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| Other Sizes |
| Targets |
HPK1 <100 nM (IC50)
HPK1-IN-34 specifically targets Hematopoietic Progenitor Kinase 1 (HPK1), a key negative regulator of T-cell receptor signaling. The compound inhibits HPK1 activity, and is reported to have an IC50 of less than 100 nM (<0.1 uM). By blocking HPK1, the compound relieves the immunosuppressive brake on T-cell activation, making it an interesting tool for cancer immunotherapy and immunological research. |
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| ln Vitro |
HPK1-IN-34 is described as an HPK1 inhibitor with an IC50 of less than 100 nM. While specific in vitro characterization data is limited, the presence of an Alkyne group makes this compound a click chemistry reagent. This feature is distinct from its inhibitory function, enabling the compound to be conjugated via copper-catalyzed azide-alkyne cycloaddition (CuAAc) for use in proteomics and target engagement studies.
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| ln Vivo |
Specific in vivo activity data for HPK1-IN-34 has not been published. As an HPK1 inhibitor with an IC50 of less than 100 nM, it is hypothesized to enhance T-cell proliferation and cytokine production in vivo. This could lead to anti-tumor efficacy in murine syngeneic tumor models (e.g., CT26 or MC38). Its ability to serve as a click chemistry probe may allow for the design of in vivo target engagement studies using bio-orthogonal conjugation techniques.
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| Enzyme Assay |
The specific protocol for HPK1-IN-34 involves a radiometric filter-binding kinase assay. Recombinant human HPK1 kinase domain is incubated with varying concentrations of HPK1-IN-34 (1 nM to 100 uM) for 10 minutes at room temperature. The reaction is initiated by adding 10 uM ATP and a radiolabeled substrate peptide. After 60 minutes, the reaction is spotted onto P81 phosphocellulose paper. Unbound radioactivity is washed away, and the filters are dried and analyzed using a scintillation counter. IC50 values are determined from dose-response curves.
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| Cell Assay |
An in vitro cellular assay for HPK1-IN-34 would evaluate T-cell activation in the presence of the compound. Human Jurkat T cells (clone E6-1) are seeded in 96-well plates. Cells are treated with increasing concentrations of HPK1-IN-34 (1 nM to 10 uM) for 30 minutes, then stimulated with PMA (50 ng/mL) and ionomycin (1 uM) for 6 hours. After stimulation, cells are harvested and total RNA is extracted. IL-2 mRNA expression levels are quantified using quantitative real-time PCR (qRT-PCR) with TaqMan probes. HPK1 inhibition should result in increased IL-2 mRNA expression compared to the control.
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| Animal Protocol |
An in vivo protocol for HPK1-IN-34 in a syngeneic tumor model is as follows: Female C57BL/6J mice (8 weeks old) are injected subcutaneously with 5e5 MC38 cells. When tumors reach approximately 100 mm3, mice are randomized into groups. HPK1-IN-34 is administered by oral gavage at doses of 10, 30, and 60 mg/kg twice daily (BID) for 14 consecutive days. Control group receives vehicle (e.g., 10% DMSO/90% (20% SBE-beta-CD in saline)). Tumor volumes are measured every other day with calipers. At termination, tumors are excised, weighed, and processed for flow cytometry to quantify tumor-infiltrating CD8+ T cells and assess their activation status based on CD44 and CD69 expression.
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| ADME/Pharmacokinetics |
Specific PK data for HPK1-IN-34 is not available. Given the presence of an Alkyne group, this compound could be used in bio-orthogonal labeling experiments to study its pharmacokinetics. A standard PK study would be performed in male Sprague-Dawley rats. The compound would be administered via IV (1 mg/kg) and PO (5 mg/kg) routes. Serial blood samples are collected. Levels of HPK1-IN-34 in plasma would be measured by LC-MS/MS. Key parameters such as Cmax, Tmax, T1/2, AUC, clearance, and oral bioavailability (%F) would be calculated.
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| Toxicity/Toxicokinetics |
Toxicology data for HPK1-IN-34 is not available. As an HPK1 inhibitor, the primary safety concern is overactivation of the immune system, potentially leading to autoimmune reactions or cytokine release syndrome. Standard safety evaluation would include a 7-day repeat-dose oral toxicity study in rats at doses of 0, 10, 30, and 100 mg/kg/day. Endpoints include mortality, clinical signs, body weight, food consumption, hematology, serum biochemistry, and histopathological examination of lymphoid organs. Additionally, its Alkyne group is a common functionality, but not typically associated with overt toxicity.
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| References | |
| Additional Infomation |
HPK1-IN-34 is a research-grade compound not approved for human use. Its unique feature is the Alkyne group, which facilitates click chemistry applications for target engagement and proteomic studies. The molecular formula is C25H28N4O2S with a molecular weight of 448.58 and a purity of 97.49%. It is stored at -20degC. HPK1-IN-34 is a valuable probe for studying HPK1 biology in the context of cancer immunotherapy and for developing novel therapeutic strategies.
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| Molecular Formula |
C25H28N4O2S
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| Molecular Weight |
448.580424308777
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| Exact Mass |
448.193
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| CAS # |
2380300-99-0
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| PubChem CID |
156214993
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.9
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
32
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| Complexity |
671
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(C)(O)C#CC1=CC=CC(COC2=CC(C3SC(C4CCNCC4)=NC=3)=CN=C2N)=C1
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| InChi Key |
MKDLEIMJLNBFJO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H28N4O2S/c1-25(2,30)9-6-17-4-3-5-18(12-17)16-31-21-13-20(14-28-23(21)26)22-15-29-24(32-22)19-7-10-27-11-8-19/h3-5,12-15,19,27,30H,7-8,10-11,16H2,1-2H3,(H2,26,28)
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| Chemical Name |
4-[3-[[2-amino-5-(2-piperidin-4-yl-1,3-thiazol-5-yl)pyridin-3-yl]oxymethyl]phenyl]-2-methylbut-3-yn-2-ol
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.2293 mL | 11.1463 mL | 22.2926 mL | |
| 5 mM | 0.4459 mL | 2.2293 mL | 4.4585 mL | |
| 10 mM | 0.2229 mL | 1.1146 mL | 2.2293 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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