PK11007 targets p53 by alkylating surface-exposed cysteines Cys182 and Cys277 in the DNA-binding domain (DBD) of p53, leading to protein stabilization. [1]
PK11007 acts on mutant p53 protein, converting it to a wild-type-like conformation as evidenced by increased PAb1620 (wild-type p53) staining and decreased PAb240 (mutant p53) staining. [2]

In the concentration range of 15 to 30 µM, treatment with PK11007 (0-120 µM; 24 hours; four p53 wild-type cell lines and four p53 mutant cell lines) significantly reduced the viability of the mutant p53 cell lines MKN1 (V143A), HUH-7 (Y220C), NUGC-3 (Y220C), and SW480 (R273H/P309S). PK11007 mostly causes cell death that is not dependent on caspase [1]. In NUGC-3 (p53-Y220C), HUH-7 (p53-Y220C), and MKN1 (p53-V143A) cells, PK11007 (0-60 µM; 3 hours or 6 hours; NUGC-4, NUGC-3, MKN1, HUH-6, and HUH-7 cancer cells) treatment upregulates protein levels of p53 target genes p21, MDM2, and p53 PUMA in a concentration-dependent manner. This suggests a partial restoration of the transcriptional activity of the unstable p53 mutant. Increased MDM2, PUMA, and p21 protein levels in HUH-6 and NUGC-4 cells demonstrate that PK11007 also boosts p53 function in these cells [1]. After treating three mutant p53 cell lines with PK11007 (15–20 µM; 4.5 or 6 hours; MKN1, HUH-7, NUGC-3, HUH-6 cells), transcription of p53 target genes increased. PUMA and p21 mRNA levels were elevated twofold following NOXA treatment of NUGC-3, MKN, and HUH-7 cells, as well as the latter two cells. MKN1 and NUGC-3 cells have halved MDM2 levels [1]. Depletion of glutathione intensifies the decline in PK11007 activity. PK11007 was incubated with NUGC-3, NUGC-4, HUH-6, HUH-7, and MKN1 cells for two hours in order to see if it also raised ROS levels. All cell lines showed an increase in ROS levels after two hours. Nevertheless, the rise in ROS at 60 µM PK11007 in mutant p53 cells MKN1, HUH-7, and NUGC-3 was greater than in NUGC-4 and HUH-6 cells, suggesting that the mutant p53 cell lines were more susceptible to PK11007. Increased induction of ROS is mediated. MKN1 cells had ROS levels that are at least twice as high as those of other cell lines, both basal and PK11007-induced [1]. In line with its capacity to reactivate mutant p53, PK11007 suppresses cell growth, triggers apoptosis, and modifies genes related to cell death [2].

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PK11007 (15-30 µM, 24 h) caused substantial viability reduction in mutant p53 cancer cell lines MKN1 (V143A), HUH-7 (Y220C), NUGC-3 (Y220C), and SW480 (R273H/P309S), while p53 wild-type lines HUH-6, NUGC-4, and fibroblast WI-38 were less sensitive (viability reduced only at 60-120 µM). [1]
PK11007 (15-30 µM) induced cell death in HUH-7 and MKN1 as early as 3-6 h, whereas p53 wild-type HUH-6 showed viability decrease only after 10 h. [1]
PK11007 (15-20 µM) strongly reduced viability in H1299 (p53-/-) and H1299-p53 R175H cells, and in isogenic HCT116 p53-/- cells compared to HCT116 p53 wild-type. p53 knockdown in HUH-6 and HUH-7 did not alter PK11007-mediated viability reduction, but in MKN1 cells p53 knockdown partially rescued viability at 15-20 µM. [1]
PK11007 up-regulated protein levels of p53 target genes p21, MDM2, and PUMA in NUGC-3, HUH-7, and MKN1 cells in a concentration-dependent manner (3-6 h treatment). Molecular weight of p53 increased by ~3 kDa at 30-60 µM in mutant p53 lines, suggesting hyperalkylation. [1]
PK11007 (15-20 µM, 6 h) increased PUMA and p21 mRNA levels by ~2-fold in NUGC-3, MKN1, and HUH-7 cells; NOXA mRNA increased in MKN1 and HUH-7. MDM2 mRNA levels decreased by half in MKN1 and NUGC-3. No significant change in p53 target gene mRNA was observed in p53 wild-type HUH-6 cells. [1]
PK11007 activated the unfolded protein response (UPR): increased spliced XBP-1 and CHOP protein levels in HUH-7, MKN1, and to a lesser degree HUH-6 cells (3-6 h treatment). [1]
PK11007 (30-60 µM, 6 h) did not significantly activate caspases 3/7 in HUH-6 and HUH-7 cells, but caused loss of membrane integrity in HUH-7 cells. In SW480, SJSA-1, and HCT116 cells, PK11007 slightly increased caspase 3/7 activity (15-60 µM). [1]
Combination of PK11007 (15 µM) with buthionine sulfoximine (BSO, 100 µM, inhibitor of glutathione synthesis) synergistically reduced viability in mutant p53 lines MKN1, HUH-7, NUGC-3, but not in wild-type HUH-6 or NUGC-4. [1]
PK11007 (30-60 µM, 2 h) increased intracellular ROS levels in all tested cell lines; the increase was higher in mutant p53 lines (MKN1, HUH-7, NUGC-3) than in wild-type lines (NUGC-4, HUH-6). Basal and induced ROS levels in MKN1 were at least twofold higher. N-acetylcysteine (NAC) prevented PK11007-mediated ROS increase. [1]
PK11007 (2.5 µM, 12 h) treatment of HCC1143 cells altered global gene expression: 390 genes up-regulated, 234 down-regulated (RNA-seq, fold change >1.4, adjusted p<0.05). GO terms enriched included regulation of cell death, regulation of apoptosis, signal transduction, and protein refolding. [2]
PK11007 (5 µM, 48 h) inhibited proliferation in a panel of 17 breast cell lines with IC50 values ranging from 2.3 to 42.2 µM. TNBC cell lines had significantly lower IC50 values than non-TNBC lines (p=0.03). p53-mutated cell lines had lower IC50 values than p53 wild-type lines (p=0.003). IC50 values correlated inversely with p53 protein levels (Spearman r=0.59, p=0.01). [2]
p53 knockdown (siRNA, 40 nM) in HCC1143, MDA-MB-468, and MDA-MB-231 cells significantly decreased the growth inhibitory effect of PK11007 (5 µM, 48 h), with 79-89% reduction in p53 protein levels. [2]
PK11007 (2.5-20 µM, 24-72 h) induced apoptosis in a time- and concentration-dependent manner in p53-mutated TNBC lines HCC1143 and MDA-MB-468, but not in p53 wild-type MCF-7 cells. It up-regulated PUMA, NOXA, and p21 mRNA and protein in a concentration-dependent manner (2.5-20 µM, 24-48 h) in HCC1143 and MDA-MB-468 cells. [2]
PK11007 (25-50 µM, 3 h) increased staining with wild-type p53 antibody PAb1620 and decreased staining with mutant p53 antibody PAb240 in SKBR3 cells (p53 Arg175His), indicating refolding of mutant p53 to wild-type-like conformation. Total p53 protein levels by ELISA did not change. [2]
Combination of PK11007 with cisplatin showed synergistic growth inhibition in MDA-MB-468 (CI=0.6) and BT549 (CI=0.8), but not in HCC1143 (CI=1.3). Combination with carboplatin, docetaxel, or eribulin was synergistic in only one of three TNBC lines tested; combination with doxorubicin showed no synergy. [2]

Reaction kinetics of PK11000 (a related compound) with glutathione were measured by 1H-NMR (second-order rate constant 1.37 L·mol⁻¹·s⁻¹) [1]

Cell viability assay [1]
Cell Types: p53 wild-type cell lines (WI-38, HUH-6, NUGC-4, SJSA-1) and p53 mutant cell lines (HUH-7, NUGC-3, SW480, MKN1) concentration ) , NUGC-3 (Y220C) and SW480 (R273H/P309S), and the p53 WT cell line SJSA-1 at concentrations ranging from 15 to 30 µM. p53 WT cancer cell lines HUH-6, NUGC-4 and WI-38 were less sensitive, with diminished cell viability only at high concentrations of the compound (60 and 120 µM).

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Western Blot Analysis[1]
Cell Types: NUGC-4, NUGC-3, MKN1, HUH-6, and HUH-7 Cancer cell
Tested Concentrations: 0 μM, 15 μM, 30 μM, 60 μM
Incubation Duration: 3 hrs (hours) or 6 hrs (hours)
Experimental Results: Protein levels of p53 target genes p21, MDM2 and PUMA in NUGC-3 (p53-Y220C), HUH-7 (p53-Y220C) and MKN1 (p53-V143A) cells were upregulated in a concentration-dependent manner. Increased MDM2, PUMA, and p21 protein levels indicated that p53 activity was also increased in HUH-6 and NUGC-4 cells.

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RT-PCR[1]
Cell Types: MKN1, HUH-7, NUGC-3, HUH-6 Cell
Tested Concentrations: 15 μM, 20 μM Incub

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Cell viability assay: Cells were seeded in 96-well plates at 7,500-15,000 cells/well, incubated overnight, then treated with PK11007 (final DMSO 0.5%) for 24 h (or indicated times). Cell viability was measured using CellTiter-Fluor cell viability assay kit (fluorescence at 400/500 nm) or CellTiter-Glo 2.0 assay kit (luminescence). Experiments performed in quadruplicate. [1]
Western blot: Cells seeded in 6-well plates (0.5-0.8 million/well), treated with PK11007 for 3-6 h, lysed in RIPA buffer with protease inhibitors. Protein (20 µg per lane) separated on NuPAGE 4-12% Bis-Tris gels, electroblotted to PVDF membrane, blocked with 5% skim milk, incubated with primary antibodies (p53 DO-1, p21 187, PUMA ab9643, MDM2 2A10, CHOP sc-793, XBP-1 sc-7160, GSTP1 3F2C2, β-actin AC-15) for 1 h or overnight at 4°C, then with HRP-conjugated secondary antibodies, detected by ECL chemiluminescence. [1]
Real-time PCR: Cells treated with PK11007 (15-20 µM) for 4.5-6 h, total RNA extracted with RNeasy Mini Kit, cDNA synthesized with iScript cDNA Synthesis Kit, real-time PCR performed with SYBR Green Kit on Rotor-Gene 6000 cycler. Relative standard curve method used to quantify mRNA levels. Samples measured in triplicate. [1]
p53 knockdown: Cells transfected with human-specific p53 siRNA (40 nM) or negative control siRNA using INTERFERin transfection reagent for 24 h, then treated with PK11007 (5-15 µM) for 24-48 h. Knockdown confirmed by Western blot. Cell viability measured by CellTiter-Glo or MTT assay. [1][2]
Caspase 3/7 activity and membrane integrity: After PK11007 treatment (6 h), caspase activity and loss of membrane integrity were measured. No specific kit details provided. [1]
ROS measurement: Cells treated with PK11007 (30-60 µM) for 2 h, stained with CellROX Deep Red dye (fluorescent upon oxidation by ROS), median fluorescence determined by flow cytometry in triplicate. [1]
Immunofluorescence for p53 conformation: SKBR3 cells seeded in 8-well chamber slides, treated with PK11007 (25-50 µM) for 3 h, fixed with 1% glutaraldehyde, blocked with 5% goat serum/0.1% Triton-X, stained overnight with PAb1620 (anti-wild-type p53) or PAb240 (anti-mutant p53) at 1:500, then with goat anti-rat secondary antibody (1:2000), nuclei stained with Dapi, visualized by fluorescence microscopy. [2]
Flow cytometry for p53 conformation: Cells treated with PK11007 (25-50 µM) for 3 h, fixed with 0.5% paraformaldehyde, permeabilized and blocked with 10% goat serum/0.1% Triton-X, stained with PAb1620 or PAb240 (1:500), then with secondary antibody (1:2000), analyzed by BD FACSCanto. Data analyzed with FlowJo. [2]
Apoptosis assay by annexin V/PI: Cells seeded in 6-well plates (2×10⁵/well), treated with PK11007 (various concentrations) in low serum (2% FBS) for indicated times, stained with annexin V-FITC and propidium iodide using Annexin V-FITC Apoptosis Detection Kit, analyzed by BD FACSCalibur. [2]
MTT cell proliferation assay: Breast cell lines seeded at 1×10³/well in 96-well plates, treated with PK11007 (0-50 µM) for 5 days, then 0.5 mg/mL MTT added for 5 h, DMSO added, absorbance measured at 550 nm. IC50 values calculated using CalcuSyn software. Experiments in triplicate. [2]
RNA-seq: HCC1143 cells treated with PK11007 (2.5 µM) or DMSO control in triplicate for 12 h, RNA extracted with RNeasy Mini Kit and MinElute Cleanup Kit, libraries prepared with Illumina TruSeq stranded mRNA library prep kit, sequenced on Illumina HiSeq 2500 (51bp single-end reads). Reads aligned to human genome version 19 using Subread, normalized by TMM method, differential expression determined by Limma (adjusted p<0.05, fold change >1.4). GO analysis using goseq. [2]

PK11007 showed low cytotoxicity toward normal cells (fibroblast WI-38) compared to cancer cells; reduced viability only at high concentrations (60-120 µM, 24 h). [1]
In breast cell lines, PK11007 had higher IC50 values in non-TNBC and p53 wild-type cells, indicating lower toxicity in those lines. [2]

[1]. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells. Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):E5271-80.

[2]. Mutant p53 as a therapeutic target for the treatment of triple-negative breast cancer: Preclinical investigation with the anti-p53 drug, PK11007. Cancer Lett. 2018 Feb 1;414:99-106.

PK11007 exerts anticancer effects via multiple mechanisms: (1) stabilization and reactivation of mutant p53 by selective alkylation of Cys182 and Cys277 without compromising DNA-binding affinity; (2) depletion of intracellular glutathione and increase of reactive oxygen species (ROS) to toxic levels; (3) induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR), as evidenced by increased spliced XBP-1 and CHOP. [1]
PK11007 is more effective in killing p53-compromised cancer cells (mutant or null p53) than p53 wild-type cells. The compound shows selectivity for triple-negative breast cancer (TNBC) cell lines, which frequently harbor p53 mutations. Response correlates with p53 protein levels and mutational status. [1][2]
PK11007 can induce caspase-independent cell death in highly sensitive lines (e.g., HUH-7, MKN1) and caspase-dependent apoptosis in other lines (e.g., SJSA-1, SW480). [1]
PK11007 up-regulates p53 target genes (PUMA, p21, NOXA) at both mRNA and protein levels, partially restoring transcriptional activity of destabilized p53 mutants. [1][2]
PK11007 alters the conformation of mutant p53 from a mutant (PAb240-positive) to a wild-type-like (PAb1620-positive) form, without changing total p53 protein levels. [2]
PK11007 in combination with cisplatin shows synergistic growth inhibition in some TNBC cell lines (MDA-MB-468, BT549). [2]

C15H11CLFN5O3S2

427.86094212532

427

C, 42.11; H, 2.59; Cl, 8.29; F, 4.44; N, 16.37; O, 11.22; S, 14.99

874146-69-7

16446482

White to off-white solid powder

2.3

1

9

5

27

629

0

CC1=NN=C(S1)NC(=O)C2=NC(=NC=C2Cl)S(=O)(=O)CC3=CC=C(C=C3)F

IVZQUWCWXYFPOQ-UHFFFAOYSA-N

InChI=1S/C15H11ClFN5O3S2/c1-8-21-22-14(26-8)20-13(23)12-11(16)6-18-15(19-12)27(24,25)7-9-2-4-10(17)5-3-9/h2-6H,7H2,1H3,(H,20,22,23)

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.86 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 20.8 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: ≥ 2.08 mg/mL (4.86 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 20.8 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.)