| Size | Price | Stock | Qty |
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| Targets |
KDM3B (lysine demethylase 3B). PFI-90 is a selective inhibitor of the histone demethylase KDM3B (also known as JMJD1B), a member of the JmjC domain-containing family of demethylases that removes methyl groups from histone H3 at lysine 9 (H3K9me1/me2). KDM3B is involved in transcriptional activation, spermatogenesis, and cancer. In FP-RMS, PFI-90 inhibits KDM3B activity, which suppresses PAX3-FOXO1 fusion oncoprotein function, an oncogenic driver in alveolar rhabdomyosarcoma.
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| ln Vitro |
In RH4, RH30, OSA-CL, and TC-32 cells, PFI-90 displays defined responses with IC50 values of 812, 3200, 1895, and 1113 nM, respectively [1]. Addition of PFI-90 (3 μM; 24 hours) to RH4 and
In cell-free biochemical assays, PFI-90 directly inhibits KDM3B demethylase activity. The compound shows the highest inhibitory selectivity for KDM3B among multiple histone demethylases (KDMs). Biophysical binding of PFI-90 to KDM3B has been demonstrated using nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR). PFI-90 exhibits no significant inhibitory activity against HDAC1, HDAC2, HDAC3, or PRMT5. It increases global H3K4 and H3K9 methylation levels in cells, consistent with KDM3B inhibition. |
| ln Vivo |
PFI-90 induces apoptosis and myogenic differentiation in FP-RMS cells. In RH4 cells, PFI-90 has an EC50 of 0.9 uM. In RH4, RH30, OSA-CL, and TC-32 cells, PFI-90 displays defined responses with IC50 values of 812, 3200, 1895, and 1113 nM, respectively. At 3 uM, PFI-90 significantly increases cell death. The compound suppresses PAX3-FOXO1 function, inducing apoptosis and reducing RNA Polymerase II activity without affecting PAX3-FOXO1 protein levels or DNA binding. PFI-90 inhibits KDM3B, KDM4B, KDM5A, and KDM6B by 46.8% to 87.1% when used at 10 uM, indicating some off-target activity at high concentrations.
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| Enzyme Assay |
KDM3B enzymatic activity is measured using a luminescent demethylase assay format. Recombinant KDM3B enzyme is incubated with a biotinylated H3K9me2 peptide substrate in the presence of alpha-ketoglutarate, Fe2+, and ascorbate. PFI-90 is added at various concentrations (0.001-100 uM) in assay buffer. After incubation, a detection reagent containing a demethylation-specific antibody and AlphaScreen or HTRF donor/acceptor beads is added. Luminescence or fluorescence signal is measured. IC50 values are calculated from dose-response curves. For binding studies, SPR is performed by immobilizing KDM3B protein and flowing PFI-90 at multiple concentrations (0-1000 nM). Sensorgrams are fitted to a 1:1 binding model to calculate KD. NMR-based binding assays use labeled protein and monitor chemical shift changes upon compound titration.
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| Cell Assay |
Western Blot analysis [1]
Cell Types: RH4 and SCMC Cell Tested Concentrations: 3 μM Incubation Duration: 24 hrs (hours) Experimental Results: Increased apoptosis in RH4 and SCMC cells. FP-RMS cell lines (RH4, RH30, SJCRH30) are cultured in RPMI 1640 or DMEM supplemented with 10-20% FBS. Cells are seeded in 96-well plates at appropriate densities (5,000-20,000 cells/well) and treated with PFI-90 at concentrations ranging from 0.1-100 uM for 48-96 hours. Cell viability is measured using CellTiter-Glo (ATP quantification) or MTT assay. EC50 values are calculated. For apoptosis assessment, cells are treated with PFI-90 (1-10 uM) for 24-48 hours, then stained with Annexin V-FITC and propidium iodide for flow cytometry. Caspase-3/7 activity is measured using luminescent substrates. Myogenic differentiation is evaluated by microscopy to observe multinucleated myotube formation and by Western blotting for myogenic markers (MyoD, myogenin, MHC). For methylation analysis, total histones are extracted from treated cells and H3K4me3, H3K9me2, H3K9me3 levels measured by Western blot or ELISA. |
| Animal Protocol |
In vivo efficacy of PFI-90 has been evaluated in FP-RMS mouse xenograft models. Mice bearing subcutaneous FP-RMS tumors (RH4 or RH30 cells) are treated with PFI-90 via intraperitoneal injection at doses typically ranging from 10-50 mg/kg daily or every other day for 2-3 weeks. Tumor volume is measured twice weekly with calipers. PFI-90 induces apoptosis and cell differentiation, resulting in delayed tumor progression. At study termination, tumors are harvested for analysis of KDM3B inhibition markers (H3K9me2 increase), apoptosis (TUNEL, cleaved caspase-3), and differentiation (myogenin expression). Body weight is monitored as a general indicator of toxicity.
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| ADME/Pharmacokinetics |
Dedicated pharmacokinetic studies for PFI-90 have not been extensively published. Based on its structure and observed in vivo activity in mouse models, PFI-90 is presumed to have sufficient exposure following intraperitoneal administration to achieve target engagement in tumors. The compound is expected to distribute to tissues including tumors. PK parameters such as half-life, Cmax, and oral bioavailability have not been reported in available literature. For research applications, standard formulation using DMSO:PEG300:water or similar vehicles can be used. Detailed PK profiling would be required for clinical development.
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| Toxicity/Toxicokinetics |
Preclinical toxicology data for PFI-90 have not been fully disclosed in publicly available sources. In mouse xenograft studies at efficacious doses (e.g., 10-50 mg/kg IP), PFI-90 is reported to be generally well-tolerated with no significant body weight loss or overt signs of toxicity. Standard laboratory safety practices should be followed. As an epigenetic enzyme inhibitor, PFI-90 may have the potential to alter gene expression profiles, and long-term effects would need to be evaluated in chronic toxicity studies. For research use, the compound should be handled as a potential investigational agent with appropriate precautions.
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| References | |
| Additional Infomation |
PFI-90 is a research-grade chemical probe for studying KDM3B function in rhabdomyosarcoma and other cancer types. The compound was developed as an improved analog of PFI-63 with enhanced solubility and potency. PFI-90 directly targets and binds to metal ions in KDMs' active sites. It suppresses PAX3-FOXO1 oncogenic activity in fusion-positive rhabdomyosarcoma, inducing myogenic differentiation and apoptosis. Combined knockdown of KDM3B and KDM1A phenocopies PFI-90 effects. The compound is derived from patent WO2021101929A1. It is not approved for human therapeutic use.
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| Molecular Formula |
C11H10N4O
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| Molecular Weight |
214.223
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| Exact Mass |
214.085
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| CAS # |
53995-62-3
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| PubChem CID |
12499590
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
1.697
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
16
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| Complexity |
234
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=NC(=C1)C(=O)NNC2=CC=CC=N2
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| InChi Key |
GCZPXNWBXDWHKM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C11H10N4O/c16-11(9-5-1-3-7-12-9)15-14-10-6-2-4-8-13-10/h1-8H,(H,13,14)(H,15,16)
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| Chemical Name |
N'-pyridin-2-ylpyridine-2-carbohydrazide
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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 : ~100 mg/mL (~466.81 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (11.67 mM) 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; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (11.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.6681 mL | 23.3405 mL | 46.6810 mL | |
| 5 mM | 0.9336 mL | 4.6681 mL | 9.3362 mL | |
| 10 mM | 0.4668 mL | 2.3340 mL | 4.6681 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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