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| Targets |
PF15 TFA targets the FLT3-ITD mutant protein by binding to it via the FLT3 kinase ligand. Simultaneously, the CRBN ligand binds to the E3 ubiquitin ligase complex. This brings the FLT3-ITD protein into close proximity with the ubiquitin ligase, leading to its ubiquitination and subsequent degradation by the proteasome. Unlike traditional kinase inhibitors that only block the enzymatic activity, PROTACs induce the complete elimination of the target protein, providing a more sustained effect. The selectivity for the FLT3-ITD mutant over wild-type FLT3 is noted. The compound also downregulates FLT3 and STAT5 phosphorylation. In vitro, PF15 TFA significantly inhibits the proliferation of FLT3-ITD positive cells. The compound also shows activity in mouse tumor models, inhibiting tumor growth.
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| ln Vitro |
In vitro, PF15 TFA acts as a potent and selective degrader of the FLT3-ITD mutant protein, with a DC50 of 76.7 nM. It significantly inhibits the proliferation of FLT3-ITD positive cells (e.g., MV4-11 human AML cells). The compound downregulates the phosphorylation of FLT3 and STAT5, indicating that the degradation of FLT3-ITD leads to the disruption of downstream signaling pathways involved in cell survival and proliferation. In cell viability assays, PF15 TFA demonstrates potency against FLT3-ITD-driven leukemia cells. The DC50 is the concentration that degrades 50% of the target protein.
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| ln Vivo |
In vivo, PF15 TFA has been shown to inhibit tumor growth in mouse models of FLT3-ITD-positive leukemia. It is typically administered via intraperitoneal (i.p.) or intravenous (i.v.) injection at doses of 10-50 mg/kg, once daily or every other day, for 2-3 weeks. Tumor volume is measured, and at the end of the study, tumors are excised and analyzed for FLT3 protein levels (by Western blot) to confirm degradation. The compound is well-tolerated at effective doses, as it selectively degrades the mutant FLT3-ITD protein.
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| Enzyme Assay |
A cell-free degradation assay is not typical for PROTACs, as they require the ubiquitin-proteasome system present in living cells. However, a binding assay can be performed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure the binding affinity of the FLT3 ligand to the FLT3 kinase domain. Similarly, the CRBN ligand's binding to CRBN can be assessed. The "hook effect" can be studied using a FRET-based assay with labeled FLT3 and CRBN in the presence of the PROTAC. But the primary mode of action is cellular degradation, not cell-free enzyme inhibition.
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| Cell Assay |
The human FLT3-ITD-positive AML cell line MV4-11 is used for this assay. Cells are cultured in IMDM or RPMI-1640 medium supplemented with 10% FBS. Cells are seeded in 96-well plates (2-3×10⁴ cells/well). PF15 TFA is dissolved in DMSO to prepare a stock solution (10-50 mM) and then serially diluted in culture medium to achieve final concentrations ranging from 0.1 nM to 10 uM (with a final DMSO concentration of <0.1%). The cells are treated for 24-72 h. Cell viability is measured using the CellTiter-Glo luminescent assay or MTT assay. The IC50 is calculated using nonlinear regression. For Western blotting, cells are seeded in 6-well plates (5×10⁵ cells/well) and treated with PF15 TFA at concentrations of 0.1, 1, 10, 100, 1000 nM for 4-24 h. Cells are collected, lysed, and processed for SDS-PAGE and Western blotting. The blot is probed with antibodies against FLT3, phospho-FLT3 (Y591), STAT5, phospho-STAT5 (Y694), and a loading control (beta-actin or GAPDH). The DC50 is determined by densitometric analysis of the FLT3 band relative to the control.
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| Animal Protocol |
Female NOD/SCID or NSG mice (6-8 weeks, n=6-10 per group) are injected intravenously (tail vein) with 1-5×10⁶ MV4-11 cells (FLT3-ITD positive) in 100 microL PBS. Engraftment is confirmed by measuring human FLT3-ITD levels in peripheral blood by qPCR or by flow cytometry for human CD45+ cells (typically 3-4 weeks). When a sufficient leukemia burden is achieved (e.g., >1% human CD45+ in blood), mice are randomized into treatment groups. PF15 TFA is dissolved in a vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween 80, 45% saline) to achieve a clear solution. The compound is administered intraperitoneally (i.p.) at doses of 10, 20, or 50 mg/kg, once daily for 14-21 days. A control group receives vehicle alone. A positive control group can receive a standard FLT3 inhibitor (e.g., gilteritinib, 10 mg/kg). Tumor burden is monitored by measuring the percentage of human CD45+ cells in peripheral blood by flow cytometry every 3-5 days. Splenomegaly (enlarged spleen) is measured at the end of the study. Survival is recorded. At the end of the study (e.g., day 21), mice are euthanized. Spleens, bone marrow (femurs), and peripheral blood are collected. The percentage of human CD45+ cells and FLT3-ITD expression are analyzed by flow cytometry and qPCR. FLT3 protein degradation is confirmed by Western blot of spleen cell lysates.
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| ADME/Pharmacokinetics |
Detailed PK data for PF15 TFA are not available. As a PROTAC molecule, it has a larger molecular weight (>800 Da) and may have lower cell permeability and oral bioavailability compared to small-molecule inhibitors. It is likely administered intravenously or intraperitoneally. The half-life in mice is expected to be short (1-4 hours). The TFA salt helps with solubility. The compound should be stored as a solid at -20degC. For in vivo use, it is formulated in a vehicle containing DMSO, PEG300, Tween 80, and saline.
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| Toxicity/Toxicokinetics |
No formal toxicity data are available. In mouse tumor models, PF15 TFA was well-tolerated at the doses used (up to 50 mg/kg) for up to 21 days, with no significant body weight loss or other signs of overt toxicity reported. As a PROTAC, off-target degradation of other proteins containing the FLT3 ligand-binding domain or CRBN-binding domain is a potential safety concern. However, no such data are available.
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| References | |
| Additional Infomation |
PF15 TFA is a research-grade PROTAC and is not approved for clinical use. It is a selective FLT3-ITD degrader with a DC50 of 76.7 nM. It significantly inhibits the proliferation of FLT3-ITD-positive cells, downregulates FLT3 and STAT5 phosphorylation, and inhibits tumor growth in mouse models. This product is for research use only and not for human therapeutic applications.
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| Molecular Formula |
C46H50F3N13O8
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| Molecular Weight |
969.97
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| Related CAS # |
PF15;2892631-70-6
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| Appearance |
White to off-white solid powder
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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, avoid exposure to moisture. |
| 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 | 1.0310 mL | 5.1548 mL | 10.3096 mL | |
| 5 mM | 0.2062 mL | 1.0310 mL | 2.0619 mL | |
| 10 mM | 0.1031 mL | 0.5155 mL | 1.0310 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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