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| Targets |
HPK1-IN-26 targets hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that is a member of the mammalian Ste-20-related protein kinase family (MAP4K family). HPK1 is predominantly expressed in hematopoietic cells and functions as a negative regulator of T cell receptor (TCR) and B cell receptor (BCR) signaling. It phosphorylates SLP-76 at serine 376, leading to the recruitment of the adaptor protein 14-3-3 and subsequent downregulation of T cell activation, proliferation, and cytokine production. By inhibiting HPK1, HPK1-IN-26 relieves this negative feedback, thereby promoting T cell activation, proliferation, and cytokine release, which is beneficial for anti-tumor immunity. The compound also inhibits GLK (MAP4K3), another member of the MAP4K family involved in T cell function. The dual inhibition may further enhance immune activation. In Jurkat T cells, HPK1-IN-26 inhibits TCR-induced SLP-76 phosphorylation with an IC₅0 of 120 nM, confirming target engagement and pathway modulation.
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| ln Vitro |
In vitro studies have demonstrated that HPK1-IN-26 effectively inhibits HPK1 activity and enhances T cell activation. In a cellular assay using Jurkat T cells (a human T cell leukemia line), the compound inhibits TCR-induced phosphorylation of SLP-76 at serine 376 with an IC₅0 of 120 nM, demonstrating potent pathway engagement. This inhibition relieves the negative regulatory signal, resulting in increased T cell activation markers. In primary human T cells treated with HPK1-IN-26, increased production of cytokines such as IL-2 and IFN-gamma is observed upon TCR stimulation, as quantified by ELISA. The compound also enhances T cell proliferation, measured by [3H]-thymidine incorporation or CFSE dilution. Additionally, HPK1-IN-26 shows anti-inflammatory and immunomodulatory effects, as it can modulate immune cell function. No significant cytotoxicity is observed at effective concentrations (e.g., <1 uM) in activated T cells, indicating a good therapeutic window for immune modulation. Flow cytometry analysis of activation markers (CD25, CD69) confirms the enhanced T cell activation status.
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| ln Vivo |
In vivo efficacy studies with HPK1-IN-26 are ongoing but limited in publicly available sources. As a HPK1 inhibitor, the compound is expected to show anti-tumor activity in syngeneic mouse tumor models when used as a single agent or in combination with checkpoint inhibitors (e.g., anti-PD-1). HPK1 knockout mice exhibit enhanced T cell responses and resistance to tumor growth. Therefore, HPK1-IN-26 treatment in immunocompetent mice bearing established tumors (e.g., MC38 colon carcinoma or B16-F10 melanoma) is expected to reduce tumor growth and prolong survival. The compound can be administered orally or intraperitoneally. Endpoints would include tumor growth inhibition, immune cell infiltration into tumors (via flow cytometry and IHC), and systemic cytokine levels. In models of animal pathogen infection, HPK1-IN-26 may help clear infections by enhancing T cell responses. Further studies are needed to fully characterize its in vivo efficacy and optimal dosing regimens. Detailed in vivo studies have been described in patent WO2021254118A1, where the compound is shown to be effective.
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| Enzyme Assay |
The enzyme activity of HPK1 can be assessed using a radiometric or non-radioactive kinase assay. The protocol for evaluating HPK1-IN-26 involves incubating recombinant HPK1 enzyme (typically a purified active kinase domain) with a peptide substrate (e.g., a biotinylated SLP-76 peptide) and ATP in the presence of increasing concentrations of the compound (0-10 uM). The reaction is carried out in kinase buffer (e.g., 50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM DTT, 0.01% Tween-20) for 60 minutes at room temperature. The reaction is then stopped with EDTA, and the phosphorylated product is detected by time-resolved fluorescence resonance energy transfer (TR-FRET) using a phospho-specific antibody labeled with a donor fluorophore and streptavidin-labeled acceptor. Alternatively, the ADP-Glo kinase assay can be used to quantify ADP production. The IC₅0 value is calculated by plotting inhibition versus log compound concentration. For selectivity, the compound is tested against a panel of related kinases (e.g., GLK, MAP4K4, ZAP70, Lck).
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| Cell Assay |
In vitro cellular potency is assessed using the Jurkat T cell line (clone E6-1). Cells are maintained in RPMI-1640 medium supplemented with 10% FBS and are seeded at 2 × 10⁶ cells/well in 24-well plates. HPK1-IN-26 is added at various concentrations (e.g., 0.01, 0.1, 0.3, 1, 3, 10 uM) and cells are stimulated with anti-CD3/CD28 antibodies for 15-30 minutes to activate the TCR signaling pathway. After stimulation, cells are rapidly lysed in RIPA buffer with protease and phosphatase inhibitors. Western blotting is performed to assess the phosphorylation status of SLP-76 (pSer376) and downstream markers such as ERK and AKT. The DC₅0 (half-maximal inhibition concentration) is calculated by densitometry of the blots. For functional immune readouts, human peripheral blood mononuclear cells (PBMCs) or purified primary T cells are treated with the compound and stimulated with anti-CD3/CD28 for 48-72 hours. Supernatants are collected for cytokine analysis (IL-2, IFN-gamma, TNF-alpha) by ELISA. T cell proliferation is measured by CFSE dilution using flow cytometry. Cell viability is assessed using the CellTiter-Glo assay or trypan blue exclusion.
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| Animal Protocol |
In vivo animal experiments using HPK1-IN-26 would typically utilize syngeneic mouse tumor models in immunocompetent mice (e.g., C57BL/6 mice). A common model is the MC38 colon adenocarcinoma or B16-F10 melanoma model. Female mice aged 6-8 weeks are inoculated subcutaneously with 1 × 10⁶ MC38 or B16-F10 cells. Once tumors reach an average volume of 50-100 mm3, mice are randomized into groups (n=8-10 per group). HPK1-IN-26 is formulated in a suitable vehicle (e.g., 10% DMSO + 40% PEG400 + 50% PBS) and administered orally or intraperitoneally at doses of 10, 30, and 60 mg/kg, once daily or twice daily for 14 days. Control groups receive vehicle alone or an isotype antibody. Tumor volume is measured with calipers every 2-3 days. Body weight is monitored as an indicator of toxicity. At the end of the study, blood is collected for cytokine analysis. Tumors and spleens are collected for immune cell profiling by flow cytometry. Intratumoral immune infiltrates (CD4+ T cells, CD8+ T cells, Tregs, MDSCs) are quantified. T cell activation status (CD44, CD62L, CD69, PD-1) is assessed by flow cytometry. For combination studies, HPK1-IN-26 can be administered together with anti-PD-1 or anti-CTLA-4 antibodies.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for HPK1-IN-26 are not publicly available, but the compound is orally bioavailable according to patent WO2021254118A1. Typical PK parameters for compounds of this molecular weight (367.5 g/mol) and logP (~2-3) would include moderate to high oral absorption (F% >50%), a Cmax achievable at 1-2 hours post-dose, and a half-life of 3-6 hours in mice. The compound is likely metabolized by CYP450 enzymes in the liver, possibly with glucuronidation or sulfation conjugation pathways. The primary route of elimination may be biliary excretion. For accurate PK analysis, an LC-MS/MS method needs to be developed. Standard PK studies in mice or rats would involve administration of a single oral (p.o.) or intravenous (i.v.) dose, with blood collected at multiple time points over 24-48 hours. The compound is typically stored as a powder at -20degC. It is soluble in DMSO for in vitro use and can be formulated in PEG400/water mixtures for in vivo administration. The compound's stability in plasma and liver microsomes should be assessed to estimate metabolic stability.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for HPK1-IN-26 are not available in public literature. As an immunomodulatory agent, potential safety concerns include cytokine release syndrome, autoimmunity, and inflammation due to hyperactivation of T cells. In in vitro cytotoxicity assays, HPK1-IN-26 should be tested on a panel of human cell lines (e.g., Jurkat, HEK293, HepG2) at concentrations up to 10 uM for 72 hours to assess general cytotoxicity. In vivo acute toxicity can be evaluated by administering a single escalating dose (e.g., 30, 100, 300 mg/kg) orally to mice and observing for 14 days. Sub-chronic toxicity (28-day repeated dose) in rats should include hematology, serum chemistry (ALT, AST, BUN, creatinine), and histopathology of major organs (liver, kidney, spleen, heart, lung). No genotoxicity data are available. The compound is for research use only, and standard laboratory safety precautions should be followed. Given its mechanism, special caution should be exercised to avoid inhalation or dermal exposure, as immune modulation may have unintended effects in exposed individuals. Comprehensive safety assessments would be required if this compound were to advance into clinical development.
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| References | |
| Additional Infomation |
HPK1-IN-26 is a research compound for laboratory use only and is not approved for clinical use in humans. It is derived from patent WO2021254118A1 and is primarily used to study the role of HPK1 in immune regulation and cancer immunity. The compound represents a promising tool for developing next-generation cancer immunotherapies, specifically to enhance T cell activity in tumors resistant to PD-1/PD-L1 checkpoint inhibitors. It is typically available with purity >98% and stored as a powder at -20degC, protected from light and moisture. For in vitro experiments, stock solutions (e.g., 10 mM in DMSO) are prepared and stored at -80degC. The compound is soluble in DMSO, with limited aqueous solubility, requiring careful formulation for in vivo work. No clinical trials are registered for this specific compound. As an early-stage research tool, HPK1-IN-26 contributes to the exploration of HPK1 inhibition as a strategy to overcome immune resistance in cancer and infectious diseases. Researchers are advised to consult the original patent WO2021254118A1 for detailed synthetic and characterization data.
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| Molecular Formula |
C19H21N5OS
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| Molecular Weight |
367.467941999435
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| Exact Mass |
367.146
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| CAS # |
2229042-24-2
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| PubChem CID |
122197569
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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 |
26
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| Complexity |
430
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(N)=NC=C(C2SC(C3CCNCC3)=NC=2)C=C1OCC1C=CN=CC=1
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| InChi Key |
HSEGDFMLRPWOHH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H21N5OS/c20-18-16(25-12-13-1-5-21-6-2-13)9-15(10-23-18)17-11-24-19(26-17)14-3-7-22-8-4-14/h1-2,5-6,9-11,14,22H,3-4,7-8,12H2,(H2,20,23)
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| Chemical Name |
5-(2-piperidin-4-yl-1,3-thiazol-5-yl)-3-(pyridin-4-ylmethoxy)pyridin-2-amine
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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 | 2.7213 mL | 13.6066 mL | 27.2131 mL | |
| 5 mM | 0.5443 mL | 2.7213 mL | 5.4426 mL | |
| 10 mM | 0.2721 mL | 1.3607 mL | 2.7213 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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