| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
HPK1-IN-8 targets Hematopoietic Progenitor Kinase 1 (HPK1, MAP4K1). It is an allosteric inhibitor that binds to a pocket consisting of residues both inside and outside the kinase domain, thereby stabilizing the inactive conformation of the full-length enzyme. This binding attenuates kinase autophosphorylation. Its mechanism is distinct from ATP-competitive inhibitors, offering an alternative strategy for blocking HPK1 function.
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| ln Vitro |
In cell-free assays, HPK1-IN-8 functions as an allosteric HPK1 inhibitor that attenuates kinase autophosphorylation. Instead of competing with ATP, it stabilizes the inactive conformation of the full-length HPK1 enzyme. It is described as an “inactive, conformation-selective” inhibitor, indicating that it preferentially binds to and locks the kinase in a catalytically inactive state, preventing its activation in signaling cascades.
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| ln Vivo |
Specific in vivo activity data for HPK1-IN-8 is not published. As an allosteric HPK1 inhibitor, it is predicted to enhance T-cell receptor signaling by preventing HPK1-mediated negative feedback. It is likely to augment IL-2 production and T-cell proliferation in response to TCR stimulation. It could be explored in mouse models of cancer, such as the B16-F10 melanoma or MC38 colon carcinoma models, to assess anti-tumor immunity as a single agent or in combination with checkpoint inhibitors.
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| Enzyme Assay |
The specific protocol for HPK1-IN-8 utilizes a kinase cascade assay as described in the Biochemistry (2021) reference. To assess the stabilization of the inactive HPK1 conformation, a differential scanning fluorimetry (DSF) or thermal shift assay (TSA) is employed. Recombinant full-length HPK1 protein is mixed with a serial dilution of HPK1-IN-8 (0.1-10 uM) and a fluorescent dye (SYPRO Orange). The temperature is increased from 25degC to 95degC at a rate of 1degC per minute. Changes in fluorescence intensity, reflecting protein denaturation, are monitored. An increase in the melting temperature (Tm) indicates binding and stabilization of the inactive conformation by the compound.
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| Cell Assay |
For in vitro cellular assays, Jurkat T cells (a human T lymphocyte cell line) are used to assess HPK1 autophosphorylation. Cells are treated with HPK1-IN-8 at concentrations of 0.1 nM to 1 uM for 2 hours. The cells are then stimulated with anti-CD3 antibody (10 ug/mL) to activate the TCR signaling pathway. After 10 minutes, cells are lysed in RIPA buffer containing protease and phosphatase inhibitors. HPK1 is immunoprecipitated from the lysates using an anti-HPK1 antibody. The immunoprecipitates are analyzed by Western blotting using an anti-phospho-tyrosine antibody (pY) to detect HPK1 autophosphorylation levels, and total HPK1 antibody is used as a loading control.
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| Animal Protocol |
An in vivo protocol to test an allosteric HPK1 inhibitor would involve an ovalbumin (OVA)-specific T-cell adoptive transfer mouse model to measure T-cell priming. CD45.1+ congenic recipient mice are injected intravenously with OVA-specific CD8+ T cells (OT-I cells). The next day, mice are immunized subcutaneously with OVA protein plus CFA adjuvant. HPK1-IN-8 is administered orally at doses of 1-30 mg/kg twice daily for 4 days. On day 4, draining lymph nodes and spleens are harvested. OT-I cell proliferation is assessed by flow cytometry analysis of CFSE dilution or Ki-67 staining. Cytokine production (IFN-gamma, TNF-alpha) is measured by intracellular cytokine staining after ex vivo restimulation with OVA peptide.
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| ADME/Pharmacokinetics |
Specific PK parameters for HPK1-IN-8 are not detailed in standard databases. As an allosteric kinase inhibitor with a distinct binding pocket, a typical oral PK study in C57BL/6 mice would be performed. HPK1-IN-8 is administered as a single oral dose (e.g., 10 mg/kg) in a formulation such as 10% DMSO/90% PEG400. Blood samples are collected at multiple time points up to 24 hours post-dose. Plasma compound concentrations are measured by LC-MS/MS. Key parameters including half-life (T1/2), peak concentration (Cmax), time to peak (Tmax), area under the curve (AUC), and oral bioavailability (%F) would be calculated.
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| Toxicity/Toxicokinetics |
Toxicology data for HPK1-IN-8 is not available. Since HPK1 is predominantly expressed in hematopoietic cells, the primary on-target toxicity would likely be immune-related. Excessive relief of HPK1-mediated negative regulation could theoretically lead to autoimmune pathology. Standard safety assessment would involve a 14-day repeat-dose oral toxicology study in rats, with endpoints including clinical chemistry, hematology (including T-cell subset analysis via flow cytometry), histopathology of lymphoid organs, and gross necropsy.
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| References | |
| Additional Infomation |
HPK1-IN-8 is a research-grade tool compound and is not approved for clinical use. Its discovery as an allosteric, inactive conformation-selective inhibitor was reported in the journal Biochemistry on October 19, 2021. The molecular formula is C19H17FN6O2S, and the molecular weight is 412.44. It is soluble in DMSO. This compound is an important pharmacological tool for dissecting HPK1 biology and validating its therapeutic potential.
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| Molecular Formula |
C19H17FN6O2S
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| Molecular Weight |
412.440685033798
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| Exact Mass |
412.111
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| CAS # |
1214561-09-7
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| PubChem CID |
46969799
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| Appearance |
White to off-white solid powder
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| LogP |
1.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
29
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| Complexity |
798
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C12NN=C(C)N1C(=O)C(CC(NC1=NC=C(CC3=CC=CC=C3F)S1)=O)=C(C)N=2
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| InChi Key |
DKCQZXASVNYNLW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H17FN6O2S/c1-10-14(17(28)26-11(2)24-25-18(26)22-10)8-16(27)23-19-21-9-13(29-19)7-12-5-3-4-6-15(12)20/h3-6,9H,7-8H2,1-2H3,(H,22,25)(H,21,23,27)
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| Chemical Name |
2-(3,7-dimethyl-5-oxo-1H-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)-N-[5-[(2-fluorophenyl)methyl]-1,3-thiazol-2-yl]acetamide
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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: 10 mg/mL (24.25 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4246 mL | 12.1230 mL | 24.2460 mL | |
| 5 mM | 0.4849 mL | 2.4246 mL | 4.8492 mL | |
| 10 mM | 0.2425 mL | 1.2123 mL | 2.4246 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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