MLKL (Kd = 9.3 μM); VEGFR2 (IC50 = 2 nM)
Nucleotide binding site within the MLKL pseudokinase domain (Kd = 9.3 µM) [1]

GW806742X (0.1-10000 nM) inhibits dose-dependently the necroptotic death of TSQ-stimulated wild-type mouse dermal fibroblasts (MDFs) (1 ng/mL TNF, 500 nM compound A (Smac mimetic), 10 μM Q-VD-OPh)[1].
GW806742X shows inhibition of VEGF induced proliferation of HUVECs with an IC50 of 5 nM[2].

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A screening of 367 small molecules using a thermal stability shift assay identified compound 1 as an interactor with the recombinant mouse MLKL pseudokinase domain. [1]
Surface Plasmon Resonance (SPR) analysis validated the binding of compound 1 to the MLKL pseudokinase domain, yielding a dissociation constant (Kd) of 9.3 µM. The kinetics suggested slow off-rates, indicating a relatively stable compound-protein complex. [1]
Saturation transfer difference NMR (STD-NMR) studies indicated that compound 1 competed with ATP or ADP for binding to the MLKL pseudokinase domain, suggesting it binds within the nucleotide binding site. [1]
Thermal shift assays showed that the binding of compound 1 to the K219M MLKL mutant (defective in ATP binding) was diminished compared to wild-type MLKL, further supporting its binding to the nucleotide binding site. [1]
In wild-type mouse dermal fibroblasts (MDFs) stimulated with the necroptotic stimulus TSQ (using 1 ng/mL TNF), compound 1 rescued cells from necroptosis with an IC50 < 50 nM, showing >50-fold greater potency than the established necroptosis inhibitor Necrostatin-1 (Nec-1). [1]
When supraphysiological TNF concentrations (100 ng/mL) were used in the TSQ stimulus, the potency of compound 1 decreased, yielding an IC50 value of 100-500 nM and providing maximal protection for approximately 50% of cells. [1]
Compound 1 affected cell viability at concentrations above 5 µM, likely due to off-target effects. Therefore, 1 µM was used in subsequent mechanistic experiments. [1]
Compound 1 did not protect MDFs from cell death induced by the expression of the constitutive death effector MLKL(1-180), which lacks the pseudokinase domain target. [1]
Compound 1 conferred no significant protection against the predominantly apoptotic death induced by TS stimulation (TNF + Smac mimetic without caspase inhibitor). [1]
Pre-incubation of wild-type MDFs with 1 µM compound 1 retarded the TSQ-induced translocation of endogenous MLKL from the cytoplasm to the membrane fraction over a 6-hour time course, as assessed by cell fractionation and immunoblotting. [1]
In vitro kinase assays using recombinant RIPK3 and MLKL showed that 10 µM compound 1 did not inhibit RIPK3 catalytic activity or RIPK3-mediated phosphorylation of MLKL. In fact, phosphorylation of the MLKL activation loop (residues S345 and S347) was enhanced in the presence of the compound, consistent with increased solvent exposure of the activation loop. [1]

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]

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]

[1]. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci U S A. 2014 Oct 21;111(42):15072-7.

[2]. Discovery of a novel and potent series of dianilinopyrimidineurea and urea isostere inhibitors of VEGFR2 tyrosine kinase. Bioorg Med Chem Lett. 2005;15(15):3519-3523.

Compound 1 (GW806742X) was selected from a library of 367 small molecules (published libraries of kinase inhibitors) based on its ability to bind to the MLKL pseudokinase domain. [1] It is described as an ATP mimic that binds to the nucleotide binding site (or “pseudo-active” site) of the MLKL pseudokinase. [1] This compound validates the concept that the nucleotide binding site of pseudokinases (a class of therapeutic targets that have not been adequately studied) can be targeted by small molecules to modulate signaling pathways. [1] Although compound 1 has previously been reported as a nanomolar inhibitor of the protein kinase VEGFR2, control experiments using sorafenib (a potent VEGFR2, Ret, and c-Kit inhibitor) showed that inhibition of these kinases did not block necroptosis in MDF cells, supporting the view that MLKL is a relevant target for its necroptosis-inhibiting effect in this context. Off-target effects at high concentrations (>5 µM) cannot be ruled out. [1]
The hypothesized mechanism of action is that the binding of compound 1 to the MLKL pseudokinase domain “blocks” the molecular switching mechanism of MLKL. This prevents RIPK3-mediated phosphorylation-induced conformational changes, thus preventing the release of the N-terminal four-helix bundle (4HB) death effector domain, thereby delaying its oligomerization, membrane translocation, and subsequent induction of necrotic cell death. [1]

C25H22F3N7O4S

573.5469

573.14

C, 52.35; H, 3.87; F, 9.94; N, 17.10; O, 11.16; S, 5.59

579515-63-2

GW806742X hydrochloride

5329829

White to off-white solid powder

1.5±0.1 g/cm3

1.666

3.13

4

12

8

40

922

0

S(C1=C([H])C([H])=C([H])C(=C1[H])N([H])C1=NC([H])=C([H])C(=N1)N(C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])OC(F)(F)F)=O)(N([H])[H])(=O)=O

SNRUTMWCDZHKKM-UHFFFAOYSA-N

InChI=1S/C25H22F3N7O4S/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)

1-[4-[methyl-[2-(3-sulfamoylanilino)pyrimidin-4-yl]amino]phenyl]-3-[4-(trifluoromethoxy)phenyl]urea

GW806742-X; GW806742 X; GW806742X

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~174.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.63 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.63 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.63 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)