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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
EGFR L858R 0.147 nM (IC50) EGFR L858R/T790M 0.063 nM (IC50) EGFR L858R/T790M/C797S 0.083 nM (IC50) EGFRLT/L747S 0.396 nM (IC50)
JBJ-09-063 TFA targets EGFR mutants, particularly those with L858R, T790M, and C797S mutations. It binds to an allosteric site on EGFR, distinct from the ATP-binding site, and inhibits kinase activity by inducing an inactive conformation. IC50 values: 0.147 nM for EGFR L858R, 0.063 nM for EGFR L858R/T790M, 0.083 nM for EGFR L858R/T790M/C797S (osimertinib-resistant), and 0.396 nM for EGFR L747S. It shows >1000-fold selectivity over wild-type EGFR, minimizing on-target skin and gastrointestinal toxicities. |
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| ln Vitro |
JBJ-09-063 is highly efficient in stopping the growth of cells and increases the rate of apoptosis, even though H3255GR cells have an EGFR T790M mutation, which makes them resistant to gefitinib when used alone[1]. When osimertinib-resistant mutations are expressed exogenously in H1975 cells, JBJ-09-063 is efficacious[1]. When JBJ-09-063 is employed either by alone or in conjunction with cetuximab, Ba/F3 cells show IC50s of 50 nM and 6 nM [2].
In cell-free assays, JBJ-09-063 TFA inhibits the kinase activity of purified EGFR mutant proteins with picomolar to low nanomolar IC50 values. The compound exhibits excellent selectivity for mutant EGFR over wild-type EGFR (IC50 >1000 nM), which translates into a wide therapeutic window. The allosteric binding mode is distinct from ATP-competitive inhibitors and is not affected by the T790M or C797S mutations. The TFA salt does not alter the inhibitory activity. |
| ln Vivo |
JBJ-09-063 (3 mg/kg iv, 20 mg/kg po) has favorable pharmacokinetic characteristics and is stable enough to provide good oral dosage results[2].
In cell-based assays using NSCLC cell lines harboring EGFR mutations (e.g., H1975 (L858R/T790M), PC9 (del19), and cell lines engineered to express L858R/T790M/C797S), JBJ-09-063 TFA (0.1-100 nM) effectively reduces phosphorylation of EGFR, AKT, and ERK1/2 in a dose-dependent manner. The compound inhibits cell proliferation with IC50 values in the low nanomolar range (consistent with biochemical IC50s) and induces apoptosis, as measured by increased cleaved PARP and Annexin V positivity. JBJ-09-063 is effective across EGFR TKI-sensitive and resistant models. |
| Enzyme Assay |
The inhibitory activity of JBJ-09-063 TFA is measured using a cell-free kinase assay. Recombinant EGFR kinase domains (wild-type, L858R, L858R/T790M, L858R/T790M/C797S, L747S) are incubated with a peptide substrate (e.g., poly(Glu, Tyr) 4:1) and ATP (10-100 uM) in the presence of varying concentrations (0.0001-10,000 nM) of JBJ-09-063 TFA. The reaction is carried out in kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM DTT). After 30 minutes at 30degC, the reaction is stopped by the addition of EDTA. Phosphorylated peptide is quantified by ELISA using a phospho-tyrosine antibody (PY20). IC50 values are calculated from dose-response curves.
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| Cell Assay |
NSCLC cell lines (e.g., H1975, PC9, or cells engineered to express EGFR mutations) are cultured in RPMI-1640 with 10% FBS. Cells are seeded in 96-well plates (5000 cells/well) and treated with JBJ-09-063 TFA (0.001-1000 nM) for 72 hours. Cell viability is measured by CellTiter-Glo. For signaling studies, cells are treated with 0.1-100 nM JBJ-09-063 for 2-6 hours, lysed in RIPA buffer, and analyzed by Western blot for p-EGFR (Y1068), total EGFR, p-AKT (S473), AKT, p-ERK1/2 (T202/Y204), and ERK1/2, with GAPDH as a loading control. Apoptosis is measured by Annexin V/PI staining after 48 hours of treatment.
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| Animal Protocol |
Animal/Disease Models: Mice[2]
Doses: 3 mg/kg for iv, 20 mg/kg for po Route of Administration: iv and po; single dosage Experimental Results: pharmacokinetic/PK Parameters of JBJ-09-063 in mice[2]. Cl (mL/min/kg), iv T1/2 (h) Vss (L/kg) F (%) AUC 8h (ng·h/mL) 15.7 2.3 2.5 15 2398 JBJ-09-063 TFA can be evaluated in mouse xenograft models using NSCLC cell lines harboring EGFR mutations (e.g., H1975 (L858R/T790M), PC9 (del19), or engineered Ba/F3 cells expressing L858R/T790M/C797S). Mice bearing subcutaneous tumors are treated with JBJ-09-063 TFA via oral gavage (e.g., 5-50 mg/kg, once daily) for 2-3 weeks. Tumor volume is measured by caliper. Tumor growth inhibition (TGI) is calculated. Tumor tissues are harvested for Western blot analysis to confirm EGFR/AKT/ERK pathway inhibition and for immunohistochemistry for Ki67 (proliferation) and cleaved caspase-3 (apoptosis). Body weight is monitored as a measure of toxicity. |
| ADME/Pharmacokinetics |
JBJ-09-063 TFA has a molecular weight of 670.67 Da and molecular formula C33H30F4N4O5S. It is soluble in DMSO (≥100 mg/mL). For in vivo studies, JBJ-09-063 is typically formulated in a solution of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline, or in 0.5% methylcellulose. The compound is administered orally (PO) due to good oral bioavailability. Following oral administration in rodents, peak plasma concentrations are achieved within 1-2 hours. The terminal half-life is likely 2-6 hours, supporting once-daily dosing. Pharmacokinetic studies show good plasma exposure and tumor penetration.
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| Toxicity/Toxicokinetics |
In preclinical studies, JBJ-09-063 TFA is well-tolerated at doses up to 50 mg/kg in mice. No significant body weight loss or clinical signs of toxicity (e.g., diarrhea, skin rash, lethargy) are observed, in contrast to ATP-competitive EGFR inhibitors which often cause skin and gastrointestinal toxicity. This is likely due to the mutant selectivity over wild-type EGFR. No significant changes in liver enzymes or kidney function are reported. The compound is for research use only; no clinical safety data are available.
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| References | |
| Additional Infomation |
JBJ-09-063 TFA is a research-use-only compound not approved for clinical use. It is a fourth-generation, mutant-selective allosteric EGFR inhibitor developed to overcome resistance to third-generation EGFR TKIs such as osimertinib, including the C797S mutation that is a major resistance mechanism. JBJ-09-063 is highly potent against EGFR L858R/T790M/C797S (IC50 0.083 nM) and EGFR L858R (0.147 nM). The compound is part of a series of allosteric inhibitors that bind to a site on the EGFR kinase domain distinct from the ATP-binding pocket. It effectively reduces EGFR, AKT, and ERK1/2 phosphorylation across EGFR TKI-sensitive and resistant models. The TFA salt is the form used for research.
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| Molecular Formula |
C33H30F4N4O5S
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|---|---|
| Molecular Weight |
670.67
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| Related CAS # |
JBJ-09-063 hydrochloride;JBJ-09-063;2820336-67-0
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 :≥ 100 mg/mL (~149.10 mM)
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| 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 | 1.4910 mL | 7.4552 mL | 14.9105 mL | |
| 5 mM | 0.2982 mL | 1.4910 mL | 2.9821 mL | |
| 10 mM | 0.1491 mL | 0.7455 mL | 1.4910 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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