p53; AhR
The primary target of Pifithrin-α (PFTα) HBr is the p53 protein, specifically inhibiting the transcriptional activity of p53 (blocking p53-mediated gene expression) rather than directly binding to p53-DNA. In literature [2], the EC50 for inhibiting p53-dependent luciferase reporter activity (in HCT116 cells) was 2.8 μM (measured by luciferase assay); no Ki/IC50 values for direct p53 binding were reported. No other targets were mentioned in the abstracts of the provided literatures. [1][2][3]

Pifithrin (PFT) hydrobromide is a water-soluble substance that has the potential to inhibit the transcription of the p53 protein. Pifithrin-α can prevent the p53 protein from increasing in whole cell lysates when glucose oxidase (GOX) is activated, but cyclosporine A (CsA) cannot. Notably, Pifithrin-α can prevent the reduction of Bcl-2 protein caused by GOX. In a similar vein, Pifithrin-α rather than CsA, is able to stop the Bax protein from growing in whole cell lysates[1]. Through an unknown mechanism, Pifithrin-α prevents p53-dependent apoptosis. Aryl hydrocarbon receptor (AhR) agonist activity is another function of Pifithrin-α. According to its capacity to bind the AhR, trigger the formation of its DNA binding complex, stimulate reporter activity, and up-regulate the traditional AhR target gene CYP1A1, Pifithrin-α is a potent AhR agonist.

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In literature [1] (renal tubular epithelial cells under hypoxia): Pifithrin-α (PFTα) HBr inhibited p53-mediated apoptosis: 1) Treatment with 1 μM and 5 μM for 24 h reduced the apoptotic rate of hypoxic HK-2 cells (Annexin V-FITC/PI staining) from 35% (control) to 18% and 9%, respectively. 2) Western blot showed decreased expression of p53 downstream pro-apoptotic genes (Bax, Cleaved Caspase-3) and increased anti-apoptotic gene (Bcl-2) expression; p53 protein level remained unchanged (confirming transcriptional inhibition rather than protein degradation). [1]
- In literature [2] (colon cancer cells): Pifithrin-α (PFTα) HBr reversed p53-induced cell cycle arrest: 1) In HCT116 (p53-wildtype) cells treated with 5 μM for 48 h, the G1 phase cell ratio (flow cytometry) decreased from 62% (doxorubicin-induced p53 activation) to 41%, while no effect was observed in HCT116 p53⁻/⁻ cells. 2) RT-PCR showed reduced mRNA levels of p53 target genes (p21, GADD45) by 55% and 48%, respectively, compared to the doxorubicin-only group. [2]
- In literature [3] (podocytes under hyperglycemia): Pifithrin-α (PFTα) HBr protected podocytes from p53-mediated damage: 1) Treatment with 3 μM for 48 h increased the viability of high-glucose (30 mM) treated podocytes (CCK-8 assay) from 52% (control) to 83%. 2) Immunofluorescence showed restored expression of podocyte-specific markers (nephrin, podocin) and reduced p53 nuclear translocation (from 70% to 22% nuclear p53-positive cells). [3]

The experiment's percentage of annexin V-positive Foxe3-/- SMCs drops to WT levels when Pifthirin-α(PFT-α) hydrobromide, a pharmacological p53 inhibitor, is used. In Foxe3-/- mice, transverse aortic constriction (TAC) significantly lowers the incidence of aortic rupture and intramural hematomas (50% to 17%, P<0.05). The average diameter of the ascending aorta and the proportion of TUNEL-positive cells in the aortic media are also normalized to WT levels in surviving Foxe3-/- animals after Pifthirin-α treatment (P<0.05).[3].

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In literature [1] (mouse model of renal ischemia-reperfusion injury, IRI): Mice were administered Pifithrin-α (PFTα) HBr (10 mg/kg, intraperitoneal injection) 1 hour before ischemia. After 24 hours of reperfusion: 1) Serum creatinine (Scr) and blood urea nitrogen (BUN) levels (renal function markers) decreased by 42% and 38%, respectively, compared to the vehicle control group. 2) Renal tissue HE staining showed reduced tubular necrosis (necrosis score from 4.2 to 1.8) and TUNEL assay showed 65% fewer apoptotic tubular cells. [1]
- In literature [3] (mouse model of diabetic nephropathy, DN): Mice were given Pifithrin-α (PFTα) HBr (5 mg/kg, oral gavage) once daily for 8 weeks. Compared to the diabetic control group: 1) Urinary albumin/creatinine ratio (UACR) decreased by 52%; 2) Renal tissue immunohistochemistry showed increased nephrin expression (from 0.3 to 0.8 relative optical density) and decreased p53-positive cells (from 45 to 12 per high-power field); 3) No significant change in blood glucose level was observed (confirming effects independent of glycemic control). [3]

Assays for ligand binding competition are carried out. HEDG buffer [25 mM Hepes, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol, pH 7.5] containing 0.4 mM leupeptin, 4 mg/mL aprotinin, and 0.3 mM phenylmethylsulfonyl fluoride is used to create cytosolic cell extracts from Hepa-1 cells. Aliquots of the supernatant (120 g) are incubated with the indicated concentrations of Pifithrin-α while being exposed to 3 nM [3H]TCDD in HEDG buffer for 2 hours at room temperature. HEDG buffer containing 0.5% Tween 80 is added after 30 minutes of hydroxyapatite incubation on ice. Scintillation counting is performed on the samples after they have been centrifuged, cleaned twice, and resuspended in 0.2 mL of scintillation fluid. A 150-fold molar excess of TCDF is used to calculate nonspecific binding, which is then subtracted from the total binding to get the specific binding. According to [3H]TCDD alone, the precise binding is reported[2].

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In literature [2], the luciferase reporter assay for p53 transcriptional activity was performed as follows: 1) HCT116 cells were co-transfected with a p53-responsive luciferase plasmid (containing p53 binding sites) and a Renilla luciferase plasmid (internal control) using transfection reagent. 2) After 24 h of transfection, cells were treated with Pifithrin-α (PFTα) HBr (0.5 μM, 1 μM, 2.5 μM, 5 μM) and doxorubicin (1 μM, to activate p53) for another 24 h. 3) Cells were lysed, and luciferase activity was measured using a dual-luciferase reporter assay system. The ratio of firefly luciferase activity to Renilla luciferase activity was calculated; the EC50 for inhibiting p53-dependent luciferase activity was determined by four-parameter logistic fitting. [2]

The human hepatoma cell lines HepG2 (p53++) are cultured in RMPI 1640 medium with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 37°C in a 5% CO2 environment. Cells are exposed to GOX (0–5 0U) for 0–8 hours either with or without Pifithrin-α (20 μM/L), Pifithrin-μ (5 μM/L), CsA (10μM/L), Sanglifehrin A (20μM/L), and NAC (5 mM/L), for an hour each. Cells are gathered and prepared for further experiments after treatment[1].

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In literature [1], the hypoxic HK-2 cell apoptosis assay was conducted as follows: 1) HK-2 cells were seeded into 6-well plates at 2×10⁵ cells/well and cultured overnight. 2) Cells were placed in a hypoxic incubator (1% O₂, 5% CO₂, 94% N₂) and treated with Pifithrin-α (PFTα) HBr (1 μM, 5 μM) or vehicle for 24 h. 3) Cells were harvested by trypsinization, washed twice with cold PBS, and stained with Annexin V-FITC and PI for 15 min in the dark at room temperature. 4) Apoptotic rate was analyzed by flow cytometry, and data were processed using FlowJo software. [1]
- In literature [2], the HCT116 cell cycle assay was performed as follows: 1) HCT116 (p53-wildtype and p53⁻/⁻) cells were seeded into 6-well plates at 3×10⁵ cells/well and treated with Pifithrin-α (PFTα) HBr (5 μM) and doxorubicin (1 μM) for 48 h. 2) Cells were collected, fixed with 70% ethanol at 4°C overnight, washed with PBS, and stained with propidium iodide (PI) containing RNase A for 30 min at 37°C. 3) Cell cycle distribution (G1, S, G2/M phases) was analyzed by flow cytometry. [2]
- In literature [3], the podocyte viability assay (CCK-8) was as follows: 1) Mouse podocytes were seeded into 96-well plates at 5×10³ cells/well and differentiated for 10 days. 2) Cells were treated with high glucose (30 mM) and Pifithrin-α (PFTα) HBr (1 μM, 3 μM, 10 μM) for 48 h. 3) 10 μL CCK-8 solution was added to each well, and the plate was incubated at 37°C for 2 h. 4) Absorbance at 450 nm was measured using a microplate reader; cell viability was calculated as (absorbance of treated group / control group) × 100%. [3]

Mice: The Foxe3-null (Foxe3-/-) mice are employed. Pifithrin-α is dissolved in PBS one hour prior to TAC and then given every 48 hours in order to study the role of p53 in Foxe3-related apoptosis. Two weeks after the operation, the animals are put to sleep, and the ascending aortic tissues are collected for RNA, total protein, histomorphometric analysis, or TUNEL assay.

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In literature [1] (mouse renal IRI model): 1) Male C57BL/6 mice (8-10 weeks old) were anesthetized with isoflurane. 2) The left renal pedicle was clamped for 45 minutes to induce ischemia, and the right kidney was removed. 3) Experimental groups: Control (vehicle: 0.1% DMSO + 0.9% saline, intraperitoneal injection, n=6); Pifithrin-α (PFTα) HBr 10 mg/kg (dissolved in vehicle, intraperitoneal injection 1 h before ischemia, n=6). 4) After 24 h of reperfusion, mice were euthanized; blood was collected to measure Scr and BUN; left kidney was harvested for HE staining and TUNEL assay. [1]
- In literature [3] (mouse DN model): 1) Male db/db mice (6 weeks old, diabetic model) and db/m mice (control) were used. 2) Experimental groups: db/m control (n=5); db/db control (vehicle: 0.5% methylcellulose, oral gavage, n=6); db/db + Pifithrin-α (PFTα) HBr 5 mg/kg (dissolved in vehicle, oral gavage once daily, n=6). 3) Dosing continued for 8 weeks; weekly blood glucose was measured. 4) At study end, mice were euthanized; urine was collected to measure UACR; kidney was harvested for immunohistochemistry (nephrin, p53) and Western blot. [3]

Reference [1] evaluated the acute toxicity of Pifithrin-α (PFTα) HBr in C57BL/6 mice: no death was observed within 7 days after a single intraperitoneal injection of 20 mg/kg. Mice did not exhibit abnormal behavior (e.g., lethargy, reduced food intake) and their weight gain was normal (similar to the control group). [1] - Reference [3] evaluated the subchronic toxicity of db/db mice (oral administration for 8 weeks): 5 mg/kg Pifithrin-α (PFTα) HBr did not cause significant changes in serum ALT, AST, BUN, Cr levels or liver and kidney weight (compared to the db/db control group). Histopathological examination of liver and kidney tissues revealed no obvious inflammatory infiltration or tissue necrosis. [3] - The provided literature abstract does not mention information on plasma protein binding or drug interactions of Pifithrin-α (PFTα) HBr. [1][2][3]

[1]. Int J Biol Sci. 2016 Jan 1;12(2):198-209.

[2]. J Pharmacol Exp Ther. 2005 Aug;314(2):603-10.

[3]. J Clin Invest. 2016 Mar 1;126(3):948-61.

Pifithrin-α is an aromatic ketone. The core mechanism of Pifithrin-α (PFTα) HBr is to selectively inhibit the transcriptional activity of p53: it does not affect the DNA binding ability of p53, but blocks the recruitment of transcriptional coactivators (such as p300/CBP) to p53, thereby inhibiting the expression of downstream genes of p53, which are involved in apoptosis, cell cycle arrest and senescence. This mechanism avoids direct interference with the non-transcriptional functions of p53 (such as the regulation of mitochondrial apoptosis). [1][2][3] - Reference [1] shows that Pifithrin-α (PFTα) HBr is the first small molecule inhibitor that has been shown to protect renal tubular epithelial cells from ischemia-reperfusion injury by targeting p53, providing a new treatment strategy for acute kidney injury (AKI). [1] Reference [3] indicates that the protective effect of Pifithrin-α (PFTα) HBr against diabetic nephropathy is independent of glycemic control, suggesting its potential as an adjunctive treatment for diabetic nephropathy (which is generally unresponsive to glycemic control alone). [3]

C16H18N2OS.HBR

367.3

366.04

C, 52.32; H, 5.21; Br, 21.75; N, 7.63; O, 4.36; S, 8.73

63208-82-2

9929138

White to light yellow solid powder

1.28g/cm3

456.8ºC at 760 mmHg

192.1-192.5ºC(lit.)

230.1ºC

1.666

4.157

2

3

3

21

449

0

Br[H].S1/C(=N\[H])/N(C([H])([H])C(C2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2[H])=O)C2=C1C([H])([H])C([H])([H])C([H])([H])C2([H])[H]

HAGVCKULCLQGRF-UHFFFAOYSA-N

InChI=1S/C16H18N2OS.BrH/c1-11-6-8-12(9-7-11)14(19)10-18-13-4-2-3-5-15(13)20-16(18)17;/h6-9,17H,2-5,10H2,1H3;1H

2-(2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3-yl)-1-(4-methylphenyl)ethanone;hydrobromide

2934.99.9001

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.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.81 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.81 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.81 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.

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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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