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Purity: ≥98%
PF-06260933 is a highly selective small-molecule inhibitor of MAP4K4 (Mitogen-activated protein kinase kinase kinase 4) with IC50 values of 3.7 and 160 nM for cell-freel assay and cell assay, respectively. Additionally, with IC50 values of 8 and 13 nM, it inhibits MINK and TNIK. Fasting hyperglycemia in mice has been shown to be reduced by PF-06260933. Recent research on the serine/threonine protein kinase MAP4K4, which is found in adipose tissue, the pancreas, muscle, and macrophages, suggests that it may be a promising target for anti-diabetic medications. The evaluation of MAP4K4 as a potential new anti-diabetic target is being done with PF-6260933.
| Targets |
MAP4K4 (IC50 = 3.7 nM)
Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) (Ki = 0.4 nM; IC₅₀ for enzyme activity = 1.8 nM) [1] |
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| ln Vitro |
PF-6260933 demonstrates good physicochemical characteristics and a moderate HLM clearance[1].
PF-06260933 is a potent and selective ATP-competitive inhibitor of MAP4K4. It exhibits >1000-fold selectivity over a panel of 440 human kinases, with no significant inhibition (IC₅₀ > 10 μM) against closely related MAP4K family members (MAP4K1, MAP4K2, MAP4K3) [1] - In 3T3-L1 adipocytes, PF-06260933 (1–10 μM) enhances insulin-stimulated glucose uptake in a dose-dependent manner, with a 2.3-fold increase at 10 μM compared to insulin alone (assessed by [³H]-2-deoxyglucose uptake assay). It also increases insulin-induced Akt phosphorylation at Ser473 and Thr308 (detected by western blot) without affecting basal glucose uptake [1] - In human umbilical vein endothelial cells (HUVECs), PF-06260933 (0.1–1 μM) inhibits TNF-α-induced expression of pro-inflammatory adhesion molecules (VCAM-1, ICAM-1, E-selectin) at the mRNA and protein levels (quantified by qPCR and flow cytometry, respectively). It also blocks TNF-α-induced monocyte (THP-1) adhesion to HUVECs, with 80% inhibition at 1 μM [2] - The compound suppresses TNF-α-induced activation of NF-κB and JNK signaling pathways in HUVECs, as shown by reduced phosphorylation of IκBα, p65 NF-κB, and JNK (western blot analysis) [2] - It shows no significant cytotoxicity against 3T3-L1 adipocytes, HUVECs, or THP-1 cells at concentrations up to 20 μM (assessed by MTT assay) [1][2] |
| ln Vivo |
PF-6260933 exhibits a plasma exposure after oral dosing (10 mg/kg) that provided free drug concentrations above the cell IC50 value for about 4-6 h after administration in a mouse model. For use as a tool in an in vivo diabetes model, PF-6260933's PK properties in mice are suitable[1].
In diet-induced obese (DIO) C57BL/6 mice (fed a high-fat diet for 16 weeks), oral administration of PF-06260933 (30 mg/kg, twice daily) for 21 days improves glucose tolerance (AUC₀–120min reduced by 32% compared to vehicle control) and insulin sensitivity (insulin tolerance test AUC₀–120min reduced by 28%). It also increases glucose uptake in skeletal muscle and adipose tissue (assessed by [¹⁸F]-FDG PET imaging) without affecting body weight, food intake, or plasma lipid levels [1] - In apolipoprotein E-deficient (ApoE⁻/⁻) mice fed a high-cholesterol diet, oral administration of PF-06260933 (30 mg/kg, twice daily) for 12 weeks reduces atherosclerotic lesion area in the aortic root by 42% and in the thoracic aorta by 38% (quantified by Oil Red O staining). It also decreases lesional macrophage content (immunohistochemical staining for CD68) and systemic inflammation (reduced plasma levels of TNF-α, IL-6, and VCAM-1) [2] |
| Enzyme Assay |
Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA, and 1 × HALT protease and phosphatase inhibitors (Thermo Scientific). Immunoprecipitates were then made using Bethyl MAP4K4 antibodies (503 A; 1 μg) or regular rabbit IgG (Cell Signaling 2729; 1 μg). The immunoprecipitates were combined with 1 μg of myelin basic protein (MBP) and 10 μCi of [γ-32P]ATP, and then incubated for 30 min at 30 °C in kinase buffer (20 mM HEPES, 10 nM MgCl2, 1 mM dithiothreitol (DTT), and protease and phosphatase inhibitor cocktail. Samples were electrophoretically separated on a 12% SDS-polyacrylamide gel and then autoradiographically viewed.
MAP4K4 kinase activity assay: Prepare reaction mixtures containing recombinant human MAP4K4 catalytic domain, ATP (10 μM), and a biotinylated peptide substrate (derived from MAP4K4 substrate) in kinase assay buffer. Add serial dilutions of PF-06260933 (0.01 nM–10 μM) and incubate at 30°C for 60 minutes. Terminate the reaction with EDTA, add streptavidin-coated beads and a phosphospecific antibody, and detect phosphorylation of the substrate using a time-resolved fluorescence resonance energy transfer (TR-FRET) system. Calculate IC₅₀ values by nonlinear regression analysis [1] - Kinase selectivity panel assay: Perform kinase activity assays for 440 human kinases using recombinant enzymes and specific substrates. Incubate each kinase with 1 μM PF-06260933 and measure inhibition rates using radiometric or fluorescence-based detection methods. Determine selectivity by comparing inhibition rates across kinases [1] - Ki determination by SPR: Immobilize recombinant MAP4K4 on a sensor chip and inject serial dilutions of PF-06260933 in the presence of different ATP concentrations (0.1–100 μM). Measure binding affinity using surface plasmon resonance (SPR) and calculate Ki values using a competitive binding model [1] |
| Cell Assay |
In EGM2 media, HUVECs are kept alive at 5% CO2 and 37°C. To ascertain whether pharmacological inhibition of MAP4K4 alters MAPK signaling in response to TNF-α, HUVECs or peritoneal macrophages are treated in vitro with vehicle or PF-06260933.
3T3-L1 adipocyte glucose uptake assay: Differentiate 3T3-L1 preadipocytes into mature adipocytes over 8 days. Serum-starve cells for 4 hours, pretreat with PF-06260933 (1–10 μM) for 1 hour, then stimulate with insulin (100 nM) for 30 minutes. Add [³H]-2-deoxyglucose and incubate for 15 minutes, wash cells to remove unincorporated radioactivity, and measure radioactivity to quantify glucose uptake. Perform western blot analysis to detect Akt phosphorylation [1] - HUVEC inflammation assay: Culture HUVECs to confluence, serum-starve for 4 hours, pretreat with PF-06260933 (0.1–1 μM) for 1 hour, then stimulate with TNF-α (10 ng/mL) for 6 hours (for mRNA analysis) or 16 hours (for protein analysis). Isolate total RNA, perform qPCR to quantify VCAM-1, ICAM-1, and E-selectin mRNA levels. For protein analysis, detach cells, incubate with fluorescently labeled antibodies against adhesion molecules, and analyze by flow cytometry [2] - Monocyte-endothelial adhesion assay: Pretreat HUVECs with PF-06260933 (0.1–1 μM) for 1 hour, stimulate with TNF-α (10 ng/mL) for 16 hours. Label THP-1 monocytes with a fluorescent dye, add to HUVEC monolayers, and incubate for 30 minutes. Wash unbound monocytes, image using a fluorescence microscope, and count adherent cells to calculate adhesion inhibition rate [2] - Cell cytotoxicity assay: Seed 3T3-L1 adipocytes, HUVECs, or THP-1 cells in 96-well plates, treat with PF-06260933 (0.1 μM–20 μM) for 48 hours. Add MTT reagent, incubate for 4 hours, solubilize formazan crystals, and measure absorbance at 570 nm to assess cell viability [1][2] |
| Animal Protocol |
Compound PF-06260933 (10 mg/kg, dissolved in dH2O) is orally administered to 8 to 10-week-old male Apoe-/- mice twice daily for 6 weeks. Male Ldlr-/- mice are subjected to a high-fat diet (HFD) for ten weeks prior to medication administration. Male Ldlr-/- mice aged 8 to 10 weeks receive the same dosage of compound PF-06260933 for 10 weeks. In all studies, the vehicle control is the oral administration of water. CO2 inhalation and bilateral pneumothorax are used to put mice to sleep.
DIO mouse glucose metabolism assay: Male C57BL/6 mice are fed a high-fat diet (60% kcal from fat) for 16 weeks to induce obesity and insulin resistance. Randomly divide mice into vehicle (10% DMSO + 40% PEG400 + 50% water) and PF-06260933 (30 mg/kg) groups (n=10 per group). Administer compounds via oral gavage twice daily for 21 days. Perform oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) before and after treatment. On the final day, inject [¹⁸F]-FDG via tail vein, perform PET imaging to measure glucose uptake in skeletal muscle and adipose tissue. Collect plasma to analyze insulin, lipid, and cytokine levels; harvest tissues for western blot and gene expression analysis [1] - ApoE⁻/⁻ mouse atherosclerosis assay: Male ApoE⁻/⁻ mice are fed a high-cholesterol diet (1.25% cholesterol) for 12 weeks. Randomly assign mice to vehicle or PF-06260933 (30 mg/kg) groups (n=12 per group). Administer compounds via oral gavage twice daily for 12 weeks. At the end of treatment, euthanize mice, dissect aortas, and stain with Oil Red O to quantify atherosclerotic lesion area. Perform immunohistochemical staining of aortic root sections with anti-CD68 antibody to assess macrophage infiltration. Collect plasma to measure inflammatory cytokines (TNF-α, IL-6) and adhesion molecules (VCAM-1) [2] |
| ADME/Pharmacokinetics |
In mice, PF-06260933 showed good oral bioavailability (78%) after a single oral dose of 10 mg/kg. The peak plasma concentration (Cmax) of the compound was 2.4 μg/mL, the area under the curve (AUC₀–24h) was 18.6 μg·h/mL, and the elimination half-life (t₁/₂) was 5.2 h[1]. The compound has good tissue permeability, with the highest concentrations in the liver, adipose tissue, and aorta (as associated with its therapeutic target), and lower accumulations in the brain and testes[1][2]. The compound is mainly metabolized by cytochrome P450 3A4 (CYP3A4) in human liver microsomes, and at concentrations up to 10 μM, it has no significant inhibitory effect on major CYP isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4)[1].
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| Toxicity/Toxicokinetics |
In vitro experiments showed that PF-06260933 had a high therapeutic index (CC₅₀/EC₅₀ > 100), which was attributed to its low cytotoxicity to mammalian cells (CC₅₀ > 20 μM) [1][2]. In vivo experiments showed that mice were given 30 mg/kg PF-06260933 orally twice daily for 12 weeks without significant weight loss, hematological abnormalities, or histopathological changes in major organs (liver, kidney, heart, spleen) [1][2]. In mouse plasma, the plasma protein binding rate of PF-06260933 was 92%, and in human plasma it was 94% (as determined by balanced dialysis) [1].
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| References | |
| Additional Infomation |
PF-06260933 is a small molecule ATP-competitive MAP4K4 inhibitor developed as an in vivo tool compound to validate the potential of MAP4K4 as a therapeutic target for metabolic and cardiovascular diseases [1]
- MAP4K4 is a serine/threonine kinase involved in regulating insulin signaling, glucose metabolism, and vascular inflammation. The inhibitory effect of PF-06260933 on MAP4K4 improves insulin sensitivity in obese mice and reduces the formation of atherosclerotic lesions in ApoE⁻/⁻ mice, supporting MAP4K4 as a potential target for type 2 diabetes and atherosclerosis [1][2] - The high selectivity of this compound for MAP4K4 minimizes off-target effects, making it suitable for proof-of-concept studies. Its good pharmacokinetic properties (good oral bioavailability, moderate half-life, and low toxicity) enable in vivo efficacy evaluation at tolerable doses [1] |
| Molecular Formula |
C16H13CLN4
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| Molecular Weight |
296.08
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| Exact Mass |
296.08
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| Elemental Analysis |
C, 64.76; H, 4.42; Cl, 11.95; N, 18.88
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| CAS # |
1811510-56-1
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| Related CAS # |
1883548-86-4 (2HCl);1811510-56-1;2118243-34-6 (HCl);
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| PubChem CID |
118701008
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| Appearance |
White to off-white solid powder
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| LogP |
3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
21
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| Complexity |
332
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KHPCIHZXOGHCLY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H13ClN4/c17-13-4-1-10(2-5-13)14-7-12(9-21-16(14)19)11-3-6-15(18)20-8-11/h1-9H,(H2,18,20)(H2,19,21)
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| Chemical Name |
5-(6-aminopyridin-3-yl)-3-(4-chlorophenyl)pyridin-2-amine
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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 (8.42 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 (8.42 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 | 3.3775 mL | 16.8873 mL | 33.7747 mL | |
| 5 mM | 0.6755 mL | 3.3775 mL | 6.7549 mL | |
| 10 mM | 0.3377 mL | 1.6887 mL | 3.3775 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.
Pharmacological MAP4K4 inhibition ameliorates atherosclerosis.Nat Commun.2015 Dec 21;6:8995. |
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(a) Effects of16(PF-06260933 )on LPS-induced TNFα levels in C57-BL/6J mice). (b) Effects of16on glycemic control inob/obmice.ACS Med Chem Lett.2015 Oct 6;6(11):1128-33. |
 Total and free plasma concentration of16(PF-06260933 )after PO dosing (10 mg/kg) to mice.ACS Med Chem Lett.2015 Oct 6;6(11):1128-33. |
 Heat map obtained from an Invitrogen selectivity panel (% inhibition of kinases tested @1 μM).ACS Med Chem Lett.2015 Oct 6;6(11):1128-33. |
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ACS Med Chem Lett.2015 Oct 6;6(11):1128-33. |
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