-proliferative activity with an IC50 of 1.9 µM against RLI (an IL-15 superagonist) and 1.1 µM against IL-2 in 32Dβ cell proliferation assays. Further modifications yielded compound 76 (ethyl ester analog of 43), which exhibited potent inhibition of Stat5 phosphorylation (p-Stat5) in NK-92 cells stimulated by IL-15 (IC50 = 57 nM) and IL-2 (IC50 = 43 nM). Compound 76 also inhibited RLI- and IL-2-induced 32Dβ cell proliferation with an IC50 of 0.8 µM for both cytokines. Other potent analogs included phenoxy derivatives 80 and 82, with p-Stat5 inhibitory IC50 values around 40-55 nM for IL-15. The compounds showed direct binding to immobilized IL-15 (or RLI) and IL-2 in SPR assays, with Kd values mostly in the range of 10-20 µM for the best leads. No significant cytotoxicity was observed for the main compounds at tested concentrations (e.g., 11 µM) in 32Dβ cells using a cytotoxicity assay.[1] |
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| Enzyme Assay |
Surface Plasmon Resonance (SPR) Binding Assay: Recombinant IL-15, RLI (IL-15/IL-15Rα fusion), or IL-2 was covalently immobilized on a CM5 sensor chip via amine coupling. IL-15-IN-1 compounds at 100 µM or in a concentration series (prepared in PBS with 1% or 5% DMSO) were flowed over the chip surface at 25°C. Binding-induced changes in reflectance units (RU) were monitored. Dissociation constants (Kd) were determined by analyzing the sensorgrams using dedicated software.[1]
Homogeneous Time-Resolved Fluorescence (HTRF) Competition Assay: Biotinylated human IL-15 was pre-incubated with IL-15-IN-1 compounds for 30 minutes. Recombinant human IL-2Rβ was then added for another 30 minutes. Subsequently, a donor (anti-IL-2Rβ antibody conjugated to a Tb cryptate) and an acceptor (streptavidin conjugated to D2) were added. After a 30-minute incubation, fluorescence was measured at 620 nm (donor emission) and 665 nm (acceptor emission, FRET signal). Inhibitor binding to IL-15 reduces the FRET signal. Data were normalized using positive and negative controls to calculate the inhibitory effect.[1] |
| Cell Assay |
Cell Proliferation Assay (32Dβ cells): 32Dβ cells (expressing IL-2Rβ/γ) were starved for 4 hours. Cells (1x10^4 per well) were cultured for 2.5 days in medium containing a fixed concentration of RLI (100 pM) or IL-2 (1.5 nM), which had been pre-incubated for 30 minutes with IL-15-IN-1 compounds or vehicle (0.1% DMSO). Cell proliferation was assessed using the Alamar Blue reduction assay. Fluorescence (ex560/em590 nm) was measured after a 6-hour incubation with the dye. Dose-response curves were generated to calculate IC50 values.[1]
Phospho-Stat5 (p-Stat5) Assay (NK-92 cells): Exponentially growing NK-92 cells were serum-starved overnight. Cells (2x10^5 per condition) were stimulated for 1 hour at 37°C with a fixed concentration of IL-15 (50 pM) or IL-2 (250 pM) that had been pre-incubated for 30 minutes with IL-15-IN-1 compound or vehicle (0.6% DMSO). Cells were then lysed, and Stat5 phosphorylation levels were quantified using a commercial AlphaScreen SureFire kit according to the manufacturer's instructions. Dose-response curves were generated to calculate IC50 values.[1] Cytotoxicity Assay (32Dβ cells): 32Dβ cells (1x10^4 per well) were cultured for 3 hours in medium containing vehicle (0.1% DMSO) or IL-15-IN-1 compounds at a concentration of 11 µM. A fluorogenic peptide substrate specific for a dead-cell protease was added. After 30 minutes, fluorescence (ex485/em520 nm) was measured. Digitoxin-treated cells served as a positive control for cytotoxicity.[1] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: The major IL-15-IN-1 compounds (e.g., 1, 43, 45, 54, 64, 66-69, 73, 74, 76, 78, 80-82) were tested for cytotoxicity in 32Dβ cells at a concentration of 11 µM using a dead cell protease activity assay. No significant cytotoxicity was observed under these conditions. [1]
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| References | |
| Additional Infomation |
This study reports the discovery of the first small molecule inhibitors targeting the protein-protein interaction (PPI) of IL-15/IL-2Rβ. IL-15-IN-1 represents a family of compounds discovered through pharmacophore-based virtual screening and optimized through structure-activity relationship (SAR) studies. Their mechanism of action is to block the interaction between IL-15 and its signaling receptor chain IL-2Rβ, thereby inhibiting downstream signaling pathways (Jak/Stat) and cytokine-dependent cell proliferation. Notably, these initial inhibitors also effectively block the closely related IL-2/IL-2Rβ interaction, indicating that they possess dual inhibitory properties of IL-15/IL-2. This combined inhibitory property may have therapeutic value for diseases involving both cytokines, such as rheumatoid arthritis and transplant rejection. This work paves the way for the development of more selective IL-15 inhibitors. [1]
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| Molecular Formula |
C30H36N6O4S
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|---|---|
| Molecular Weight |
576.709645271301
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| Exact Mass |
576.251
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| CAS # |
1831830-20-6
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| PubChem CID |
129320517
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| Appearance |
White to off-white solid powder
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| LogP |
4.4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
41
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| Complexity |
919
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C1=NN=C(CC2C3C=CC=CC=3C(N(C)N=2)=O)N1C)CCCCCCCC(NC1C=CC=C(C(=O)OCC)C=1)=O
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| InChi Key |
GWNFQAKCJYEJEW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H36N6O4S/c1-4-40-29(39)21-13-12-14-22(19-21)31-27(37)17-8-6-5-7-11-18-41-30-33-32-26(35(30)2)20-25-23-15-9-10-16-24(23)28(38)36(3)34-25/h9-10,12-16,19H,4-8,11,17-18,20H2,1-3H3,(H,31,37)
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| Chemical Name |
ethyl 3-[8-[[4-methyl-5-[(3-methyl-4-oxophthalazin-1-yl)methyl]-1,2,4-triazol-3-yl]sulfanyl]octanoylamino]benzoate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~216.75 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.61 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.61 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7340 mL | 8.6699 mL | 17.3397 mL | |
| 5 mM | 0.3468 mL | 1.7340 mL | 3.4679 mL | |
| 10 mM | 0.1734 mL | 0.8670 mL | 1.7340 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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