| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
| Targets |
MLKL (Kd = 9.3 μM); VEGFR2 (IC50 = 2 nM)
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|---|---|
| ln Vitro |
In a dose-dependent manner, GW806742X (0.1-10000 nM) prevents the necroptotic mortality of wild-type mouse dermal fibroblasts (MDFs) treated with TSQ (1 ng/mL TNF, 500 nM compound A (Smac mimic), and 10 μM Q-VD-OPh)[1]. GW806742X exhibits an IC50 of 5 nM for VEGF-induced suppression of HUVEC proliferation[2].
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| Enzyme Assay |
A thermal stability shift assay was performed to screen for small molecule interactors with the MLKL pseudokinase domain. Recombinant mouse MLKL pseudokinase domain (residues 179-464) at 2.6 µM was used. ATP (positive control) was added at 0.2 mM, and small molecules from the screening library, including compound 1, were added at a final concentration of 40 µM. Changes in protein thermal stability upon ligand binding were monitored. [1]
Surface Plasmon Resonance (SPR) was used to characterize the binding kinetics of compound 1 to the MLKL pseudokinase domain. The recombinant MLKL protein was captured on a sensor chip via Ni2+/NTA chelation. Compound 1, at concentrations ranging from 6.25 to 200 µM, was flowed over the chip. Sensorgrams were obtained, and the binding data were globally fitted to a two-state kinetic interaction model to determine the equilibrium dissociation constant (Kd). [1]
Saturation transfer difference NMR (STD-NMR) experiments were conducted to probe the binding site of compound 1 on MLKL. The competition between compound 1 and nucleotides (ATP or ADP) for binding to the MLKL pseudokinase domain was assessed by observing changes in the NMR signals. [1]
Thermal shift assays were also used to compare the binding of compound 1 to wild-type and mutant (K219M) MLKL pseudokinase domains, which has impaired nucleotide binding. Reduced thermal stabilization of the K219M mutant by the compound supported binding site specificity. [1]
In vitro kinase assays were performed to test if compound 1 affected the activity of the upstream kinase RIPK3. Recombinant RIPK3 kinase domain was incubated with recombinant MLKL protein in the presence or absence of 10 µM compound 1 and ATP. The reactions were analyzed by autoradiography or mass spectrometry to assess RIPK3 autophosphorylation and MLKL phosphorylation. [1]
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| Cell Assay |
Wild-type mouse dermal fibroblasts (MDFs) were used to assess the inhibitory effect of compound 1 on necroptosis. Cells were pre-treated with varying concentrations of compound 1 (or vehicle control) and then stimulated with the necroptotic stimulus TSQ (TNF, Smac mimetic, and the pan-caspase inhibitor Q-VD-OPh). Cell death was quantified after 24 hours by measuring propidium iodide (PI) uptake using flow cytometry. Dose-response curves were generated to determine IC50 values. [1]
To test specificity, MDFs were also treated with the apoptotic stimulus TS (TNF + Smac mimetic, without caspase inhibitor) in the presence of compound 1, and cell death was similarly quantified by PI uptake and flow cytometry. [1]
To determine if compound 1 acted upstream or downstream of MLKL activation, MDFs stably expressing an inducible construct of the constitutively active MLKL N-terminal domain (MLKL(1-180)) were used. Cells were induced to express MLKL(1-180) with doxycycline in the presence or absence of compound 1, and cell death was measured by PI uptake and flow cytometry. [1]
To investigate the mechanism of action, wild-type MDFs were pre-incubated with 1 µM compound 1 or DMSO control for 1 hour, then stimulated with TSQ. At various time points (over 6 hours), cells were harvested and subjected to subcellular fractionation using digitonin to separate cytoplasmic and crude membrane fractions. The distribution of endogenous MLKL in these fractions was analyzed by immunoblotting, with GAPDH and VDAC1 serving as markers for cytoplasmic and membrane fractions, respectively. [1]
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| References |
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| Additional Infomation |
Compound 1 (GW806742X) was identified from a library of 367 small molecules (the Published Kinase Inhibitor Set) based on its ability to bind the MLKL pseudokinase domain. [1]
It is described as an ATP mimetic that binds the nucleotide binding site (or "pseudoactive" site) of the MLKL pseudokinase. [1]
The compound provides proof-of-concept that the nucleotide binding sites of pseudokinases, a largely unexplored class of therapeutic targets, can be targeted by small molecules to modulate signaling pathways. [1]
Although compound 1 has been previously reported as a nanomolar inhibitor of the protein kinase VEGFR2, control experiments with sorafenib (a potent VEGFR2, Ret, and c-Kit inhibitor) showed that inhibition of these kinases did not block necroptosis in MDFs, supporting MLKL as the relevant target for its necroptosis-inhibitory activity in this context. Off-target effects at high concentrations (>5 µM) cannot be excluded. [1]
The proposed mechanism of action is that binding of compound 1 to the pseudokinase domain "jams" the molecular switch mechanism of MLKL. This prevents the RIPK3-mediated phosphorylation from inducing the conformational change necessary to unleash the N-terminal four-helix bundle (4HB) death effector domain, thereby retarding its oligomerization, membrane translocation, and subsequent induction of necroptotic cell death. [1]
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| Molecular Formula |
C25H23CLF3N7O4S
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|---|---|
| Molecular Weight |
610.01
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| Exact Mass |
609.1172856
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| Related CAS # |
GW806742X;579515-63-2
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| PubChem CID |
155882743
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
41
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| Complexity |
922
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN(C1=CC=C(C=C1)NC(=O)NC2=CC=C(C=C2)OC(F)(F)F)C3=NC(=NC=C3)NC4=CC(=CC=C4)S(=O)(=O)N.Cl
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| InChi Key |
RXKWLRYRNSVTJY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H22F3N7O4S.ClH/c1-35(22-13-14-30-23(34-22)31-18-3-2-4-21(15-18)40(29,37)38)19-9-5-16(6-10-19)32-24(36)33-17-7-11-20(12-8-17)39-25(26,27)28;/h2-15H,1H3,(H2,29,37,38)(H,30,31,34)(H2,32,33,36);1H
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| Chemical Name |
1-[4-[methyl-[2-(3-sulfamoylanilino)pyrimidin-4-yl]amino]phenyl]-3-[4-(trifluoromethoxy)phenyl]urea;hydrochloride
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| Synonyms |
GW806742X hydrochloride; GW806742X HCl; GW-806742X HCl; GW 806742X; NSC756366 HCl; NSC 756366 HCl; NSC-756366; GW806742X (hydrochloride); 2930350-95-9;
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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 (~163.93 mM)
H2O :< 0.1 mg/mL |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.10 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 (4.10 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 | 1.6393 mL | 8.1966 mL | 16.3932 mL | |
| 5 mM | 0.3279 mL | 1.6393 mL | 3.2786 mL | |
| 10 mM | 0.1639 mL | 0.8197 mL | 1.6393 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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