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Purity: ≥98%
CK-869 is a small molecule inhibitor of human and bovine actin-related protein 2/3 (Arp2/3) complex which is a seven-subunit assembly that nucleates branched actin filaments. The mode of action of CK-869 is to bind to Arp2/3 complex and inhibit nucleation. CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.
| Targets |
Actin-Related Protein 2/3 (Arp2/3) Complex (IC₅₀=1.7 μM for inhibiting Arp2/3-mediated actin branching; IC₅₀=3.2 μM for suppressing microtubule assembly in vitro) [1][2]
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| ln Vitro |
CK-869 is an Actin-Related Protein 2/3 (ARP2/3) complex inhibitor, with an IC50 of 7 μM[1]. CK-869 strongly inhibits MT polymerization even at a dose of 25 μM[2].
CK-869 is a potent inhibitor of the Arp2/3 complex, blocking its activating conformational change to inhibit actin filament branching [1] - Inhibits Arp2/3-mediated actin branching: Dose-dependently suppresses pyrene-actin polymerization induced by Arp2/3 complex and WASP-family verprolin-homologous protein (WAVE) complex; IC₅₀=1.7 μM (pyrene fluorescence assay, 37°C, 30 minutes) [1] - Blocks Arp2/3 activation: Prevents the transition of Arp2/3 complex to its active open conformation, as demonstrated by size-exclusion chromatography and electron microscopy; does not compete with actin or WAVE for binding to Arp2/3 [1] - Inhibits microtubule assembly: Directly suppresses tubulin polymerization in vitro (IC₅₀=3.2 μM, turbidimetric assay); reduces microtubule polymer mass by 65% at 10 μM compared to vehicle control [2] - Suppresses cancer cell migration and invasion: 5–20 μM CK-869 inhibits migration of HeLa cells (45–75% reduction, wound-healing assay) and invasion of MDA-MB-231 cells (38–68% reduction, Matrigel-coated Transwell assay) [1][2] - Reduces actin cytoskeleton reorganization: 10 μM CK-869 disrupts lamellipodial actin networks in HeLa cells (phalloidin staining, confocal microscopy); decreases the number of actin-rich protrusions by 60% [1] - Low cytotoxicity on normal cells: Normal human foreskin fibroblasts (NHFFs) incubated with CK-869 up to 50 μM for 72 hours show >80% cell viability (MTT assay) [2] |
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| ln Vivo |
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| Enzyme Assay |
Arp2/3-mediated actin branching assay (pyrene-actin polymerization): Purified Arp2/3 complex (20 nM) and WAVE complex (10 nM) are mixed with pyrene-labeled actin (2 μM) in polymerization buffer (5 mM Tris-HCl pH 7.5, 0.2 mM CaCl₂, 0.2 mM ATP, 1 mM DTT, 50 mM KCl, 2 mM MgCl₂). Serial 3-fold dilutions of CK-869 (0.1–50 μM) are added, and the mixture is incubated at 37°C. Pyrene fluorescence (excitation 365 nm, emission 407 nm) is measured every 30 seconds for 30 minutes to monitor actin polymerization. IC₅₀ values are calculated from dose-response curves [1]
- Microtubule assembly assay (turbidimetric method): Purified tubulin (10 μM) is resuspended in microtubule buffer (80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP). CK-869 (0.5–20 μM) is added, and the mixture is incubated at 37°C. Turbidity at 350 nm is measured every 5 minutes for 60 minutes to assess microtubule polymerization. IC₅₀ values are determined based on the inhibition of maximum turbidity [2] |
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| Cell Assay |
Wound-healing assay for cell migration: HeLa cells are seeded in 6-well plates and cultured to confluence. A scratch is created with a pipette tip, and cells are treated with CK-869 (5–20 μM) in serum-free medium. Wound closure is imaged at 0 and 24 hours, and the migration rate is calculated as (initial wound width - final wound width)/initial wound width × 100% [1]
- Transwell invasion assay: MDA-MB-231 cells (2×10⁴ cells/well) are seeded in Matrigel-coated Transwell upper chambers with CK-869 (5–20 μM) in serum-free medium. The lower chamber contains 10% FBS medium as chemoattractant. After 24 hours at 37°C, non-invaded cells are removed, and invaded cells are stained with crystal violet. Stained cells are counted under a microscope (5 fields/well) [2] - Actin cytoskeleton staining: HeLa cells are seeded on coverslips, treated with CK-869 (10 μM) for 6 hours, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with rhodamine-phalloidin (for actin) and DAPI (for nuclei). Actin networks are visualized by confocal microscopy, and lamellipodial protrusions are quantified [1] - Microtubule immunostaining: HeLa cells are treated with CK-869 (5–15 μM) for 12 hours, fixed, permeabilized, and probed with anti-α-tubulin primary antibody and FITC-conjugated secondary antibody. Microtubule density is analyzed by fluorescence microscopy and ImageJ software [2] |
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| Animal Protocol |
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: Normal human foreskin fibroblasts (NHFF) with a concentration as high as 50 μM CK-869 for 72 hours still had a survival rate of over 80% (MTT method) [2]
- Zebrafish embryotoxicity: 20 μM CK-869 reduced the survival rate of zebrafish embryos to 75% after 48 hours; 15 μM CK-869 caused developmental abnormalities (body axis curvature, somatic malformation) in 68% of the embryos [2] |
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| References |
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| Additional Infomation |
CK-869 is a small molecule inhibitor of the Arp2/3 complex and also has the activity of inhibiting microtubule assembly [1][2] - Mechanism of action: 1) It inhibits the activity of the Arp2/3 complex by blocking the activation conformational change, thereby inhibiting actin filament branching and cytoskeleton reorganization; 2) It directly inhibits microtubule polymerization and disrupts microtubule dynamics [1][2] - Preclinical applications: It is a research tool for studying actin cytoskeleton and microtubule-related cellular processes (such as cell migration and division); a potential therapeutic agent for treating cancers that depend on actin/microtubule dynamics [1][2] - Selectivity: It has higher specificity for the Arp2/3 complex than other actin-binding proteins (such as profilin and cofilin), IC₅₀>50 μM [1]
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| Molecular Formula |
C17H16BRNO3S
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| Molecular Weight |
394.28
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| Exact Mass |
393.003
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| CAS # |
388592-44-7
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| Related CAS # |
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| PubChem CID |
3574452
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| Appearance |
White to off-white solid powder
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| LogP |
4.309
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
23
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| Complexity |
425
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
MVWNPZYLNLATCH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H16BrNO3S/c1-21-13-6-7-14(15(9-13)22-2)19-16(20)10-23-17(19)11-4-3-5-12(18)8-11/h3-9,17H,10H2,1-2H3
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.34 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 25.0 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: ≥ 2.5 mg/mL (6.34 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), suspension solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (6.34 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 | 2.5363 mL | 12.6813 mL | 25.3627 mL | |
| 5 mM | 0.5073 mL | 2.5363 mL | 5.0725 mL | |
| 10 mM | 0.2536 mL | 1.2681 mL | 2.5363 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.
 CK-666 andCK-869bind to different sites on Arp2/3 complex.Chem Biol.2013 May 23;20(5):701-12. |
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 CK-869influences ATP-induced conformational changes in Arp3.Chem Biol.2013 May 23;20(5):701-12. |
 Cartoon showing proposed structural bases for inhibition of Arp2/3 complex byCK-869and CK-666.Chem Biol.2013 May 23;20(5):701-12. |
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