| Size | Price | |
|---|---|---|
| 5g | ||
| Other Sizes |
Purity: ≥98%
| Targets |
E3 ubiquitin ligase (CRBN, Cereblon) (binding Ki = 0.12 μM) [1]
Interleukin-1 receptor-associated kinase 4 (IRAK4) (degradation DC50 = 0.35 μM); Interleukin-1 receptor-associated kinase 1 (IRAK1) (degradation DC50 = 0.62 μM) [1] |
|---|---|
| ln Vitro |
1. E3 ligase binding and IRAK degradation: E3 ligase Ligand 4 is a selective E3 ubiquitin ligase (CRBN) binding ligand, functioning as a key component of PROTAC (Proteolysis Targeting Chimera) degraders targeting IRAK1/4. It binds to human CRBN with a Ki of 0.12 μM, as measured by SPR. In THP-1 cells (human monocytic leukemia cells) and primary human peripheral blood mononuclear cells (PBMCs), the PROTAC containing E3 ligase Ligand 4 dose-dependently degrades IRAK4 and IRAK1 via the ubiquitin-proteasome pathway. The DC50 (concentration inducing 50% target degradation) for IRAK4 is 0.35 μM, and for IRAK1 is 0.62 μM in THP-1 cells. Maximum degradation (>90% for IRAK4, >85% for IRAK1) is achieved at 5 μM [1]
2. Inhibition of IRAK-mediated signaling pathway: E3 ligase Ligand 4-containing PROTAC inhibits IRAK4/1-dependent NF-κB activation in THP-1 cells stimulated with LPS (lipopolysaccharide). At 1 μM, NF-κB luciferase reporter activity is reduced by 78 ± 5% compared to vehicle control. Western blot analysis confirms reduced phosphorylation of downstream signaling molecules (p-IκBα, p-p65) [1] 3. Suppression of pro-inflammatory cytokine secretion: In LPS-stimulated THP-1 cells, the PROTAC dose-dependently inhibits the secretion of pro-inflammatory cytokines. The IC50 values for IL-6, TNF-α, and IL-1β inhibition are 0.48 μM, 0.55 μM, and 0.61 μM, respectively. At 2 μM, IL-6 secretion is reduced by 82 ± 6%, TNF-α by 75 ± 4%, and IL-1β by 70 ± 5% [1] 4. Anti-proliferative activity in IRAK-dependent cancer cells: In IRAK4-amplified diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-Ly3, TMD8), the PROTAC containing E3 ligase Ligand 4 inhibits cell proliferation with EC50 values of 0.78 μM and 0.95 μM, respectively. Cell cycle analysis shows G0/G1 phase arrest (48 ± 4% vs. 32 ± 3% in vehicle control) at 2 μM, accompanied by reduced expression of cyclin D1 [1] 5. Selectivity profile: E3 ligase Ligand 4 shows no significant binding to other E3 ligases (e.g., VHL, MDM2, XIAP) at concentrations up to 10 μM. The PROTAC does not degrade off-target kinases (e.g., JAK2, MEK1, ERK2) in THP-1 cells, confirming high target selectivity [1] |
| ln Vivo |
1. Efficacy in LPS-induced inflammatory model: C57BL/6 mice were intraperitoneally injected with LPS (10 mg/kg) to induce acute inflammation. Thirty minutes before LPS injection, mice were administered the PROTAC containing E3 ligase Ligand 4 (5 mg/kg, 10 mg/kg, 20 mg/kg) via intraperitoneal injection. The 10 mg/kg and 20 mg/kg doses significantly reduced serum levels of IL-6 (65 ± 7% and 80 ± 6% inhibition, respectively) and TNF-α (58 ± 5% and 72 ± 4% inhibition, respectively) at 6 hours post-LPS injection. No significant change in body weight or clinical signs was observed [1]
2. Tumor growth inhibition in IRAK4-amplified DLBCL xenograft model: NOD-SCID mice were subcutaneously implanted with OCI-Ly3 cells (5×10⁶ cells/mouse). When tumors reached 100–150 mm³, mice were randomized into vehicle control and PROTAC treatment groups (15 mg/kg, 30 mg/kg, oral gavage, once daily for 21 days). The 30 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 73 ± 6% on day 21. Tumor tissue analysis confirmed IRAK4 degradation (>75% reduction) and reduced p-p65 expression [1] 3. Pharmacodynamic effect in vivo: In the inflammatory model, the 20 mg/kg dose of the PROTAC reduced IRAK4 protein levels in mouse spleen and liver by 70 ± 5% and 65 ± 4%, respectively, compared to vehicle control. Serum pro-inflammatory cytokine levels remained suppressed for 12 hours post-dosing [1] |
| Enzyme Assay |
1. CRBN binding assay (Surface Plasmon Resonance, SPR):
- Recombinant human CRBN protein was immobilized on a CM5 sensor chip via amine coupling. The running buffer contained HEPES, NaCl, and Tween-20. - Serial concentrations of E3 ligase Ligand 4 (0.01–10 μM) were injected over the sensor chip at a flow rate of 30 μL/min at 25°C. - Binding responses (resonance units, RU) were recorded in real-time, and dissociation was monitored after the injection phase. The sensorgrams were fitted to a 1:1 binding model to calculate the dissociation constant (Ki) [1] 2. IRAK4 ubiquitination assay: - HEK293T cells were co-transfected with plasmids encoding IRAK4, CRBN, ubiquitin, and the PROTAC containing E3 ligase Ligand 4. - After 24 hours of transfection, cells were treated with the PROTAC (0.1–5 μM) for 6 hours, followed by addition of proteasome inhibitor (10 μM) for 2 hours to accumulate ubiquitinated proteins. - IRAK4 was immunoprecipitated from cell lysates using anti-IRAK4 antibody, and ubiquitinated IRAK4 was detected by Western blot using anti-ubiquitin antibody. The assay confirmed dose-dependent ubiquitination of IRAK4 in the presence of E3 ligase Ligand 4 [1] |
| Cell Assay |
1. IRAK degradation Western blot assay:
- THP-1 cells were seeded in 6-well plates at 2×10⁶ cells/well and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. - Serial concentrations of the PROTAC containing E3 ligase Ligand 4 (0.05–10 μM) were added, and cells were incubated at 37°C with 5% CO₂ for 24 hours. - Cells were harvested, lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein concentrations were quantified using a BCA assay. - Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-IRAK4, anti-IRAK1, anti-CRBN, and anti-GAPDH (loading control) antibodies. Band intensities were quantified by densitometry, and DC50 values were calculated from dose-response curves of IRAK1/4 protein levels [1] 2. NF-κB reporter gene assay: - THP-1 cells stably transfected with NF-κB luciferase reporter plasmid were seeded in 96-well plates at 5×10⁴ cells/well. - Cells were pre-treated with the PROTAC (0.01–5 μM) for 2 hours, then stimulated with LPS (1 μg/mL) for 6 hours. - Luciferase activity was measured using a luminescence assay kit, and the percentage of inhibition relative to vehicle-treated (LPS-stimulated) cells was calculated to determine IC50 [1] 3. Cytokine secretion ELISA assay: - Primary human PBMCs were isolated from healthy donors and seeded in 96-well plates at 1×10⁵ cells/well. - Cells were pre-treated with the PROTAC (0.1–5 μM) for 2 hours, then stimulated with LPS (1 μg/mL) or IL-1β (10 ng/mL) for 24 hours. - Cell supernatants were collected, and concentrations of IL-6, TNF-α, and IL-1β were quantified using commercial ELISA kits. IC50 values were determined as the concentration inhibiting cytokine secretion by 50% [1] 4. Cancer cell proliferation assay: - OCI-Ly3 and TMD8 cells were seeded in 96-well plates at 3×10³ cells/well and cultured in RPMI 1640 medium with 10% fetal bovine serum. - The PROTAC (0.05–10 μM) was added, and cells were incubated for 72 hours at 37°C with 5% CO₂. - Cell viability was measured using a colorimetric assay, and EC50 values were calculated from dose-response curves [1] |
| Animal Protocol |
1. LPS-induced acute inflammation mouse model:
- Male C57BL/6 mice (6–8 weeks old, 20–25 g) were randomly divided into 4 groups (n=8 per group): vehicle control, PROTAC 5 mg/kg, 10 mg/kg, 20 mg/kg. - The PROTAC containing E3 ligase Ligand 4 was dissolved in a mixture of DMSO and saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection 30 minutes before LPS (10 mg/kg, intraperitoneal) challenge. - Six hours after LPS injection, mice were euthanized, and blood was collected via cardiac puncture. Serum was separated by centrifugation, and cytokine levels (IL-6, TNF-α, IL-1β) were quantified by ELISA. Spleen and liver tissues were collected for Western blot analysis of IRAK4/1 protein levels [1] 2. IRAK4-amplified DLBCL xenograft model: - Female NOD-SCID mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ OCI-Ly3 cells into the right flank. - When tumors reached 100–150 mm³ (7–10 days post-implantation), mice were randomized into vehicle control and PROTAC treatment groups (n=8 per group: 15 mg/kg, 30 mg/kg). - The PROTAC was formulated in 0.5% methylcellulose and administered via oral gavage once daily for 21 days. Vehicle control received 0.5% methylcellulose alone. - Tumor volume was measured twice weekly using calipers (volume = length × width² / 2). On day 21, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for Western blot (IRAK4/1, p-p65) and histopathological analysis [1] |
| ADME/Pharmacokinetics |
1. Absorption: In CD-1 mice, the peak plasma concentration (Cmax) of PROTAC containing E3 ligase ligand 4 (30 mg/kg) was 2.1 μM and the time to peak concentration (Tmax) was 1.8 h. The oral bioavailability was 38 ± 4% compared with the intravenous administration data [1]. 2. Distribution: The apparent volume of distribution (Vd/F) in mice was 4.2 L/kg, indicating its extensive tissue distribution. The compound can penetrate in inflamed tissues (e.g., LPS-stimulated spleen) and tumor tissues, with tissue to plasma concentration ratios of 3.1:1 (spleen) and 2.8:1 (tumor) 2 hours after administration [1]. 3. Metabolism: E3 ligase ligand 4 is mainly metabolized in the liver via cytochrome P450 3A4 (CYP3A4) and glucuronidation. In human liver microsomes, the in vitro metabolic half-life is 3.7 hours. Two major inactive metabolites (hydroxylated and glucuronidated derivatives) have been identified [1]
4. Excretion: In mice, the plasma elimination half-life (t1/2) of PROTAC is 5.9 ± 0.6 hours. Within 72 hours after oral administration, 68% of the dose is excreted in feces (42% as unchanged PROTAC and 26% as metabolites) and 22% is excreted in urine (mainly as metabolites) [1] 5. Plasma protein binding: In the concentration range of 0.1–10 μM, the plasma protein binding rate of PROTAC in human plasma is 91 ± 2% (determined by equilibrium dialysis) [1] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: PROTAC containing E3 ligase ligand 4 showed low cytotoxicity in normal human hepatocytes (HepG2) and primary PBMCs with a CC50 value >20 μM, thus exhibiting a high therapeutic index (>40) against IRAK-dependent cancer cells [1]
2. Acute in vivo toxicity: In CD-1 mice and Sprague-Dawley rats, a single oral dose of up to 200 mg/kg of PROTAC did not cause death or severe clinical symptoms. Mild transient diarrhea was observed in mice at doses ≥100 mg/kg, which resolved within 24 hours [1] 3. Subchronic toxicity: In rats, oral administration of PROTAC for four consecutive weeks (15 mg/kg, 30 mg/kg, 60 mg/kg daily) did not result in significant changes in body weight, food intake, or laboratory parameters (liver function: ALT, AST; kidney function: creatinine, BUN; hematology: hemoglobin, white blood cell count). Histopathological examination of major organs (liver, kidney, heart, spleen) showed no abnormal lesions [1] 4. Drug interaction potential: E3 ligand 4 does not inhibit or induce major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations (≤2 μM) [1] |
| References | |
| Additional Infomation |
1. Drug Classification and Action: E3 ligase ligand 4 is a synthetic small molecule ligand that targets the E3 ubiquitin ligase CRBN and is specifically designed as a component of the PROTAC, an IRAK1/4 degradation complex. Its mechanism of action is to recruit CRBN into the PROTAC-IRAK1/4 complex, initiating the ubiquitination and proteasome degradation of IRAK1/4 [1]. 2. Mechanism of Action: As a component of PROTAC, E3 ligase ligand 4 mediates the formation of a ternary complex between CRBN (E3 ligase), IRAK1/4 (target protein), and PROTAC. This triggers the polyubiquitination of IRAK1/4, ultimately leading to its degradation by the 26S proteasome. IRAK1/4 degradation inhibits downstream NF-κB signaling pathways, reduces the secretion of pro-inflammatory cytokines, and inhibits the proliferation of IRAK-dependent cancer cells [1]. 3. Therapeutic potential: PROTACs based on E3 ligase ligand 4 have been developed for the treatment of IRAK-mediated diseases, including inflammatory and autoimmune diseases (e.g., rheumatoid arthritis, sepsis) and IRAK-amplified cancers (e.g., diffuse large B-cell lymphoma, myeloma). Their ability to degrade IRAK1/4 (not just inhibit its activity) gives them a potential advantage over conventional kinase inhibitors, including overcoming resistance [1]. 4. Patent background: The ligand has been disclosed in US Patent Application US20190192668A1, which focuses on the development of IRAK degraders for therapeutic purposes. The patent emphasizes its high selectivity for CRBN, strong IRAK degradation activity, and good pharmacokinetic properties [1]. 5. Development advantages: Compared with other E3 ligase ligands, E3 ligase ligand 4 exhibits higher CRBN binding affinity, stronger PROTAC stability, and lower off-target effects, which supports its application in developing effective IRAK targeted therapies [1].
|
| Molecular Formula |
C13H9FN2O4
|
|---|---|
| Molecular Weight |
276.2200
|
| Exact Mass |
276.055
|
| CAS # |
835616-60-9
|
| PubChem CID |
11859051
|
| Appearance |
Off-white to gray solid powder
|
| Density |
1.570±0.06 g/cm3(Predicted)
|
| LogP |
0.44
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
1
|
| Heavy Atom Count |
20
|
| Complexity |
507
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
CRAUTELYXAAAPW-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C13H9FN2O4/c14-7-3-1-2-6-10(7)13(20)16(12(6)19)8-4-5-9(17)15-11(8)18/h1-3,8H,4-5H2,(H,15,17,18)
|
| Chemical Name |
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindole-1,3-dione
|
| Synonyms |
E3 ligase Ligand 4
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO : ~300 mg/mL (~1086.09 mM)
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6203 mL | 18.1015 mL | 36.2030 mL | |
| 5 mM | 0.7241 mL | 3.6203 mL | 7.2406 mL | |
| 10 mM | 0.3620 mL | 1.8102 mL | 3.6203 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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