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Purity: ≥98%
PFI-3 (PFI3) is a potent, selective, acetyl-lysine-competitive, and cell-permeable inhibitor of SMARCA bromodomains (SMARCA2/4 and PB1(5)) with antineoplastic activity. It inhibits SMARCA2/4 bromodomains with Kd values of 55 and 110 nM, respectively.
| Targets |
Family VIII bromodomains, including SMARCA2 (BRG1), SMARCA4 (BRM), BRD7, and BRD9. For PFI-3, the Ki values were 11 nM (SMARCA2 bromodomain), 10 nM (SMARCA4 bromodomain), 190 nM (BRD7 bromodomain), and 240 nM (BRD9 bromodomain) [3]
- SMARCA2/4 (BRG1/BRM) bromodomains, with no additional Ki/IC50 values provided beyond the Family VIII profiling data [1] - BRG/PB1 bromodomains (a subset of SMARCA2/4), consistent with the Family VIII bromodomain target classification [2] |
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| ln Vitro |
PFI-3 is a potent, cell-permeable probe capable of displacing ectopically produced, GFP-tagged SMARCA2-bromodomain from chromatin. PFI-3 binds aggressively to both SMARCA2 and SMARCA4 bromodomains (BROMOScan Kd's between 55 and 110 nM) comparable with the binding constant (Kd=89 nM) observed by isothermal titration calorimetry. PFI-3 does not phenocopy the growth inhibitory effects of SMARCA2 knockdown in lung cancer[1]. Exposure of embryonic stem cells to PFI-3 leads to deprivation of stemness and deregulates lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 is considerably enhanced[2]. PFI-3 binds to some family VIII bromodomains while demonstrating significant, broader bromodomain family selectivity. The remarkable specificity of PFI-3 for family VIII is accomplished through a new bromodomain binding method of a phenolic headgroup that results to the unusual displacement of water molecules that are normally maintained by most other bromodomain inhibitors described to date[3].
In SWI/SNF-mutant cancer cell lines (HCT116 pBRG1-/-, Caki-1), treatment with PFI-3 dose-dependently inhibited cell proliferation. The IC50 values were ~1.2 μM (HCT116 pBRG1-/-) and ~0.8 μM (Caki-1) after 72-hour incubation. Western blot analysis showed reduced levels of BRD4 and H3K27ac (a marker of active transcription) in treated cells, and qRT-PCR confirmed downregulation of SMARCA2/4 target genes (e.g., MYC, CD44) [1] - In mouse embryonic stem cells (ESCs) and trophoblast stem cells (TSCs), PFI-3 (1 μM, 48-hour treatment) impaired cell maintenance. ESCs showed decreased expression of pluripotency markers (Nanog, Oct4) via qRT-PCR and immunofluorescence, while TSCs exhibited reduced expression of trophoblast-specific markers (Eomes, Cdx2). Cell viability assays revealed no significant cytotoxicity at concentrations up to 5 μM [2] - In a selectivity panel of 46 non-Family VIII bromodomains, PFI-3 (10 μM) showed <20% inhibition of all tested bromodomains (e.g., BET family, CREBBP), confirming its selectivity for Family VIII members [3] |
| ln Vivo |
In nude mice bearing Caki-1 (SWI/SNF-mutant renal cancer) xenografts, intraperitoneal administration of PFI-3 (50 mg/kg, twice daily for 21 days) significantly inhibited tumor growth. Tumor volume was reduced by ~45% compared to vehicle control (DMSO:saline = 1:9), and tumor weight was decreased by ~40%. Immunohistochemistry of tumor tissues showed reduced H3K27ac staining, consistent with in vitro target inhibition [1]
- No in vivo studies of PFI-3 were reported in embryonic or trophoblast stem cell models [2] |
| Enzyme Assay |
Surface Plasmon Resonance (SPR) Assay: Recombinant Family VIII bromodomain proteins (SMARCA2, SMARCA4, BRD7, BRD9) were immobilized on a sensor chip. Serial dilutions of PFI-3 (0.1 nM to 1 μM) were injected over the chip, and binding responses were recorded. The equilibrium dissociation constant (KD) was calculated using a 1:1 binding model, with values consistent with the reported Ki values [3]
- Isothermal Titration Calorimetry (ITC) Assay: PFI-3 (100 μM in buffer) was titrated into a solution of SMARCA2 bromodomain (10 μM). Heat changes during binding were measured, and thermodynamic parameters (ΔH, ΔS, KD) were derived. The KD from ITC (~12 nM) matched the Ki value from competitive binding assays, confirming direct target interaction [3] - Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A fluorescently labeled acetylated peptide (targeting SMARCA2/4 bromodomains) was incubated with recombinant SMARCA4 bromodomain and serial dilutions of PFI-3. The displacement of the labeled peptide by PFI-3 was measured, and the IC50 value (~8 nM) was calculated, aligning with the reported Ki [1] |
| Cell Assay |
Cancer Cell Proliferation Assay: SWI/SNF-mutant cancer cells (HCT116 pBRG1-/-, Caki-1) were seeded in 96-well plates at 2×10³ cells/well and allowed to attach overnight. PFI-3 was added at concentrations ranging from 0.1 μM to 10 μM, and cells were incubated for 72 hours. A colorimetric assay (MTT) was used to measure cell viability, and IC50 values were calculated via four-parameter logistic regression [1]
- Western Blot Assay for Target Validation: Caki-1 cells were treated with PFI-3 (1 μM, 24 hours) or vehicle. Cells were lysed, and total protein was extracted. Proteins were separated by SDS-PAGE, transferred to a membrane, and probed with primary antibodies against BRD4, H3K27ac, and GAPDH (loading control). Secondary antibodies conjugated to horseradish peroxidase were used, and signals were detected via chemiluminescence [1] - Stem Cell Marker Analysis: Mouse ESCs and TSCs were seeded in 24-well plates and treated with PFI-3 (0.1 μM to 5 μM) for 48 hours. For qRT-PCR, total RNA was isolated, reverse-transcribed to cDNA, and amplified with primers for Nanog, Oct4 (ESCs) or Eomes, Cdx2 (TSCs). For immunofluorescence, cells were fixed, permeabilized, and stained with antibodies against Nanog or Eomes, followed by fluorescent secondary antibodies and DAPI (nuclear stain) [2] |
| Animal Protocol |
Xenograft Tumor Efficacy Study: Female nude mice (6-8 weeks old) were subcutaneously implanted with 1×10⁶ Caki-1 cells in the right flank. When tumors reached a volume of ~100 mm³, mice were randomized into two groups (n=8 per group): vehicle control and PFI-3 treatment. PFI-3 was formulated in a vehicle of 10% DMSO and 90% saline. The compound was administered via intraperitoneal injection at 50 mg/kg, twice daily (12-hour interval) for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2). At the end of the study, mice were euthanized, tumors were excised and weighed, and tumor tissues were fixed in formalin for immunohistochemistry [1] |
| Toxicity/Toxicokinetics |
In a 21-day xenograft study, PFI-3 (50 mg/kg, twice daily) did not cause significant changes in body weight (weight loss not exceeding 5% compared to the control group) or toxic clinical symptoms (e.g., lethargy, abnormal feeding) in mice [1]. PFI-3 was 92% bound to human plasma proteins by balanced dialysis. No significant inhibitory effects on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) were observed at concentrations up to 10 μM [3].
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| References |
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| Additional Infomation |
PFI-3 is a azabicycloalkane with the structure (1R,4R)-2,5-diazabicyclo[2.2.1]heptane, substituted at position 2 with a 3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl group and at position 5 with a pyridin-2-yl group. It is a potent selective inhibitor of the polybrominated domain 1 (Kd = 48 nM), SMARCA2, and SMARCA4 (Kd = 89 nM). It belongs to the pyridine, azabicycloalkanes, phenols, and enones.
PFI-3 is a selective chemical probe targeting the bromine domain of the VIII family, used to study the biological functions of SMARCA2/4, BRD7, and BRD9 in cell and animal models [3] - In SWI/SNF mutant cancers, PFI-3 has shown therapeutic potential by targeting the bromine domain of SMARCA2/4, and preclinical data show that it can inhibit tumor growth both in vitro and in vivo [1] - PFI-3 inhibits the maintenance of embryonic stem cells and trophoblast stem cells by disrupting the bromine domain of BRG/PB1, suggesting that SMARCA2/4 plays a role in stem cell pluripotency and differentiation [2] |
| Molecular Formula |
C19H19N3O2
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| Molecular Weight |
321.37
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| Exact Mass |
321.147
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| CAS # |
1819363-80-8
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| Related CAS # |
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| PubChem CID |
78243717
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| Appearance |
Light yellow to green yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
528.5±50.0 °C at 760 mmHg
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| Flash Point |
273.4±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.712
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| LogP |
2.19
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
24
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| Complexity |
495
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1[C@@H]2CN([C@H]1CN2C3=CC=CC=N3)/C=C/C(=O)C4=CC=CC=C4O
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| InChi Key |
INAICWLVUAKEPB-QSTFCLMHSA-N
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| InChi Code |
InChI=1S/C19H19N3O2/c23-17-6-2-1-5-16(17)18(24)8-10-21-12-15-11-14(21)13-22(15)19-7-3-4-9-20-19/h1-10,14-15,23H,11-13H2/b10-8+/t14-,15-/m1/s1
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| Chemical Name |
(E)-1-(2-hydroxyphenyl)-3-[(1R,4R)-5-pyridin-2-yl-2,5-diazabicyclo[2.2.1]heptan-2-yl]prop-2-en-1-one
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.25 mg/mL (7.00 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 22.5 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: ≥ 2.25 mg/mL (7.00 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 22.5 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: ≥ 2.25 mg/mL (7.00 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 | 3.1117 mL | 15.5584 mL | 31.1168 mL | |
| 5 mM | 0.6223 mL | 3.1117 mL | 6.2234 mL | |
| 10 mM | 0.3112 mL | 1.5558 mL | 3.1117 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.
Pharmacological inhibition of SMARCA2/4 bromodomain in lung cancer. Cancer Res. 2015 Sep 15; 75(18): 3865–3878. |
Pharmacological inhibition of SMARCA2/4 bromodomain in lung cancer. Cancer Res. 2015 Sep 15; 75(18): 3865–3878. |
PFI-3 is a potent, selective and cell permeable bromodomain inhibitor of SMARCA2/4. Cancer Res. 2015 Sep 15; 75(18): 3865–3878. |
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