| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg |
|
||
| 500mg |
|
||
| 1g | |||
| Other Sizes |
Purity: ≥98%
WZ4003 is a novel, potent and highly specific inhibitor of NUAK kinases. It exhibits little inhibition against 139 other kinases but inhibits NUAK1 and NUAK2 with IC50s of 20 nM and 100 nM in cell-base assays.
| Targets |
NUAK1 (IC50 = 20 nM); NUAK2 (IC50 = 100 nM)
NUAK1 (AMPK-related kinase 5): WZ4003 exhibits selective inhibition with an IC50 of 14 nM against recombinant human NUAK1; no Ki/EC50 values reported [1] - NUAK2 (AMPK-related kinase 6): WZ4003 shows selective inhibition with an IC50 of 13 nM against recombinant human NUAK2; no Ki/EC50 values reported [1] - Other AMPK-related kinases/kinases: WZ4003 has minimal inhibitory activity (IC50 > 10,000 nM) against AMPKα1, AMPKα2, MARK1, MARK2, MARK3, MARK4, BRSK1, BRSK2, SNRK, QIK, QSK, SIK, PANK4, and MELK, confirming its selectivity for NUAK1/2 [1] |
|---|---|
| ln Vitro |
WZ4003 (3–10 μM) significantly reduces MYPT1 phosphorylation caused by NUAK1 in HEK-293 cells that are overexpressing wild-type NUAK1. Furthermore, WZ4003 (10 μM) inhibits MYPT1 Ser445 phosphorylation as well as cell migration, invasion, and proliferation to a similar extent as NUAK1 knockout in MEFs or knockdown in U2OS cells.[1] While having a significantly lower cellular IC50 against T790M-containing Ba/F3 cells, WZ4003 also shows a high, specific affinity to the L858R/T790M mutant EGFR. [2]
Recombinant kinase activity inhibition: WZ4003 potently inhibits NUAK1 and NUAK2 (IC50 = 14 nM and 13 nM, respectively) in a dose-dependent manner. In a panel of 46 related kinases (e.g., AMPK, MARK family), WZ4003 (tested at 1 μM and 10 μM) showed >90% inhibition only for NUAK1/2, demonstrating high target selectivity [1] - HEK293 cell-based NUAK1 downstream signaling inhibition: HEK293 cells transiently transfected with FLAG-tagged NUAK1 were treated with WZ4003 (0.1–3 μM for 2 hours). Western blot analysis revealed a concentration-dependent reduction in phosphorylation of MYPT1 (Thr696), a direct downstream substrate of NUAK1. At 1 μM, WZ4003 inhibited MYPT1 phosphorylation by ~70% compared to vehicle control, with no effect on total MYPT1 or FLAG-NUAK1 expression [1] - A549 lung cancer cell proliferation inhibition: A549 cells (which endogenously express NUAK1/2) were treated with WZ4003 (0.1–10 μM) for 72 hours. Cell viability assays (MTT-based) showed a dose-dependent decrease in proliferation, with an IC50 of ~1.2 μM. This antiproliferative effect was associated with reduced MYPT1 phosphorylation (Thr696) in A549 cells, confirming on-target activity [1] - EGFR-related activity: No in vitro data reported; WZ4003 is not evaluated in the EGFR T790M inhibitor study [2] |
| ln Vivo |
NA
|
| Enzyme Assay |
The reactions take place in a 50 μL reaction volume for 30 min at 30°C, and they are stopped by sprinkling 40 μL of the reaction mix onto P81 paper and then immediately submerging it in 50 mM orthophosphoric acid. Three 50 mM orthophosphoric acid washings are performed on the samples, followed by one acetone rinse and air drying. Cerenkov counting is used to measure the [γ-32P]ATP incorporation into Sakamototide caused by kinases. One unit of activity is the amount of time it took to catalyze the incorporation of 1 nmol of [32P]phosphate into the substrate over the course of one hour.
Recombinant NUAK1/2 kinase activity assay: Purified recombinant human NUAK1 (residues 1–522) or NUAK2 (residues 1–513) was incubated in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 200 μM ATP, 0.1 mg/mL BSA, and a synthetic peptide substrate (KKKLRRASLG, a NUAK-specific substrate). WZ4003 was added at serial concentrations (0.1 nM–10 μM) and pre-incubated with the enzyme for 10 minutes at room temperature before initiating the reaction with ATP/substrate. The reaction was incubated at 30°C for 60 minutes, then terminated with 3% phosphoric acid. Phosphorylated substrate was detected using a colorimetric assay (via binding to a phosphospecific antibody), and IC50 values were calculated from dose-response curves [1] - Kinase selectivity panel assay: WZ4003 was tested at 1 μM and 10 μM against a panel of 46 recombinant kinases (including AMPKα1, AMPKα2, MARK1–4, BRSK1/2, etc.). Each kinase assay followed a similar format to the NUAK1/2 assay (with kinase-specific substrates and reaction conditions). Inhibition efficiency was calculated as (1 – (phosphorylated substrate signal with WZ4003 / signal without WZ4003)) × 100%. Only NUAK1/2 showed >90% inhibition at 1 μM, confirming selectivity [1] |
| Cell Assay |
In 96-well plates, colorimetric cell proliferation assays are conducted. U2OS cells and MEFs are initially seeded with 2000 and 3000 cells, respectively, per well. Over the course of five days, the proliferation assays are performed with or without 10 M HTH-01-015 or WZ4003.
HEK293 cell NUAK1 signaling assay: HEK293 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded in 6-well plates (5 × 105 cells/well) and transfected with 2 μg of FLAG-NUAK1 plasmid using a transfection reagent. Twenty-four hours post-transfection, cells were treated with WZ4003 (0.1, 0.3, 1, 3 μM) or vehicle (DMSO, 0.1% final concentration) for 2 hours. Cells were then lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-MYPT1 (Thr696), total MYPT1, and FLAG (to detect NUAK1). Chemiluminescent signals were quantified using densitometry, and the ratio of phospho-MYPT1 to total MYPT1 was calculated [1] - A549 cell proliferation assay: A549 cells were maintained in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin. Cells were seeded in 96-well plates (2 × 103 cells/well) and allowed to adhere overnight. WZ4003 was added at serial concentrations (0.1, 0.3, 1, 3, 10 μM) in triplicate, with vehicle (DMSO, 0.1%) as control. After 72 hours of incubation at 37°C (5% CO2), MTT reagent (5 mg/mL) was added to each well (10 μL/well), and incubation continued for 4 hours. The medium was removed, and 100 μL of DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured, and cell viability was calculated as (absorbance of treated wells / absorbance of control wells) × 100%. IC50 values were derived from non-linear regression of dose-response curves [1] |
| Animal Protocol |
NA;
|
| References | |
| Additional Infomation |
WZ4003 is the first reported selective small molecule inhibitor that inhibits NUAK1 and NUAK2, both of which are AMPK-associated kinases activated by the LKB1 tumor suppressor. WZ4003’s selectivity for NUAK1/2 is much higher than that for other AMPK family members, making it a valuable tool for studying the physiological and pathological effects of NUAK kinases (e.g., their role in cell survival, metabolism, and cancer progression)[1]. NUAK1/2 is overexpressed in a variety of human cancers, including lung cancer, as observed in A549 cells. The inhibitory effect of WZ4003 on these cancers suggests potential therapeutic value in cancers with dysregulated NUAK signaling pathways, but there is currently no clinical development data available[1].
|
| Molecular Formula |
C25H29CLN6O3
|
|---|---|
| Molecular Weight |
496.9892
|
| Exact Mass |
496.199
|
| Elemental Analysis |
C, 60.42; H, 5.88; Cl, 7.13; N, 16.91; O, 9.66
|
| CAS # |
1214265-58-3
|
| Related CAS # |
1214265-58-3
|
| PubChem CID |
72200024
|
| Appearance |
White to beige solid powder
|
| Density |
1.3±0.1 g/cm3
|
| Index of Refraction |
1.639
|
| LogP |
4.1
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
8
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
35
|
| Complexity |
667
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
SDGJBAUIGHSMRI-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C25H29ClN6O3/c1-4-23(33)28-17-6-5-7-19(14-17)35-24-20(26)16-27-25(30-24)29-21-9-8-18(15-22(21)34-3)32-12-10-31(2)11-13-32/h5-9,14-16H,4,10-13H2,1-3H3,(H,28,33)(H,27,29,30)
|
| Chemical Name |
N-[3-[5-chloro-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]propanamide
|
| Synonyms |
WZ 4003; WZ4003; WZ-4003
|
| 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: ~7 mg/mL (~14.1 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.03 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0121 mL | 10.0606 mL | 20.1211 mL | |
| 5 mM | 0.4024 mL | 2.0121 mL | 4.0242 mL | |
| 10 mM | 0.2012 mL | 1.0061 mL | 2.0121 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.
|
|
|
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
