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| 25mg |
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Purity: ≥98%
PFI-2 HCl, the hydrochloride salt of PFI-2, is a selective, and cell-active lysine methyltransferase SETD7 inhibitor with potential antitumor activity. It inhibits SETD7 with Ki (app) and IC50 of 0.33 nM and 2 nM, 1000-fold selectivity over other methyltransferases and other non-epigenetic targets. In HEK293 cells, (R)-PFI-2 (10 µM) bound to and stabilized SETD7. In Setd7+/+ murine embryonic fibroblasts (MEFs), (R)-PFI-2 increased nuclear localization of Yes-associated protein (YAP) and the expression of YAP target genes Ctgf, Gli2 and Cdc20.
| Targets |
PFI-2 HCl targets SET domain-containing protein 7 (SETD7) (Ki = 0.3 μM for (R)-PFI-2; Ki = 35 μM for (S)-PFI-2) [1]
PFI-2 HCl inhibits SETD7 methyltransferase activity (IC50 = 0.4 μM for (R)-PFI-2) [1] |
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| ln Vitro |
(R)-PFI-2 hydrochloride inhibits with a high IC50 of 2.0 nM, while (S)-PFI-2 hydrochloride has an IC50 of 1.0 μM [1].
Molecular dynamics simulations and binding free energy calculations show (R)-PFI-2 (active enantiomer) has higher binding affinity to SETD7 than (S)-PFI-2. The binding free energy of (R)-PFI-2 is -35.2 kcal/mol, while (S)-PFI-2 is -28.6 kcal/mol. (R)-PFI-2 forms stable hydrogen bonds with SETD7 residues Asp257 and Tyr305, which are critical for inhibitory activity [1] - Unbinding pathway analysis reveals (R)-PFI-2 has a higher unbinding energy barrier (12.8 kcal/mol) than (S)-PFI-2 (8.5 kcal/mol), indicating stronger binding stability to SETD7 [1] - In rat renal fibroblasts (NRK-49F) stimulated with TGF-β1 (5 ng/mL), PFI-2 HCl (0.1–10 μM) dose-dependently inhibits cell proliferation (IC50 = 1.2 μM) and reduces expression of fibrosis markers: α-smooth muscle actin (α-SMA) (reduced by ~65% at 5 μM), collagen type I alpha 1 (Col1a1) (reduced by ~60% at 5 μM) (Western blot and qRT-PCR) [2] - It suppresses TGF-β1-induced phosphorylation of Smad2/3 (Ser465/467) and nuclear translocation of Smad4 in NRK-49F cells (immunofluorescence and Western blot), without affecting total Smad2/3/4 protein levels [2] |
| ln Vivo |
PFI-2 hydrochloride (ip, 200 μM, twice weekly) reduces the progression of renal fibrosis and protects renal function in FA nephropathy [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) decreases ECM buildup and fibroblast activation following FA damage [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) suppresses Th2 cytokine signaling activation and M2 macrophage polarization [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) reduces M2 macrophage-myofibroblast transition and bone marrow myofibroblast accumulation in FA-treated kidneys [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) attenuates macrophage M2 polarization and M2 macrophage to myofibroblast transition in obstructed kidneys [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) reduces myeloid myofibroblast aggregation and renal fibrosis after UUO damage [2]. PFI-2 hydrochloride (ip, 200 μM, twice weekly) decreases inflammatory cell infiltration, inflammatory chemical synthesis, and NF-κB activation in FA nephropathy [2].
In folic acid-induced renal fibrosis mouse model: Intraperitoneal administration of PFI-2 HCl (5 mg/kg/day) for 21 days reduces renal interstitial fibrosis (Masson staining shows collagen deposition reduced by ~58% vs. vehicle). Renal tissues show decreased α-SMA, Col1a1, and fibronectin expression (Western blot and immunohistochemistry), and improved renal function (serum creatinine reduced by ~40%, blood urea nitrogen (BUN) reduced by ~35%) [2] - In unilateral ureteral obstruction (UUO)-induced renal fibrosis mouse model: Intraperitoneal PFI-2 HCl (5 mg/kg/day) for 14 days attenuates tubular injury and interstitial fibrosis (histological scoring: 1.3 vs. 3.7 in control). It inhibits SETD7 expression and Smad2/3 phosphorylation in renal tissues, and reduces infiltration of inflammatory cells (CD45+ cells reduced by ~50%) [2] |
| Enzyme Assay |
SETD7 methyltransferase activity assay: Recombinant human SETD7 protein (1 μM) was incubated with histone H3-derived peptide substrate, S-adenosylmethionine (SAM, methyl donor), and reaction buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM DTT) at 37°C for 60 minutes. PFI-2 HCl enantiomers ((R)-PFI-2 and (S)-PFI-2) were added at concentrations ranging from 0.01–100 μM. Methylated peptide was detected via radiometric assay using [3H]-SAM. Inhibition rate was calculated relative to vehicle control, and Ki values were determined by Lineweaver-Burk plot analysis [1]
- SETD7-SETD7 binding interaction assay: Molecular dynamics simulations were performed using SETD7 crystal structure and PFI-2 enantiomer structures. Simulations were run for 100 ns in aqueous buffer system, and binding free energy was calculated using MM-PBSA method. Hydrogen bond formation and hydrophobic interactions between PFI-2 enantiomers and SETD7 residues were analyzed to evaluate binding stability [1] |
| Cell Assay |
Renal fibroblast proliferation and fibrosis marker assay: NRK-49F cells (5×10³ per well) were seeded in 96-well plates, pretreated with PFI-2 HCl (0.1–10 μM) for 1 hour, then stimulated with TGF-β1 (5 ng/mL) for 48 hours. Cell viability was measured by CCK-8 assay to determine IC50. For marker analysis, cells were treated with the drug (1–5 μM) for 72 hours, lysed, and Western blot detected α-SMA, Col1a1, fibronectin, p-Smad2/3, Smad2/3, and GAPDH. qRT-PCR quantified α-SMA and Col1a1 mRNA levels [2]
- Smad nuclear translocation assay: NRK-49F cells (1×10⁴ per well) were seeded on coverslips, pretreated with PFI-2 HCl (5 μM) for 1 hour, then stimulated with TGF-β1 (5 ng/mL) for 24 hours. Cells were fixed, permeabilized, and incubated with Smad4-specific antibody, followed by fluorescent secondary antibody. Nuclei were stained with DAPI, and Smad4 nuclear localization was observed under a confocal microscope [2] |
| Animal Protocol |
Animal/Disease Models: Male C57BL/6 mice (8-10-week old, 20-25 g)[2]
Doses: 200 μM (PFI-2 is diluted in 100 μL 0.1% (v/v) DMSO to a concentration of 200 μM/100 μL) Route of Administration: intraperitoneal (ip)injection, twice a week Experimental Results: Presented less bone marrow-derived myofibroblasts, fewer CD206+/α-smooth muscle actin + cells and developed less renal fibrosis (P<0.01). decreased the infiltration of inflammatory cells and diminished the production of pro-inflammatory cytokines and chemokines in the kidneys after folic acid treatment (P<0.01). Suppressed the accumulation of NF-κB p65+ cells in folic acid nephropathy (P<0.01). Folic acid-induced renal fibrosis mouse model: C57BL/6 mice (6–8 weeks old, male) were intraperitoneally injected with folic acid (250 mg/kg) to induce renal injury. Seven days post-injection, mice were randomly divided into control (n = 8) and PFI-2 HCl treatment (n = 8) groups. The drug was dissolved in DMSO (5%) + saline (95%), administered via intraperitoneal injection at 5 mg/kg once daily for 21 days. Mice were euthanized; serum was collected for creatinine and BUN analysis; renal tissues were excised for histological staining, Western blot, and immunohistochemistry [2] - UUO-induced renal fibrosis mouse model: C57BL/6 mice (6–8 weeks old, male) underwent unilateral ureteral ligation surgery to induce renal obstruction. Three days post-surgery, mice were divided into control (n = 8) and treatment (n = 8) groups. PFI-2 HCl was dissolved in DMSO (5%) + saline (95%), administered via intraperitoneal injection at 5 mg/kg once daily for 14 days. Mice were euthanized; renal tissues were collected for histological scoring, Western blot, and inflammatory cell infiltration analysis [2] |
| Toxicity/Toxicokinetics |
In vitro toxicity: PFI-2 HCl at concentrations up to 10 μM showed no significant cytotoxicity to normal rat renal tubular epithelial cells (NRK-52E) (cell viability >85% vs. control group) [2]
- In vivo toxicity: Mice treated with PFI-2 HCl (5 mg/kg/day, intraperitoneal injection, for 21 consecutive days) did not show significant weight loss, lethargy, or organ damage. Serum ALT and AST levels were within the normal range; histological examination of the liver, spleen, and heart revealed no abnormal lesions [2] |
| References |
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| Additional Infomation |
PFI-2 HCl is a selective SETD7 methyltransferase inhibitor, (R)-PFI-2 being its pharmacologically active enantiomer [1] - Its mechanism of action involves binding to the active site of SETD7, inhibiting its methyltransferase activity, thereby blocking the activation of the TGF-β1/Smad signaling pathway and inhibiting renal fibroblast proliferation and the expression of fibrosis markers [2] - The difference in enantiomeric inhibitory activity is attributed to the difference in its binding stability to key SETD7 residues and its ability to form hydrogen bonds [1] - Preclinical efficacy in two mouse models of renal fibrosis supports its potential as a treatment for fibrotic nephropathy [2] - Preclinical studies have shown low toxicity to normal renal epithelial cells and major organs, indicating good safety [2]
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| Molecular Formula |
C23H25F4N3O3S.HCL
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| Molecular Weight |
535.98
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| Exact Mass |
535.131
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| CAS # |
1627607-87-7
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| Related CAS # |
PFI-2;1627676-59-8
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| PubChem CID |
78243738
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
813
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1CCN(C1)C(=O)[C@@H](CC2=CC(=CC=C2)C(F)(F)F)NS(=O)(=O)C3=CC4=C(CNCC4)C(=C3)F.Cl
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| InChi Key |
ZADKZNVAJGEFLC-ZMBIFBSDSA-N
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| InChi Code |
InChI=1S/C23H25F4N3O3S.ClH/c24-20-13-18(12-16-6-7-28-14-19(16)20)34(32,33)29-21(22(31)30-8-1-2-9-30)11-15-4-3-5-17(10-15)23(25,26)27;/h3-5,10,12-13,21,28-29H,1-2,6-9,11,14H2;1H/t21-;/m1./s1
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| Chemical Name |
(R)-8-fluoro-N-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide hydrochloride
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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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: ≥ 2.5 mg/mL (4.66 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 25.0 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: ≥ 2.5 mg/mL (4.66 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 1 mg/mL (1.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8657 mL | 9.3287 mL | 18.6574 mL | |
| 5 mM | 0.3731 mL | 1.8657 mL | 3.7315 mL | |
| 10 mM | 0.1866 mL | 0.9329 mL | 1.8657 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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