| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
IC50: 0.061 nM (HPK1)[1].
HPK1-IN-4 specifically targets Hematopoietic Progenitor Kinase 1 (HPK1/MAP4K1). It is a kinase inhibitor that demonstrates an IC50 of 0.061 nM, indicating picomolar potency. HPK1 is a serine/threonine kinase that acts as a negative feedback regulator of T-cell receptor (TCR) and B-cell receptor (BCR) signaling. It phosphorylates and activates the adaptor protein SLP-76, leading to downstream signals that dampen immune cell activation. |
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| ln Vitro |
HPK1-IN-4 is an extremely potent inhibitor in cell-free enzymatic assays. It inhibits HPK1 activity with an IC50 of 0.061 nM (61 picomolar). This high potency suggests that the compound binds with very high affinity to the HPK1 kinase domain. It is a tool compound for probing HPK1's role in immunological signaling pathways. The compound is used in research to study the effects of HPK1 inhibition on T-cell proliferation.
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| ln Vivo |
Specific in vivo activity data for HPK1-IN-4 has not been published. However, as a preclinical immunotherapy tool compound, it is hypothesized that HPK1-IN-4, by relieving the negative regulatory function of HPK1 on T cells, could enhance T-cell proliferation and cytokine production in vivo. It would likely augment anti-tumor immune responses in syngeneic mouse tumor models, such as MC38 colon carcinoma or B16-F10 melanoma, when used as a single agent or in combination with checkpoint inhibitors.
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| Enzyme Assay |
The specific in vitro protocol for HPK1-IN-4 uses a kinase activity assay, such as the ADP-Glo™ Kinase Assay. Recombinant full-length human HPK1 (MAP4K1) enzyme is incubated with a serial dilution of HPK1-IN-4 (e.g., 0.01 pM to 10 uM) in kinase reaction buffer containing ATP (at Km concentration) and a myelin basic protein (MBP) substrate. After 60 minutes, the reaction is stopped and ADP production is quantified by adding the ADP-Glo reagent followed by Kinase Detection Reagent. Luminescence is measured, and IC50 values are calculated from the concentration-response curves.
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| Cell Assay |
In a typical in vitro cell-based assay, human peripheral blood mononuclear cells (PBMCs) are isolated from healthy donor blood via Ficoll-Paque density gradient centrifugation. PBMCs are seeded in 96-well plates and treated with HPK1-IN-4 (0.1 nM to 1 uM) in the presence of anti-CD3/CD28 antibodies to stimulate T-cell activation. After 72 hours, cell supernatants are collected, and cytokine levels such as IL-2, IFN-gamma, and TNF-alpha are measured using a multiplex ELISA kit. T-cell proliferation is assessed by adding [3H]-thymidine for the last 18 hours of culture, followed by measuring incorporation using a scintillation counter.
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| Animal Protocol |
A representative in vivo protocol for an HPK1 inhibitor involves a syngeneic mouse tumor model. Female C57BL/6 mice (6-8 weeks) are inoculated subcutaneously with MC38 colon carcinoma cells. When tumors reach an average size of 100 mm3, HPK1-IN-4 is administered orally at doses of 1-30 mg/kg twice daily for 14 days. Tumor volumes are measured every 2-3 days with calipers. At the end of the study, tumors and spleens are collected for flow cytometry analysis to assess CD8+ T-cell infiltration and activation.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic parameters for HPK1-IN-4 are not available. As a preclinical tool compound, its properties are likely optimized for in vivo use. A typical oral PK study would be conducted in male C57BL/6 mice. Mice receive a single oral dose of HPK1-IN-4 (e.g., 10 mg/kg). Blood samples are collected via tail vein at multiple time points (0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose). Plasma concentrations are determined by LC-MS/MS. Non-compartmental analysis is used to calculate T1/2, Cmax, Tmax, and oral bioavailability.
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| Toxicity/Toxicokinetics |
Specific toxicity data for HPK1-IN-4 is not available. As an HPK1 inhibitor, the primary safety concern is immune-related adverse events (irAEs) due to hyperactivation of the immune system, similar to checkpoint inhibitors. Standard preclinical toxicology studies would include a 7- or 28-day repeat-dose oral toxicity study in rodents and non-rodents (e.g., beagle dogs). Assessments include clinical observations, body weight, food consumption, hematology (with differential), serum chemistry, and histopathological examination of all major organs, with special focus on lymphoid tissues.
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| References | |
| Additional Infomation |
HPK1-IN-4 is a research-grade tool compound and is not approved for human therapeutic use. It was developed by researchers for preclinical immunology research. Its molecular formula is C23H26N6O3 with a molecular weight of 434.49 and a purity of 99.42%. HPK1-IN-4 is stored as a powder at -20degC. This compound is an important tool for exploring HPK1 as a cancer immunotherapy target.
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| Molecular Formula |
C23H26N6O3
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|---|---|
| Molecular Weight |
434.49
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| Exact Mass |
434.206
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| CAS # |
2739844-28-9
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| PubChem CID |
155925915
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
32
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| Complexity |
625
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(NC2=CC3=C(C=C2OC)CCN(C)C3)=NC=C(C(N)=O)C(NC2=CC=CC=C2CO)=N1
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| InChi Key |
BNZFLUIBCNIAJW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H26N6O3/c1-29-8-7-14-10-20(32-2)19(9-16(14)12-29)27-23-25-11-17(21(24)31)22(28-23)26-18-6-4-3-5-15(18)13-30/h3-6,9-11,30H,7-8,12-13H2,1-2H3,(H2,24,31)(H2,25,26,27,28)
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| Chemical Name |
4-[2-(hydroxymethyl)anilino]-2-[(6-methoxy-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidine-5-carboxamide
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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 |
| 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 (230.15 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.79 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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. Solubility in Formulation 2: ≥ 0.83 mg/mL (1.91 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. View More
Solubility in Formulation 3: ≥ 0.83 mg/mL (1.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3015 mL | 11.5077 mL | 23.0155 mL | |
| 5 mM | 0.4603 mL | 2.3015 mL | 4.6031 mL | |
| 10 mM | 0.2302 mL | 1.1508 mL | 2.3015 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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