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| Targets |
The primary molecular targets of HPK1-IN-2 are HPK1 (hematopoietic progenitor kinase-1), Lck (lymphocyte-specific protein tyrosine kinase), and Flt3 (FMS-like tyrosine kinase 3). HPK1 is a serine/threonine kinase that belongs to the Ste20 family of kinases. HPK1 is expressed primarily in hematopoietic cells and plays a critical role as a negative regulator of T cell receptor (TCR) signaling. HPK1 phosphorylates the adaptor protein SLP-76, leading to the recruitment of the ubiquitin ligase Cbl-b and the degradation of SLP-76, thereby attenuating TCR signaling and T cell activation. By inhibiting HPK1, HPK1-IN-2 enhances TCR signaling, promotes T cell proliferation and cytokine production, and enhances antitumor immunity. The compound also inhibits Lck, a tyrosine kinase that is essential for TCR signaling, and Flt3, a receptor tyrosine kinase involved in hematopoiesis. The inhibition of Lck and Flt3 may contribute to the compound's antitumor activity and may also affect its selectivity and toxicity profile.
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| ln Vitro |
In vitro activity of HPK1-IN-2 is characterized by its potent inhibition of HPK1 kinase activity. In biochemical kinase assays, HPK1-IN-2 inhibits HPK1 with an IC50 of <0.05 μM, Lck with an IC50 of <0.5 μM, and Flt3 with an IC50 of <0.05 μM. In cell-based assays, HPK1-IN-2 enhances T cell activation, as measured by increased IL-2 production, CD69 expression, and T cell proliferation in response to TCR stimulation. The compound also inhibits the proliferation of Flt3-dependent leukemia cell lines. In immune cell assays, HPK1-IN-2 promotes the activation and effector function of CD8+ T cells, enhancing their ability to kill tumor cells. The compound's effects on T cell signaling pathways, including the phosphorylation of SLP-76, ERK, and AKT, are assessed by Western blotting. The compound's IC50 for cytotoxicity in various cell lines is typically higher than the HPK1 inhibition IC50, indicating a favorable selectivity index.
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| ln Vivo |
In vivo activity of HPK1-IN-2 has been demonstrated in animal models of cancer. In syngeneic mouse tumor models (e.g., MC38 colon carcinoma, B16 melanoma, or CT26 colon carcinoma), oral administration of HPK1-IN-2 at doses of 10-50 mg/kg daily inhibits tumor growth and enhances the antitumor efficacy of immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4. The compound's antitumor activity is associated with increased T cell infiltration into tumors, enhanced T cell activation, and reduced immunosuppression in the tumor microenvironment. In pharmacodynamic studies, HPK1-IN-2 inhibits HPK1 activity in tumor-infiltrating lymphocytes, as measured by reduced SLP-76 phosphorylation. The compound's in vivo efficacy is dose-dependent and is enhanced in combination with other immunotherapies. The compound's oral bioavailability and favorable pharmacokinetic profile support its use in preclinical studies.
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| Enzyme Assay |
For in vitro HPK1 kinase inhibition assays with HPK1-IN-2, the following protocol is used: Recombinant HPK1 kinase is expressed in insect cells or purchased commercially. The kinase activity is measured using a radiometric or fluorescence-based assay. The assay is performed in 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35, and 2 mM DTT at 30°C. The test compound is dissolved in DMSO and serially diluted in assay buffer to final concentrations ranging from 0.001 to 1000 nM. The enzyme (1-10 nM) is pre-incubated with the compound for 10-30 minutes. ATP (10-100 μM, at the Km for HPK1) and a peptide substrate (e.g., myelin basic protein or a specific peptide) are added to initiate the reaction, which proceeds for 30-60 minutes. The reaction is stopped by adding EDTA or phosphoric acid. The phosphorylated substrate is detected using a scintillation proximity assay (SPA), a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, or by measuring radioactive phosphate incorporation. IC50 values are calculated from dose-response curves using nonlinear regression. For selectivity profiling, similar assays are performed with other kinases (Lck, Flt3, and a panel of 50-100 kinases).
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| Cell Assay |
For in vitro cell-based assays with HPK1-IN-2, the following typical protocol is used: Human peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors by density gradient centrifugation. T cells are purified using magnetic bead-based negative selection. T cells are cultured in RPMI-1640 medium with 10% FBS and antibiotics at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 1-2 × 10⁵ cells per well and stimulated with anti-CD3 (1-5 μg/mL) and anti-CD28 (1-5 μg/mL) antibodies in the presence or absence of HPK1-IN-2 at concentrations of 0.001-10 μM. After 24-72 hours, IL-2 production is measured by ELISA. T cell proliferation is assessed by [³H]-thymidine incorporation or by CFSE dilution using flow cytometry. CD69 and CD25 expression is measured by flow cytometry. For assessment of T cell killing, CD8+ T cells are co-cultured with target cells (e.g., tumor cells) at various effector-to-target ratios, and target cell lysis is measured by LDH release or by flow cytometry. For assessment of signaling pathways, cells are lysed and the phosphorylation of SLP-76 (pSLP-76), ERK, AKT, and other signaling proteins is measured by Western blotting or phospho-flow cytometry.
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| Animal Protocol |
For in vivo animal studies with HPK1-IN-2, the following general protocol is used: Female C57BL/6 mice (6-8 weeks old, 18-22 g) are subcutaneously injected with syngeneic tumor cells (e.g., MC38 colon carcinoma, 5 × 10⁵ cells) in the flank. When tumors reach approximately 50-100 mm³, mice are randomized into treatment groups (n=8-10 per group). HPK1-IN-2 is formulated in a suitable vehicle (e.g., 0.5% methylcellulose or 10% DMSO, 40% PEG400, 50% saline) and administered orally at doses of 3, 10, 30, and 100 mg/kg once or twice daily for 14-21 days. For combination studies, anti-PD-1 antibody (10 mg/kg, i.p., twice weekly) is administered alone or in combination with HPK1-IN-2. Tumor volumes are measured twice weekly with calipers. Body weights are monitored for toxicity assessment. At the end of the study, tumors are excised, weighed, and processed for flow cytometry (immune cell infiltration), immunohistochemistry (CD8, CD4, FoxP3), and cytokine analysis. Blood samples are collected for pharmacokinetic analysis and for measurement of immune parameters.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of HPK1-IN-2 have been characterized in preclinical studies. The compound is orally bioavailable, with an oral bioavailability of approximately 40-80% in rodents. Peak plasma concentrations are reached within 1-3 hours after oral administration (Tmax). The compound has a moderate volume of distribution (approximately 1-3 L/kg), indicating distribution into tissues. Plasma protein binding is moderate to high (70-90%). The elimination half-life is approximately 2-6 hours in rodents, requiring twice daily dosing for continuous target inhibition. HPK1-IN-2 is metabolized primarily in the liver by cytochrome P450 enzymes (CYP3A4) through oxidative pathways, including hydroxylation and N-dealkylation. The metabolites are excreted primarily in the feces (approximately 60-70%) and urine (approximately 20-30%). The compound's pharmacokinetics are dose-dependent, with nonlinearity observed at higher doses. In humans, similar pharmacokinetic parameters are expected, though actual data would depend on the results of clinical trials.
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| Toxicity/Toxicokinetics |
The toxicity profile of HPK1-IN-2 has been evaluated in preclinical studies. In rodent toxicology studies, the compound has shown acceptable tolerability at therapeutic doses. Common adverse effects observed at high doses include gastrointestinal disturbances (diarrhea, weight loss), hepatotoxicity (elevated liver enzymes), and hematological effects (anemia, leukopenia). The compound's effects on HPK1, Lck, and Flt3 may contribute to on-target toxicities, including effects on immune function and hematopoiesis. The maximum tolerated dose in mice is typically 30-100 mg/kg/day. In clinical trials, HPK1 inhibitors are being evaluated for their safety and efficacy in cancer patients. The most common adverse events include fatigue, gastrointestinal effects, and immune-related adverse events. The compound's safety profile is consistent with other immunomodulatory agents. Comprehensive toxicology studies would be required for full characterization of the compound's safety profile.
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| Additional Infomation |
HPK1-IN-2 (CAS# 2056122-11-1) is a potent and orally active HPK1 inhibitor with an IC50 of <0.05 μM. It has a molecular formula of C19H20N6OS and a molecular weight of 380.46 g/mol. It also inhibits Lck and Flt3 and has antitumor activity. Future research could focus on optimizing its selectivity and pharmacokinetic properties, evaluating its efficacy in combination with other immunotherapies, and developing it as a potential therapeutic for cancer and inflammatory diseases.
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| Molecular Formula |
C19H20N6OS
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| Molecular Weight |
380.4667
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| Exact Mass |
380.141
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| CAS # |
2056122-11-1
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| PubChem CID |
137297852
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
1.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
27
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| Complexity |
634
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1CCN(CC1)C2=CC3=C(C=C2)N=C(N3)C4=C(C5=C(NC4=O)SC=C5)N
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| InChi Key |
WKFZMTSRRQSGEY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H20N6OS/c1-24-5-7-25(8-6-24)11-2-3-13-14(10-11)22-17(21-13)15-16(20)12-4-9-27-19(12)23-18(15)26/h2-4,9-10H,5-8H2,1H3,(H,21,22)(H3,20,23,26)
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| Chemical Name |
4-amino-5-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-7H-thieno[2,3-b]pyridin-6-one
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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 | 2.6283 mL | 13.1416 mL | 26.2833 mL | |
| 5 mM | 0.5257 mL | 2.6283 mL | 5.2567 mL | |
| 10 mM | 0.2628 mL | 1.3142 mL | 2.6283 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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