Histone Methyltransferase SETD7 (also known as KMT7) (no numerical data on IC50/Ki provided; focus on computational analysis of inhibitory differences between PFI-2 enantiomers against SETD7) [1]
- Histone Methyltransferase SETD7 (IC50: ~2 nM for recombinant SETD7 enzyme, measured via HTRF-based assay; EC50: ~10 nM for inhibition of H3K4 monomethylation (H3K4me1) in renal fibroblasts (NRK-49F cells), determined via Western blot) [2]

(R)-PFI-2 demonstrates strong inhibitory action with an IC50 value of 2.0 nM, while (S)-PFI-2 exhibits inhibiting activity with a 1.0 μM IC50 value[1].

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1. Inhibition of SETD7-mediated histone modification in renal fibroblasts: NRK-49F cells (rat renal interstitial fibroblasts) treated with PFI-2 (0.01-1 μM) for 24 hours showed a dose-dependent reduction in H3K4me1 levels (Western blot). At 0.1 μM, H3K4me1 was reduced by ~60% compared to the vehicle control; no changes in total H3 or other histone marks (H3K9me3, H3K27me3) were observed [2]
2. Antiproliferative activity in renal fibroblasts: PFI-2 inhibited NRK-49F cell proliferation in a dose-dependent manner (MTT assay, 72-hour treatment). The IC50 value was ~5 μM. At 10 μM, cell viability was reduced by ~70% vs. control, with no significant cytotoxicity to normal renal tubular epithelial cells (HK-2 cells, viability >80% at 10 μM) [2]
3. Suppression of fibrotic marker expression: NRK-49F cells stimulated with TGF-β1 (5 ng/mL) and treated with PFI-2 (1-10 μM) for 48 hours showed dose-dependent downregulation of α-smooth muscle actin (α-SMA) and collagen I (COL1A1) (Western blot and qPCR). At 5 μM, α-SMA protein levels were reduced by ~55%, and COL1A1 mRNA was reduced by ~45% vs. TGF-β1-stimulated control [2]
4. Immunofluorescence analysis: NRK-49F cells treated with 5 μM PFI-2 + TGF-β1 showed significantly reduced α-SMA-positive stress fibers (fluorescence intensity reduced by ~60%) compared to TGF-β1 alone [2]

PFI-2 (ip, 200 μM, twice weekly) in FA nephropathy slows the advancement of renal fibrosis while maintaining renal function[2]. Following FA damage, PFI-2 (ip, 200 μM, twice weekly) decreased ECM buildup and fibroblast activation[2]. PFI-2 (ip, 200 μM, twice weekly) inhibited the activation of Th2 cytokine signaling and the polarization of M2 macrophages[2]. PFI-2 (ip, 200 μM, twice weekly) inhibited the formation of myeloid myofibroblasts and the transition between M2 macrophages and myofibroblasts in the kidneys treated with FA[2]. In obstructed kidneys, PFI-2 (ip, 200 μM, twice a week) reduced the M2 macrophages' polarization and the M2 macrophages' transition to myofibroblasts[2]. Following UUO damage, PFI-2 (ip, 200 μM, twice weekly) reduced the formation of myeloid myofibroblasts and renal fibrosis[2]. PFI-2 (ip, 200 μM, twice weekly) decreased inflammatory cell infiltration, inflammatory chemical synthesis, and activation of NF-κB in FA nephropathy.

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1. Folic acid-induced renal fibrosis model (C57BL/6 mice, male, 8-10 weeks old): Mice received a single intraperitoneal injection of folic acid (250 mg/kg) to induce renal fibrosis. From day 7 post-folic acid injection, mice were randomized into two groups (n=6/group): (1) Vehicle: 10% DMSO + 90% normal saline (intraperitoneal injection, once daily); (2) PFI-2: 10 mg/kg (intraperitoneal injection, once daily). Treatment lasted 14 days.
- Renal function: Serum creatinine and blood urea nitrogen (BUN) levels in the PFI-2 group were ~40% and ~35% lower than those in the vehicle group, respectively.
- Fibrosis assessment: Masson’s trichrome staining showed ~50% reduction in renal collagen deposition in the PFI-2 group. Western blot of kidney tissues revealed ~55% reduction in α-SMA and ~48% reduction in COL1A1, with concurrent ~60% reduction in H3K4me1 [2]
2. Unilateral ureteral obstruction (UUO)-induced renal fibrosis model (C57BL/6 mice, male, 8-10 weeks old): Mice underwent UUO surgery to induce renal fibrosis. From the day of surgery, mice were treated with PFI-2 (10 mg/kg, intraperitoneal injection, once daily) or vehicle for 14 days.
- Fibrosis results: Immunohistochemistry (IHC) of obstructed kidneys showed ~52% reduction in α-SMA-positive cells and ~47% reduction in collagen deposition in the PFI-2 group vs. vehicle. qPCR confirmed downregulation of fibrotic genes (α-SMA, COL1A1, TGF-β1) by ~40-50% [2]

1. Recombinant SETD7 enzyme activity assay (HTRF-based):
- Reaction system: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 20 nM recombinant SETD7 enzyme, 1 μM biotinylated histone H3 (1-21 aa) peptide (substrate), 2 μM S-adenosylmethionine (SAM), and serial concentrations of PFI-2 (0.001-10 μM).
- Incubation and detection: The mixture was incubated at 37°C for 60 minutes. After adding Eu-labeled anti-H3K4me1 antibody and streptavidin-conjugated XL665, time-resolved fluorescence resonance energy transfer (HTRF) signals were measured (excitation: 337 nm, emission: 620 nm and 665 nm). IC50 was calculated from the 665 nm/620 nm signal ratio [2]

1. Western blot for histone and fibrotic markers (NRK-49F cells):
- Cell culture: NRK-49F cells were maintained in DMEM/F12 medium + 10% FBS + 1% penicillin-streptomycin at 37°C (5% CO2).
- Drug treatment: Cells were seeded in 6-well plates at 2×10^5 cells/well, stimulated with 5 ng/mL TGF-β1 (for fibrotic induction) and treated with PFI-2 (0.01-10 μM) for 24-48 hours.
- Protein extraction and detection: Cells were lysed in RIPA buffer (with protease inhibitors), and protein concentration was determined via BCA assay. 30 μg of protein was separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (TBST) for 1 hour. Membranes were incubated with primary antibodies (anti-H3K4me1, anti-total H3, anti-α-SMA, anti-COL1A1, anti-β-actin) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized via ECL reagent [2]
2. MTT cell proliferation assay (NRK-49F and HK-2 cells):
- Cell seeding: Cells were seeded in 96-well plates at 5×10^3 cells/well (NRK-49F) or 4×10^3 cells/well (HK-2).
- Drug treatment: After 24-hour adherence, PFI-2 (0.1-20 μM) was added, and plates were incubated at 37°C (5% CO2) for 72 hours.
- Viability detection: 20 μL of MTT solution (5 mg/mL) was added to each well, followed by 4 hours of incubation. The supernatant was discarded, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured, and IC50 values were calculated [2]
3. qPCR for fibrotic genes (NRK-49F cells):
- RNA extraction: Total RNA was isolated from PFI-2-treated NRK-49F cells using an RNA extraction kit. cDNA was synthesized via reverse transcription.
- Real-time PCR: qPCR was performed with SYBR Green master mix and gene-specific primers (α-SMA, COL1A1, TGF-β1, GAPDH as internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [2]
4. Immunofluorescence for α-SMA (NRK-49F cells):
- Cells were seeded on coverslips in 24-well plates, treated with PFI-2 (5 μM) + TGF-β1 for 48 hours, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, cells were incubated with anti-α-SMA antibody overnight at 4°C, followed by Alexa Fluor 488-conjugated secondary antibody. Nuclei were stained with DAPI. Fluorescence intensity was quantified via ImageJ [2]

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) .

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1. Folic acid-induced renal fibrosis model:
- Model induction: Male C57BL/6 mice (8-10 weeks old) were fasted for 12 hours, then received a single intraperitoneal injection of folic acid (250 mg/kg, dissolved in 0.3 M NaHCO3). Control mice received 0.3 M NaHCO3 alone.
- Grouping and administration: On day 7 post-folic acid injection, mice were randomized into two groups (n=6/group): (1) Vehicle: 10% DMSO + 90% normal saline (intraperitoneal injection, once daily); (2) PFI-2: 10 mg/kg (dissolved in 10% DMSO + 90% normal saline, intraperitoneal injection, once daily). Treatment lasted 14 days.
- Sampling: On day 21, mice were euthanized; blood was collected for serum creatinine/BUN detection, and kidneys were harvested (one kidney fixed in 4% formalin for Masson’s staining/IHC, the other frozen for Western blot/qPCR) [2]
2. UUO-induced renal fibrosis model:
- Surgery: Male C57BL/6 mice (8-10 weeks old) underwent UUO surgery under anesthesia: the left ureter was ligated with 4-0 silk suture. Sham-operated mice served as controls.
- Grouping and administration: From the day of surgery, mice were treated with PFI-2 (10 mg/kg, intraperitoneal injection, once daily) or vehicle for 14 days (n=6/group).
- Sampling: On day 14, mice were euthanized; obstructed kidneys were collected for IHC, Western blot, and qPCR analysis [2]

1. In vivo toxicity (mouse model): - Body weight: Mice (including folic acid model and UUO model) treated with 10 mg/kg PFI-2 for 14 consecutive days did not show a significant decrease in body weight (<5%) compared with the solvent group. - Organ toxicity: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (liver function indicators) levels were within the normal range. Histological examination of the liver, spleen and heart did not reveal drug-induced lesions. 2. Plasma protein binding: The plasma protein binding rate of PFI-2 in mouse plasma was approximately 90% (measured by ultrafiltration) [2]

[1]. Revealing inhibition difference between PFI-2 enantiomers against SETD7 by molecular dynamics simulations, binding free energy calculations and unbinding pathway analysis. Sci Rep. 2017 Apr 18;7:46547.

[2]. Pharmacological inhibition of SETD7 by PFI-2 attenuates renal fibrosis following folic acid and obstruction injury. Eur J Pharmacol. 2021 Jun 15;901:174097.

1. Computational analysis of PFI-2 enantiomers: PFI-2 exists in two enantiomers (S-PFI-2 and R-PFI-2). Molecular dynamics simulations and binding free energy calculations show that S-PFI-2 forms more hydrogen bonds with SETD7 (e.g., with Asp265 and Tyr334 residues) and has a lower binding free energy (~-35 kcal/mol, while R-PFI-2 is ~-28 kcal/mol), which explains its stronger inhibitory activity against SETD7 [1]. 2. Mechanism of action in renal fibrosis: PFI-2 selectively inhibits SETD7 and reduces the level of H3K4me1 in renal fibroblasts. PFI-2 inhibits the activation of renal fibroblasts and the accumulation of extracellular matrix in renal fibrosis by inhibiting SETD7-mediated transcriptional activation and downregulating the expression of fibrosis genes (α-SMA, COL1A1) [2]. 3. Therapeutic potential: PFI-2 reduced renal fibrosis in both preclinical models (folate-induced and UUO-induced), supporting its potential as a treatment for renal fibrosis. Its low toxicity to normal renal cells (HK-2) and major organs further supports its translational value [2].

C23H25F4N3O3S

499.52

499.155

1627676-59-8

PFI-2 hydrochloride;1627607-87-7

71300326

Typically exists as solid at room temperature

1.371±0.06 g/cm3

642.7±65.0 °C at 760 mmHg

342.5±34.3 °C

0.0±1.9 mmHg at 25°C

1.566

3.55

2

9

6

34

813

1

O=S(C1=CC2=C(CNCC2)C(F)=C1)(N[C@H](CC3=CC=CC(C(F)(F)F)=C3)C(N4CCCC4)=O)=O

ZADKZNVAJGEFLC-ZMBIFBSDSA-N

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

(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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)