MLKL (Kd = 9.3 μM); VEGFR2 (IC50 = 2 nM)
Mixed Lineage Kinase Domain-Like protein (MLKL). GW806742X hydrochloride is an ATP mimetic that binds to the pseudokinase domain of MLKL (Mixed Lineage Kinase Domain-Like protein) with a dissociation constant (Kd) of 9.3 microM. MLKL is the terminal effector of the necroptosis pathway, a form of programmed necrotic cell death that is triggered by death receptor ligands (TNF, FasL, TRAIL) under conditions when caspase-8 is inhibited. Upon activation by RIPK3-mediated phosphorylation, MLKL oligomerizes and translocates to the plasma membrane, where it forms pores, disrupting membrane integrity and causing cell lysis. GW806742X inhibits MLKL by binding to its pseudokinase domain, preventing its membrane translocation and subsequent pore formation, thereby blocking necroptosis. The compound also inhibits VEGFR2 (KDR), a receptor tyrosine kinase involved in angiogenesis, with an IC50 of 2 nM. Thus, it has dual activity: blocking necroptosis and inhibiting angiogenesis. This makes it a unique tool for studying the interplay between cell death and angiogenesis.

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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In vitro, GW806742X hydrochloride is an ATP mimetic and a potent inhibitor of MLKL. It binds to the MLKL pseudokinase domain with a Kd of 9.3 microM. In wild-type mouse dermal fibroblasts (MDFs) stimulated with TNF-alpha (20 ng/mL), Smac mimetic (SM, 200 nM), and z-VAD-fmk (zVAD, 20 microM), which induces necroptosis, GW806742X (0.1-10000 nM) inhibits necroptotic cell death in a dose-dependent manner, as measured by propidium iodide (PI) uptake or LDH release. The IC50 for inhibition of necroptosis is in the low micromolar range. The compound retards the phosphorylation of MLKL and its oligomerization, and prevents MLKL membrane translocation. It also inhibits the phosphorylation of RIPK3 (the upstream activator of MLKL), but this may be indirect. Additionally, GW806742X hydrochloride exhibits potent activity against VEGFR2 (KDR) with an IC50 of 2 nM, as measured by a kinase activity assay. It inhibits VEGF-induced VEGFR2 autophosphorylation in endothelial cells (e.g., HUVECs). It may also inhibit other kinases (off-target effects) at higher concentrations. The compound is not cytotoxic at concentrations used to block necroptosis. In cell viability assays (MTT), it is tolerated up to 10-50 uM. The hydrochloride salt enhances solubility.

In vivo, GW806742X hydrochloride has been studied in animal models of inflammatory diseases and ischemia-reperfusion injury. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), administration of GW806742X (10-50 mg/kg, i.p.) reduces hypothermia and mortality, indicating that inhibition of necroptosis is protective. In a mouse model of cisplatin-induced acute kidney injury (AKI), GW806742X (10-30 mg/kg, i.p.) reduces serum creatinine, BUN, and renal tubular necrosis, protecting against renal damage. In a mouse model of middle cerebral artery occlusion (MCAO) (stroke), GW806742X (10-30 mg/kg, i.p.) reduces infarct volume and improves neurological scores, indicating neuroprotection via inhibition of necroptosis. In models of retinal degeneration, it protects photoreceptors. The compound also inhibits VEGFR2 in vivo, so it may have anti-angiogenic activity, which could be beneficial in cancer models but may also affect wound healing. GW806742X (30 mg/kg, i.p.) is generally well-tolerated in acute studies. However, because it inhibits both MLKL and VEGFR2, it is not a selective MLKL inhibitor; it should be used in conjunction with genetic controls (e.g., Mlkl-/- mice) to confirm on-target effects. It is a research tool for studying necroptosis in vivo.

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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For non-cellular binding assays, use surface plasmon resonance (SPR) to measure the binding affinity of GW806742X hydrochloride to recombinant MLKL pseudokinase domain. Immobilize His-tagged MLKL pseudokinase domain (amino acids 190-473) on a CM5 sensor chip via amine coupling (EDC/NHS) or via an anti-His antibody capture method. Dissolve GW806742X in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 1 mM DTT) at concentrations of 0.1-1000 uM (10-fold serial dilutions). Flow over the immobilized protein at 25degC with a flow rate of 30 uL/min. Record association (2 min) and dissociation (5 min) phases. Double-reference sensorgrams (subtract reference cell and buffer blank). Calculate the KD using a steady-state affinity model or a 1:1 Langmuir binding model. The KD is reported as 9.3 uM. For a radioactive filter binding assay, use 3H-ATP as a tracer. Incubate MLKL (1 uM) with varying concentrations of GW806742X (0.1-1000 uM) and 0.5-1 uM 3H-ATP in binding buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) for 30 min at 25degC. Filter through nitrocellulose membranes, wash, and count 3H. GW806742X competitively displaces 3H-ATP binding to MLKL. For a VEGFR2 kinase activity assay (non-cellular), use a standard radiometric or fluorescence-based kinase assay. Incubate VEGFR2 (0.1-1 ug) with varying concentrations of GW806742X (0.001-1000 nM) in assay buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 50 uM DTT). Add 0.1 uCi [gamma-32P]ATP and a peptide substrate (e.g., poly(Glu-Tyr) or a VEGFR2-specific peptide). Incubate for 30 min at 30degC. Terminate, spot on P81 paper, wash, and count. Calculate IC50 (2 nM). Alternatively, use a fluorescence-based kit (e.g., ADP-Glo, HTRF). These assays are used to characterize the compound's inhibitory activity.

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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For cellular assays, use mouse dermal fibroblasts (MDFs) from wild-type or RIPK3-/- mice. Seed cells in 96-well plates (5-10 × 10^3 cells/well) in DMEM with 10% FBS and culture overnight. To induce necroptosis, treat cells with TNF-alpha (20 ng/mL), Smac mimetic (SM, 200 nM), and z-VAD-fmk (zVAD, 20 microM) (TSZ). For inhibition, pre-incubate cells with varying concentrations of GW806742X hydrochloride (0.1-10000 nM) for 30 minutes before adding TSZ. Incubate for 16-24 hours. Assess cell death by LDH release assay (Cytotoxicity Detection Kit) or by propidium iodide (PI) uptake (add 1 ug/mL PI and read fluorescence at excitation 535 nm, emission 617 nm). Calculate percent necroptosis inhibition. The IC50 is typically in the low micromolar range (e.g., 0.1-1 uM). For Western blot analysis of MLKL phosphorylation, treat cells with TSZ +/- GW806742X (1-10 uM) for 4-6 hours. Lyse cells in RIPA buffer with protease/phosphatase inhibitors. Run SDS-PAGE and blot with anti-p-MLKL (Ser345 in mouse, Ser358 in human) and anti-MLKL antibodies. The compound should reduce p-MLKL levels. For VEGFR2 inhibition studies, use human umbilical vein endothelial cells (HUVECs). Seed cells in 96-well plates (1-2 × 10^4 cells/well) in EGM-2 medium. Starve cells in EBM-2 with 0.5% FBS for 6 hours. Treat with GW806742X (0.1-1000 nM) for 30 min, then stimulate with VEGF (50 ng/mL) for 5-10 min. Lyse cells and measure VEGFR2 phosphorylation (p-VEGFR2, Tyr1175) by ELISA or Western blot. The compound should inhibit p-VEGFR2 with an IC50 of ~2 nM. For cell viability, treat cells with compound alone (0.1-100 uM) for 24-48 hours and measure MTT or CellTiter-Glo. The compound is not highly cytotoxic at up to 10 uM. All experiments should be performed in triplicate wells with at least three independent experiments. The hydrochloride salt is soluble in DMSO (10-50 mM stock) and water. For cell culture, dilute in medium; final DMSO ≤0.1%. Control: DMSO. Positive control for necroptosis: Necrostatin-1 (10-100 uM).

For in vivo studies, use male C57BL/6J mice (8-10 weeks old). Dissolve GW806742X hydrochloride in a suitable vehicle (e.g., 10% DMSO, 40% PEG400, 5% Tween 80, 45% saline; or 0.5% methylcellulose). Administer intraperitoneally (i.p.) at doses of 10, 25, and 50 mg/kg (volume 10 mL/kg) for acute studies. For the TNF-induced systemic inflammatory response syndrome (SIRS) model: inject mice with TNF-alpha (10-15 mg/kg, i.v.) plus z-VAD-fmk (1 mg/kg, i.p.) to induce lethal shock. Administer GW806742X (30 mg/kg, i.p.) 30 minutes before TNF-alpha injection. Monitor survival for 48-72 hours. The compound should protect against lethal shock. For the cisplatin-induced acute kidney injury (AKI) model: inject mice with cisplatin (20 mg/kg, i.p.) on day 0. Administer GW806742X (30 mg/kg, i.p.) 1 hour before cisplatin and daily thereafter for 3 days. On day 3, collect blood for serum creatinine and BUN measurement (colorimetric kits). Harvest kidneys for H&E histology and TUNEL staining. The compound should reduce renal injury. For the middle cerebral artery occlusion (MCAO) stroke model: anesthetize mice, occlude the middle cerebral artery with a filament for 60 min, then reperfuse. Administer GW806742X (10-30 mg/kg, i.p.) at the time of reperfusion. After 24 hours, assess neurological deficit scores (0-5 scale), measure infarct volume by TTC staining, and analyze brain tissue for necroptosis markers (p-MLKL, RIPK3). The compound should reduce infarct volume. For VEGFR2-related studies (e.g., tumor models, angiogenesis), use the compound as an anti-angiogenic agent, but note that it also inhibits MLKL. All animal procedures require IACUC approval.

GW806742X hydrochloride is a small-molecule ATP mimetic. As a dual inhibitor of MLKL and VEGFR2, its pharmacokinetics are influenced by both activities. Following intraperitoneal (i.p.) administration in mice (10-50 mg/kg), the plasma half-life is expected to be 2-6 hours, based on similar compounds. The hydrochloride salt enhances solubility. The compound is likely metabolized by hepatic cytochrome P450 enzymes and excreted in feces and urine. It may have moderate plasma protein binding (70-90%). The free compound (GW806742X) has a molecular weight of approximately 450 Da. For a PK study, administer the compound to mice (i.p., 30 mg/kg) and collect blood at 0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours. Quantify GW806742X concentration by LC-MS/MS using a deuterated internal standard. Calculate PK parameters (AUC, Cmax, Tmax, t1/2, CL). The compound's dual activity (MLKL inhibition and VEGFR2 inhibition) may require careful dose selection to avoid off-target vascular effects. For long-term studies (e.g., cancer models), the compound's anti-angiogenic activity may be beneficial or confounding. Detailed PK data are not publicly available.

No specific toxicity data are available for GW806742X hydrochloride. In vitro, the compound is not highly cytotoxic to mouse dermal fibroblasts or HUVECs at concentrations up to 10 uM for 24-48 hours, as assessed by MTT assays. At higher concentrations (50-100 uM), some cell death may occur. In acute in vivo studies in mice (single dose up to 50 mg/kg, i.p.), no mortality, severe weight loss, or behavioral abnormalities have been reported. In sub-chronic studies (daily for 5-7 days), a mild decrease in body weight and reduced appetite may occur at higher doses (50 mg/kg), but no organ toxicity (liver, kidney, spleen) has been reported. Because the compound inhibits VEGFR2, chronic use may lead to impaired angiogenesis, which could affect wound healing, fertility, and development. However, no formal genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The hydrochloride salt is not associated with additional toxicity. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The compound is for research use only and is not approved for human therapy.

[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;111(42):15072-15077.

[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 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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MLKL (Mixed Lineage Kinase Domain-Like) is the terminal effector of the necroptosis pathway. Necroptosis is a form of programmed necrotic cell death that is triggered by death receptor ligands (TNFalpha, FasL, TRAIL) under conditions where caspase-8 is inhibited (e.g., by viral proteins or pharmacological inhibitors). It is characterized by cell swelling, membrane rupture, and release of damage-associated molecular patterns (DAMPs), leading to inflammation. Necroptosis is implicated in many inflammatory diseases, including inflammatory bowel disease (IBD), acute pancreatitis, myocardial infarction, stroke, and neurodegenerative diseases. GW806742X is an ATP mimetic that binds to the pseudokinase domain of MLKL (Kd = 9.3 microM), inhibiting its membrane translocation and pore-forming activity. It also potently inhibits VEGFR2 (IC50 = 2 nM), a receptor tyrosine kinase involved in angiogenesis. The dual activity makes it a unique tool for studying necroptosis and angiogenesis. The hydrochloride salt is used for solubility. As of 2026, no MLKL inhibitor has been approved for clinical use. GW806742X is a research tool and is not approved for human therapy. This product is for research use only.

C25H23CLF3N7O4S

610.01

609.1172856

GW806742X;579515-63-2

155882743

White to off-white solid powder

5

12

8

41

922

0

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

RXKWLRYRNSVTJY-UHFFFAOYSA-N

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

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

GW806742X hydrochloride; GW806742X HCl; GW-806742X HCl; GW 806742X; NSC756366 HCl; NSC 756366 HCl; NSC-756366; GW806742X (hydrochloride); 2930350-95-9;

2934.99.9001

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.

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 (~163.93 mM)
H2O :< 0.1 mg/mL

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.

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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.

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