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| Targets |
CTX-0294885 hydrochloride is a broad-spectrum kinase inhibitor that does not have a single specific target; instead, it binds to the ATP-binding pocket of many protein kinases. Through quantitative proteomics, CTx-0294885-bound beads were shown to selectively enrich kinases from cell lysates. In MDA-MB-231 cells, CTX-0294885 captured 235 protein kinases, including all members of the AKT family.
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| ln Vitro |
Large-scale kinome profiling research using CTX-0294885 hydrochloride as a kinase capture method have discovered 185 kinases as total protein and 179 as phosphopeptides from TiO2 enrichment in MDA-MB-231 cells (by quantitative MS)[1]. All members of the AKT family that have not been identified by earlier research employing alternative kinase capture reagents are captured by CTX -0294885 hydrochloride[1].
In vitro, CTX-0294885 hydrochloride is not typically used for cell viability assays; it is used as a kinase capture reagent. Addition of CTX-0294885 to a mixture of three commonly used kinase inhibitors increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases. |
| ln Vivo |
CTX-0294885 hydrochloride is not studied for direct in vivo efficacy as a therapeutic agent. Its primary application is in ex vivo proteomics: it is immobilized on Sepharose beads to create an affinity matrix for kinome profiling from cell lysates or tissue homogenates. Thus, in vivo activity data is not applicable for this compound. However, the identified kinase targets may be relevant for therapeutic development.
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| Enzyme Assay |
For in vitro enzyme-binding assays (non-cell-based, bead-based affinity capture): Immobilization of CTX-0294885: The compound is conjugated to NHS-activated Sepharose beads via primary amine coupling. Following the manufacturer's instructions for NHS-activated Sepharose (e.g., from Cytiva), CTX-0294885 hydrochloride is dissolved in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) at a concentration of 1-5 mg/mL. The NHS-Sepharose beads are washed and incubated with the CTX-0294885 solution for 2-4 hours at room temperature or overnight at 4degC with end-over-end mixing. Unreacted NHS groups are quenched with 0.1 M Tris-HCl (pH 8.0) or 1 M ethanolamine (pH 8.0) for 1 hour at room temperature. The CTX-0294885-Sepharose beads are washed extensively with PBS and then stored at 4degC in PBS with 0.02% sodium azide as a preservative. For kinase capture from cell lysates: cells (e.g., MDA-MB-231, 1×10⁸ cells) are lysed in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM Na3VO4, 1 mM NaF, and protease/phosphatase inhibitor cocktails). Protein concentration is quantified (typically 5-10 mg/mL). Equal amounts of lysate (e.g., 5 mg total protein) are incubated with 50-100 microL (settled bead volume) of CTX-0294885-Sepharose beads overnight at 4degC with gentle rotation. Beads are washed with lysis buffer (3×) and then with PBS (2×). Bound proteins are eluted by heating the beads in SDS-PAGE sample buffer (e.g., 2% SDS, 50 mM DTT) at 95degC for 5 minutes. Eluted proteins are digested with trypsin, and the resulting peptides are analyzed by LC-MS/MS for kinase identification and quantification. A quantitative proteomics approach (e.g., SILAC or TMT labeling) is used to compare kinome enrichment profiles across different samples. This protocol enables the identification of kinase targets and the assessment of selectivity. Non-specific binding is assessed by using control beads (e.g., mock-coupled Sepharose or inhibitor-negative Sepharose).
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| Cell Assay |
For cell-based assays, CTX-0294885 hydrochloride is used for kinome profiling from intact cells. Cells are treated with the compound prior to lysis, and then affinity capture is performed as described above. However, CTX-0294885 is primarily used as an immobilized affinity reagent rather than as a soluble treatment. For cellular target engagement studies: cells are treated with varying concentrations of CTX-0294885 hydrochloride (e.g., 0.1-10 microM) for 1-4 hours. Cells are then lysed, and the lysates are incubated with CTX-0294885-Sepharose beads. A reduction in kinase binding to the beads compared to vehicle-treated controls indicates competitive binding of the soluble compound, allowing assessment of cellular target engagement and estimation of EC50 values. Cell viability assays (MTT or CellTiter-Glo) can be performed on cells treated with CTX-0294885 (0-20 microM for 24-72 hours) to assess cytotoxicity; however, this is not the primary application. Typically, CTX-0294885 is not highly cytotoxic as it is a broad-spectrum ATP-competitive inhibitor at lower concentrations, but at high concentrations it may inhibit essential kinases. For reference, the compound is used to identify kinases that can be targeted for therapeutic strategies; any identified kinase targets should be validated with more selective inhibitors.
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| Animal Protocol |
CTX-0294885 hydrochloride is not used for direct in vivo animal studies as a therapeutic agent; it is an affinity proteomics reagent. However, if a kinase target of interest is identified using CTX-0294885, in vivo efficacy studies would be performed with a selective inhibitor of that target in appropriate animal models (e.g., xenograft models for cancer). CTX-0294885 itself is not administered to animals for efficacy studies. For pharmacokinetic studies of the compound, if needed, CTX-0294885 hydrochloride could be administered to rodents (e.g., mice, 10-50 mg/kg via oral gavage or intraperitoneal injection) to assess plasma levels by LC-MS/MS, but such studies are not standard for this reagent. The primary use is ex vivo affinity capture from lysates of cells or tissues from animals treated with other agents. For example, tissues from tumor-bearing mice treated with a novel drug can be lysed, and the lysate can be incubated with CTX-0294885-Sepharose beads to profile kinome changes in response to treatment.
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| ADME/Pharmacokinetics |
CTX-0294885 hydrochloride has a molecular weight of 437.93 Da (free base). CAS number: 1439934-41-4 (free base). The hydrochloride salt form enhances water solubility. The compound is supplied as a solid (powder) and should be stored at -20degC (for long-term storage, powder: -20degC for 3 years, 4degC for 2 years). Stock solutions should be stored at -80degC for up to 6 months or at -20degC for up to 1 month. Solubility: DMSO ≥ 90 mg/mL (fully soluble); water: soluble as the hydrochloride salt (to be tested). For bead immobilization, the compound is typically dissolved in DMSO as a stock solution (e.g., 10-50 mM) and then diluted into aqueous coupling buffer. For cellular treatment, CTX-0294885 is dissolved in DMSO and diluted into culture medium; the final DMSO concentration should be ≤0.1% to avoid cytotoxicity. For kinome profiling, the compound is covalently attached to NHS-activated Sepharose to generate a stable affinity matrix. The immobilization protocol must be optimized to preserve the compound's binding activity for a broad range of kinases.
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| Toxicity/Toxicokinetics |
CTX-0294885 hydrochloride is a research-grade compound for laboratory use only, not for human diagnostic or therapeutic applications. At typical research concentrations (microM range for cellular treatment), the compound is considered non-toxic; however, as a broad-spectrum kinase inhibitor, it may affect cell proliferation and survival at higher concentrations. Standard safety practices (gloves, lab coat, eye protection) should be followed. The compound is not for in vivo administration without appropriate toxicological evaluation. Acute toxicity data are not available; for animal handling, standard chemical safety protocols apply.
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| References | |
| Additional Infomation |
CTX-0294885 hydrochloride (CAS 1439934-41-4) is a broad-spectrum kinase inhibitor and affinity capture reagent developed for kinome-wide profiling of protein kinases. Key features: (1) Novel bisanilino pyrimidine scaffold; (2) Broad-spectrum kinase inhibitory activity; (3) Immobilized on Sepharose beads for affinity purification of kinases from cell lysates; (4) Enables identification of 235-261 kinases from a single cancer cell line; (5) Has been used to identify phosphosites on kinases, including previously unreported ones on BMP2K, MELK, HIPK2, and PRKDC. Common applications: kinome profiling, kinase target discovery, proteomics, and biomarker identification. Purity: >98.0% by HPLC. This product is a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies.
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| Molecular Formula |
C22H25CL2N7O
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| Molecular Weight |
474.39
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| Related CAS # |
CTX-0294885;1439934-41-4
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| Appearance |
Light yellow to yellow 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) |
H2O :~55 mg/mL (~115.94 mM)
DMSO :~9.17 mg/mL (~19.33 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.92 mg/mL (1.94 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 9.2 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: ≥ 0.92 mg/mL (1.94 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 9.2 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: ≥ 0.92 mg/mL (1.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 50 mg/mL (105.40 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1080 mL | 10.5399 mL | 21.0797 mL | |
| 5 mM | 0.4216 mL | 2.1080 mL | 4.2159 mL | |
| 10 mM | 0.2108 mL | 1.0540 mL | 2.1080 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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