| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| Other Sizes |
| Targets |
BTK (Bruton‘s Tyrosine Kinase). CNX-500 is a biotinylated covalent BTK inhibitor probe. The CC-292 moiety binds irreversibly to Cys481 in the ATP-binding pocket of BTK, forming a covalent bond. The biotin tag allows the probe to be captured by streptavidin-conjugated beads (e.g., streptavidin agarose or streptavidin magnetic beads) for the pull-down and enrichment of BTK from cell lysates. CNX-500 cannot bind to BTK that is already occupied by other covalent inhibitors (e.g., ibrutinib or CC-292 itself), allowing the detection of free, uninhibited BTK.
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|---|---|
| ln Vitro |
In competition assays, CNX-500 is utilized to identify free, unrestrained Btk and repair responses to Btk that was bound by CC-292 in the past. Over a variety of CC-292 concentrations, the quantity of Btk collected by the endpoint was compared to untreated samples in Ramos cells, indicating that the degree of Btk binding is proportionate to the CC-292 drug concentration [1].
CNX-500 retains the potent BTK inhibitory activity of CC-292, with an IC50 of 0.5 nM for BTK. It covalently binds to Cys481 of BTK. The probe has low inhibitory effects on off-target kinases, including the epidermal growth factor receptor (EGFR) and upstream Src-family kinases such as Syk and Lyn. In competition assays, the probe enables direct detection of free, uninhibited BTK, as it cannot bind to BTK already occupied by CC-292. This allows the determination of target occupancy in cells and tissues. |
| ln Vivo |
Not applicable (chemical probe for in vitro target engagement studies; not intended for in vivo efficacy as a therapeutic). In cell-based competition assays, Ramos B-cells are treated with a test covalent BTK inhibitor (e.g., ibrutinib or CC-292) for 2-6 hours. Cells are then lysed, and the lysates are incubated with CNX-500 (1-10 microM) to label free BTK. Biotinylated BTK is captured on streptavidin beads, eluted, and quantified by Western blot with an anti-BTK antibody. A reduction in CNX-500 pull-down signal indicates BTK target occupancy by the test inhibitor.
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| Enzyme Assay |
Not applicable (probe for target engagement, not for direct enzyme inhibition studies in a cell-free format). CNX-500 is used in pull-down assays to measure BTK occupancy. For a typical assay, cells (e.g., Ramos B-cells, 1 × 10⁶ cells/mL) are treated with various concentrations of a test BTK inhibitor for 2 hours. Cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. Cell lysates (500-1000 microg protein) are incubated with CNX-500 (1-10 microM, or biotinylated probe) for 2 hours at 4degC. Streptavidin-agarose beads are added, and the mixture is incubated for 2-4 hours at 4degC. Beads are washed extensively, and bound proteins are eluted by boiling in SDS-PAGE loading buffer. Eluted proteins are separated by SDS-PAGE, transferred to PVDF, and probed with an anti-BTK antibody. The signal intensity (relative to total BTK input) reflects the amount of free, unoccupied BTK.
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| Cell Assay |
Ramos B-cells (human Burkitt‘s lymphoma, 1 × 10⁶ cells/mL) are seeded in 6-well plates in RPMI-1640 with 10% FBS. Cells are treated with varying concentrations (0.1-1000 nM) of a test covalent BTK inhibitor (e.g., ibrutinib, CC-292, or acalabrutinib) for 2 hours at 37degC, 5% CO2. Untreated control cells are included for total BTK signal. Cells are harvested by centrifugation, washed with PBS, and lysed in RIPA buffer (with protease and phosphatase inhibitors) on ice for 30 minutes. Lysates are clarified by centrifugation at 14,000 rpm for 15 minutes at 4degC. Protein concentration is quantified by BCA assay. Not applicable (chemical probe for in vitro target engagement; not administered in animal efficacy studies). For ex vivo target occupancy studies, mice are dosed orally with a test BTK inhibitor (e.g., ibrutinib at 10-50 mg/kg). At various time points (1-24 hours post-dose), mice are euthanized, and whole blood, spleen, or tumor tissues are collected. Red blood cells are lysed, and leukocytes are isolated. Cells are lysed, and the lysates are subjected to the CNX-500 pull-down assay as described above. The percent BTK occupancy is calculated as 1 − (signal from treated sample / signal from untreated control) × 100%. CNX-500 is not used for in vivo administration; it is an ex vivo probe.
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| ADME/Pharmacokinetics |
CNX-500 is a chemical probe with a molecular weight of approximately 961 g/mol. It is soluble in DMSO and should be stored at -20degC protected from light. The probe is designed for in vitro and ex vivo use only; it is not administered to animals or humans for therapeutic purposes. Its primary application is in target engagement studies for covalent BTK inhibitors, enabling the determination of the relationship between drug concentration and target occupancy.
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| Toxicity/Toxicokinetics |
CNX-500 is a research chemical probe; toxicity data are not applicable as it is not intended for in vivo administration. The probe should be handled with standard laboratory safety precautions (gloves, lab coat, goggles) as it contains a covalent inhibitor moiety that may be reactive. Avoid inhalation, skin contact, and ingestion. The compound is for research use only and not for human or veterinary therapeutic use.
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| References | |
| Additional Infomation |
CNX-500 is a biotinylated covalent BTK inhibitor probe developed for target engagement studies. It enables the direct quantification of free, unoccupied BTK in cells and tissues following treatment with covalent BTK inhibitors (e.g., ibrutinib, acalabrutinib, zanubrutinib, orelabrutinib). This probe is a valuable tool for determining the relationship between drug concentration, target occupancy, and pharmacodynamic effects (e.g., inhibition of downstream signaling). CNX-500 is also useful for studying the selectivity of covalent inhibitors. The probe is for research use only.
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| Molecular Formula |
C48H68N10O9S
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|---|---|
| Molecular Weight |
961.180130004883
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| Exact Mass |
960.489
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| CAS # |
1202758-21-1
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| PubChem CID |
59174509
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| Appearance |
White to yellow solid powder
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| LogP |
3.4
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| Hydrogen Bond Donor Count |
8
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| Hydrogen Bond Acceptor Count |
14
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| Rotatable Bond Count |
34
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| Heavy Atom Count |
68
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| Complexity |
1520
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| Defined Atom Stereocenter Count |
3
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| SMILES |
S1C[C@H]2[C@@H]([C@@H]1CCCCC(NCCCOCCOCCOCCCNC(CCCC(NCCCOC1C=CC=C(C=1)NC1N=CC(C)=C(N=1)NC1C=CC=C(C=1)NC(C=C)=O)=O)=O)=O)NC(N2)=O
|
| InChi Key |
SBOPEFHTOFMNSD-IAWMPOBQSA-N
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| InChi Code |
InChI=1S/C48H68N10O9S/c1-3-41(59)53-35-12-6-13-36(30-35)54-46-34(2)32-52-47(58-46)55-37-14-7-15-38(31-37)67-25-11-22-51-44(62)19-8-18-43(61)50-21-10-24-65-27-29-66-28-26-64-23-9-20-49-42(60)17-5-4-16-40-45-39(33-68-40)56-48(63)57-45/h3,6-7,12-15,30-32,39-40,45H,1,4-5,8-11,16-29,33H2,2H3,(H,49,60)(H,50,61)(H,51,62)(H,53,59)(H2,56,57,63)(H2,52,54,55,58)/t39-,40-,45-/m0/s1
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| Chemical Name |
N-[3-[2-[2-[3-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]propoxy]ethoxy]ethoxy]propyl]-N'-[3-[3-[[5-methyl-4-[3-(prop-2-enoylamino)anilino]pyrimidin-2-yl]amino]phenoxy]propyl]pentanediamide
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| Synonyms |
CNX500; CNX 500
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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 : ~50 mg/mL (~52.02 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (1.30 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 12.5 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: ≥ 1.25 mg/mL (1.30 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 12.5 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. View More
Solubility in Formulation 3: ≥ 1.25 mg/mL (1.30 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0404 mL | 5.2019 mL | 10.4039 mL | |
| 5 mM | 0.2081 mL | 1.0404 mL | 2.0808 mL | |
| 10 mM | 0.1040 mL | 0.5202 mL | 1.0404 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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