| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Targets |
GNE-1858 specifically targets Hematopoietic Progenitor Kinase 1 (HPK1, also known as MAP4K1). It acts as an ATP-competitive inhibitor. The compound demonstrates sub-nanomolar potency against wild-type HPK1 with an IC50 of 1.9 nM. It also potently inhibits the active mimetic mutants HPK1-TSEE (IC50 = 1.9 nM) and HPK1-SA (IC50 = 4.5 nM). HPK1 functions as a negative-feedback regulator that dampens T-cell activation.
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| ln Vitro |
Hematopoietic progenitor kinase-1 (HPK1) is a type of protein kinase that is linked to Ste20 and is expressed only in the hematopoietic compartment, which includes T cells, B cells, and dendritic cells. It is a serine/threonine kinase. HPK1 inhibits T cell proliferation and effector function as a negative-feedback regulator of T cell receptor signaling[1].
In cell-free biochemical assays, GNE-1858 demonstrates potent inhibitory activity against wild-type HPK1 (IC50 = 1.9 nM). It also inhibits active mimetic mutants HPK1-TSEE (IC50 = 1.9 nM) and HPK1-SA (IC50 = 4.5 nM). These data confirm its ATP-competitive mechanism and high selectivity for the HPK1 kinase domain. The compound is inactive against a panel of other kinases, underscoring its selectivity for the HPK1 family. |
| ln Vivo |
Specific in vivo activity data for GNE-1858 is not detailed in standard databases. As a potent and ATP-competitive HPK1 inhibitor, it is hypothesized to enhance T-cell activation and anti-tumor immunity in vivo. Typically, HPK1 inhibitors are expected to augment IL-2 production and T-cell proliferation in response to TCR stimulation. It may be used in syngeneic tumor models to evaluate single-agent anti-tumor efficacy or in combination with checkpoint inhibitors like anti-PD-1.
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| Enzyme Assay |
The specific in vitro protocol for GNE-1858 uses a homogeneous time-resolved fluorescence (HTRF) kinase assay. Recombinant human HPK1 protein is incubated with a serial dilution of GNE-1858 (0.001 nM to 10 uM) in assay buffer containing ATP (at Km concentration) and a biotinylated substrate peptide. After a 60-minute incubation at room temperature, the reaction is stopped. Streptavidin-XL665 and an anti-phospho-substrate antibody labeled with Europium cryptate are added. The HTRF signal is measured after a 60-minute incubation, and IC50 values are calculated using a non-linear regression algorithm.
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| Cell Assay |
For in vitro cellular assays, primary human CD4+ or CD8+ T cells are isolated from healthy donor PBMCs using magnetic bead-based negative selection. T cells are cultured in 96-well plates pre-coated with anti-CD3 antibody (1 ug/mL) and soluble anti-CD28 antibody (1 ug/mL). GNE-1858 is added at concentrations ranging from 0.1 nM to 1 uM. After 72 hours of culture, cell culture supernatants are collected, and IL-2 and IFN-gamma levels are measured by ELISA. T-cell proliferation is assessed by [3H]-thymidine incorporation over the final 18 hours of culture.
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| Animal Protocol |
An in vivo efficacy protocol would involve the MC38 syngeneic mouse model. C57BL/6 mice are implanted subcutaneously with 5e5 MC38 colon carcinoma cells. When tumors reach a mean volume of 100-150 mm3, mice are randomized into treatment groups. GNE-1858 is administered orally at doses of 10-100 mg/kg twice daily (BID) for 14-21 days. Control groups receive vehicle or an isotype antibody. Tumor volume is measured every 2-3 days with calipers. At study termination, tumors and spleens are collected for flow cytometry analysis to evaluate CD8+ T-cell infiltration, activation markers (CD44, CD69), and cytokine production after ex vivo restimulation.
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| ADME/Pharmacokinetics |
Detailed PK data for GNE-1858 in published literature is limited. As a small molecule ATP-competitive HPK1 inhibitor, a standard PK study would be conducted in male C57BL/6 mice. The compound is administered orally at a dose of 10 mg/kg. Plasma samples are collected at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose). GNE-1858 concentrations are quantified using a sensitive LC-MS/MS method. Non-compartmental analysis is used to determine T1/2, Cmax, Tmax, AUC, and oral bioavailability to guide in vivo efficacy study dosing.
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| Toxicity/Toxicokinetics |
Specific toxicology data for GNE-1858 is not available. As an HPK1 inhibitor that relieves a negative checkpoint in T cells, the primary class-related toxicity is immune-mediated inflammation. Overactivation of the immune system could potentially lead to cytokine release syndrome or autoimmunity. Standard exploratory toxicology would involve a 7-day repeat-dose oral toxicity study in mice to identify the maximum tolerated dose (MTD), and a broader kinase selectivity panel to evaluate off-target inhibition of related kinases.
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| References | |
| Additional Infomation |
GNE-1858 is a research-grade compound and is not approved for clinical use. Its molecular formula is C21H26F2N8 with a molecular weight of 428.48. It is soluble in DMSO (20 mg/mL). HPK1 is a target of interest for cancer immunotherapy, and GNE-1858 serves as a tool to validate this approach. The compound's structure and activity are published in the journal Structure (2019).
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| Molecular Formula |
C21H26F2N8
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| Molecular Weight |
428.48
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| Exact Mass |
428.224
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| CAS # |
2680616-96-8
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| PubChem CID |
146026186
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
31
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| Complexity |
622
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=NC(NC2C=C(C3CCNC3)N=C(N3CCC(F)(F)C3)N=2)=CC2N(C(C)C)N=CC1=2
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| InChi Key |
JKPAJCPZQIJPPS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H26F2N8/c1-13(2)31-17-8-18(25-10-15(17)11-26-31)28-19-7-16(14-3-5-24-9-14)27-20(29-19)30-6-4-21(22,23)12-30/h7-8,10-11,13-14,24H,3-6,9,12H2,1-2H3,(H,25,27,28,29)
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| Chemical Name |
N-[2-(3,3-difluoropyrrolidin-1-yl)-6-pyrrolidin-3-ylpyrimidin-4-yl]-1-propan-2-ylpyrazolo[4,3-c]pyridin-6-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) |
DMSO: 20 mg/mL (46.68 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2 mg/mL (4.67 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 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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. Solubility in Formulation 2: ≥ 2 mg/mL (4.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.0 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. View More
Solubility in Formulation 3: ≥ 2 mg/mL (4.67 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 | 2.3338 mL | 11.6692 mL | 23.3383 mL | |
| 5 mM | 0.4668 mL | 2.3338 mL | 4.6677 mL | |
| 10 mM | 0.2334 mL | 1.1669 mL | 2.3338 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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