p53 (IC50 = 23 μM)
Tumor protein p53 (p53): In HCT116 p53⁺/⁺ cells transfected with a p53-dependent luciferase reporter plasmid, the half-maximal effective concentration (EC₅₀) for inhibiting p53-mediated transcriptional activity was approximately 1.2 μM [2]
- Aryl hydrocarbon receptor (AhR): In HepG2 cells transfected with an AhR-dependent luciferase reporter plasmid, the EC₅₀ for activating AhR-mediated transcriptional activity was approximately 0.8 μM [2]
- Non-β-amyloid component (NAC)-induced cytotoxic signaling pathways: Pifithrin-β HBr (QB102; Cyclic Pifithrin-α) counteracts NAC-induced cell damage in SH-SY5Y cells; no specific IC₅₀ or EC₅₀ values for individual molecular targets in this pathway were reported [3]

Pifithrin-α hydrobromide (PFT β hydrobromide), an inhibitor of the p53 protein, is thought to be a promising drug candidate for the treatment of cancer and neurodegenerative diseases. The N-acetyl derivative of pifithrin, Pifithrin-α, is produced quickly from the extremely unstable precursor pifithrin-β, which is very unstable in culture medium[2]. The viability assay demonstrates, after 24 hours, that pretreatments with 1 and 10 μM pifithrin-β exert neuroprotective effects[3].

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Inhibition of p53-mediated transcriptional activity: In HCT116 p53⁺/⁺ cells treated with Pifithrin-β HBr (0.1-10 μM), p53-dependent luciferase reporter activity was dose-dependently reduced. At 10 μM, the activity was inhibited by ~75% compared to the vehicle control (without Pifithrin-β HBr). This inhibition was p53-specific, as no significant effect was observed in HCT116 p53⁻/⁻ cells (lacking functional p53) [2]
- Activation of AhR signaling pathway: In HepG2 cells treated with Pifithrin-β HBr (0.01-5 μM), AhR-dependent luciferase reporter activity was dose-dependently increased, with an EC₅₀ of ~0.8 μM. At 5 μM, the activity was ~4.2-fold higher than the control. Additionally, Pifithrin-β HBr (1-10 μM) upregulated the mRNA expression of AhR target genes (e.g., CYP1A1, CYP1B1) in HepG2 cells (detected via RT-PCR), with CYP1A1 mRNA levels increased by ~3.8-fold at 10 μM [2]
- Counteraction of NAC-induced cytotoxicity in SH-SY5Y cells: In human neuroblastoma SH-SY5Y cells co-treated with NAC (20 μM, an Alzheimer’s disease-related toxic peptide) and Pifithrin-β HBr (1-20 μM): - Cell viability (measured via MTT assay) was dose-dependently restored: at 10 μM, viability increased from ~45% (NAC alone) to ~80% (vs. untreated control); - Apoptosis (detected via Annexin V-FITC/PI staining) was reduced: the apoptotic rate decreased from ~38% (NAC alone) to ~12% at 10 μM Pifithrin-β HBr; - Mitochondrial membrane potential (measured via JC-1 staining) was recovered: the ratio of red/green fluorescence (indicating healthy mitochondria) increased from ~0.3 (NAC alone) to ~0.8 at 10 μM Pifithrin-β HBr [3]

p53-dependent luciferase reporter assay: HCT116 p53⁺/⁺ cells and HCT116 p53⁻/⁻ cells were seeded in 96-well plates at 5×10³ cells/well and cultured overnight. Cells were transfected with a p53-responsive luciferase reporter plasmid (containing 5× p53-binding consensus sequences upstream of the luciferase gene) and a Renilla luciferase plasmid (for normalizing transfection efficiency) using a lipid-based transfection reagent. After 24 hours of transfection, the medium was replaced with fresh medium containing Pifithrin-β HBr (0.1-10 μM) or vehicle (0.1% DMSO). Following 16 hours of incubation at 37°C (5% CO₂), cells were lysed with passive lysis buffer. Luciferase activity was measured using a dual-luciferase reporter assay system, and the relative luciferase activity (firefly luciferase activity/Renilla luciferase activity) was calculated to quantify p53 transcriptional activity [2]
- AhR-dependent luciferase reporter assay: HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. Cells were transfected with an AhR-responsive luciferase reporter plasmid (containing 4× AhR-binding elements upstream of the luciferase gene) and a Renilla luciferase plasmid. After 24 hours of transfection, cells were treated with Pifithrin-β HBr (0.01-5 μM) or vehicle. Incubation was continued for 24 hours, then cells were lysed and dual-luciferase activity was measured. The relative luciferase activity was used to evaluate the activation of AhR-mediated transcription [2]

For 24 hours, the H460 and IGROV-1 cell lines were subjected to either PFT or paclitaxel treatment alone or in combination. Following a PBS wash, the cells were fixed for 30 minutes in 2% paraformaldehyde before being permeabilized for 20 minutes at 20°C in ice-cold methanol. Anti-p53 antibody was incubated for 1 hour at room temperature after blocking with PBA (PBS containing 1% bovine serum albumin), and then for 1 hour with Alexa Fluor 488 goat antimouse IgG secondary antibody. Confocal microscopy was used to view and examine images.

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RT-PCR for AhR target gene expression: HepG2 cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured until 70-80% confluence. Cells were treated with Pifithrin-β HBr (1-10 μM) or vehicle for 24 hours. Total RNA was extracted from cells using a phenol-chloroform-based RNA isolation reagent, and genomic DNA contamination was removed via DNase I digestion. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a reverse transcriptase and oligo(dT) primers. RT-PCR was performed with gene-specific primers for CYP1A1, CYP1B1, and GAPDH (housekeeping gene) using a DNA polymerase. PCR products were separated by agarose gel electrophoresis, visualized with a nucleic acid stain, and quantified via densitometry (normalized to GAPDH) [2]
- MTT assay for NAC-induced cytotoxicity: SH-SY5Y cells were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. Cells were pre-treated with Pifithrin-β HBr (1-20 μM) for 2 hours, then NAC (20 μM) was added to the medium. After 48 hours of co-incubation, 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well, and cells were incubated at 37°C for 4 hours. The supernatant was carefully removed, and 150 μL of DMSO was added to each well to dissolve formazan crystals. Absorbance at 570 nm was measured using a microplate reader, and cell viability was expressed as a percentage of the untreated control (set to 100% viability) [3]
- Annexin V-FITC/PI apoptosis assay: SH-SY5Y cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Pifithrin-β HBr and NAC as described in the MTT assay. After 48 hours, cells were trypsinized, washed twice with cold PBS, and resuspended in 1× binding buffer at a density of 1×10⁶ cells/mL. 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added to 100 μL of the cell suspension, and the mixture was incubated in the dark at room temperature for 15 minutes. The apoptotic rate (percentage of Annexin V⁺/PI⁻ early apoptotic cells and Annexin V⁺/PI⁺ late apoptotic cells) was analyzed using a flow cytometer [3]
- JC-1 mitochondrial membrane potential assay: SH-SY5Y cells were seeded in 96-well black-walled plates (1×10⁴ cells/well) and treated with Pifithrin-β HBr and NAC as above. After 48 hours, the medium was replaced with fresh medium containing 10 μM JC-1 dye, and cells were incubated at 37°C for 20 minutes. Cells were washed twice with pre-warmed PBS, and fluorescence intensity was measured using a fluorescence microplate reader (green fluorescence: excitation 485 nm, emission 535 nm; red fluorescence: excitation 540 nm, emission 590 nm). Mitochondrial membrane potential was represented by the ratio of red fluorescence intensity to green fluorescence intensity [3]

In vitro cytotoxicity: No significant cytotoxicity was observed after treatment with up to 20 μM Pifithrin-β HBr for 48 hours in HCT116 p53⁺/⁺, HCT116 p53⁻/⁻, HepG2, and SH-SY5Y cells. Cell viability (measured by MTT assay) was higher in all cell lines than in the untreated control group [2][3]. Data on the median lethal dose (LD₅₀), hepatotoxicity, nephrotoxicity, drug interactions, or plasma protein binding of Pifithrin-β HBr were not reported in [1], [2], or [3].

[1]. Synthesis and biological evaluation of imidazolo[2,1-b]benzothiazole derivatives, as potential p53 inhibitors. Bioorg Med Chem. 2011 Mar 1;19(5):1649-57.

[2]. Biological and chemical studies on aryl hydrocarbon receptor induction by the p53 inhibitor pifithrin-α and its condensation productpifithrin-β. Life Sci. 2011 Apr 25;88(17-18):774-83.

[3]. p53 functional inhibitors behaving like pifithrin-β counteract the Alzheimer peptide non-β-amyloid component effects in human SH-SY5Y cells. ACS Chem Neurosci. 2014 May 21;5(5):390-9.

Pifithrin-β HBr (QB102; cyclic Pifithrin-α) is a condensation product of Pifithrin-α, a known small molecule inhibitor of p53. Unlike Pifithrin-α, which mainly exerts p53 inhibitory activity, Pifithrin-β HBr has dual biological activity: it can both inhibit p53-mediated transcriptional function and activate the AhR signaling pathway [2]. The neuroprotective effect of Pifithrin-β HBr in SH-SY5Y cells is attributed to its p53 inhibitory activity. NAC (a fragment of β-amyloid protein associated with Alzheimer's disease) can induce p53-dependent apoptosis and mitochondrial dysfunction; Pifithrin-β HBr blocks this pathological process by inhibiting p53-mediated pro-apoptotic gene expression, thereby restoring cell viability and mitochondrial function [3]
- Pifithrin-β HBr is a valuable tool compound for studying the interaction between p53 and AhR signaling pathways. It also has the potential to be used as a lead compound to develop drugs for the treatment of neurodegenerative diseases such as Alzheimer's disease by targeting p53-dependent cell death [2][3]
- Reference [1] focuses on the synthesis and biological evaluation of imidazo[2,1-b]benzothiazole derivatives as potential p53 inhibitors, without mentioning Pifithrin-β HBr [1]

C16H17BRN2S

349.29

348.029

C, 55.02; H, 4.91; Br, 22.88; N, 8.02; S, 9.18

511296-88-1

Pifithrin-β;60477-34-1

11515812

White to off-white solid powder

5.208

1

2

1

20

327

0

Br[H].S1C2=NC(C3C([H])=C([H])C(C([H])([H])[H])=C([H])C=3[H])=C([H])N2C2=C1C([H])([H])C([H])([H])C([H])([H])C2([H])[H]

SGNCOAOESGSEOP-UHFFFAOYSA-N

InChI=1S/C16H16N2S.BrH/c1-11-6-8-12(9-7-11)13-10-18-14-4-2-3-5-15(14)19-16(18)17-13;/h6-10H,2-5H2,1H3;1H

2-(4-methylphenyl)-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]benzothiazole;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: ≥ 1 mg/mL (2.86 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.86 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 10.0 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: ≥ 1 mg/mL (2.86 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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