R175 (p53 = 8 nM)
Mutant p53 protein (specifically p53R175 conformational mutant); zinc ion; cellular redox state. No IC50/Ki/EC50 values for target binding are reported. [1]

NSC319726 is a p53(R175) mutant reactivator, exhibits growth inhibition in cells expressing mutant p53, with an IC50 for p53(R175) mutant of 8 nM, showing no inhibition for p53 wild-type cells, and exhibiting selectivity of 10- to 100-fold over other hotspot p53 mutants. p53(R175)-dependent apoptosis is induced by NSC319276. The p53(R175) mutant protein undergoes a conformational change similar to that of the WT after being treated with NSC319726, which restores sequence-specific p53 transcription. NSC319726's ability to chelate zinc ions and undergo redox changes are both necessary for the compound to be active. [1]

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NSC319726 exhibited growth inhibition with markedly lower IC50 values in cells expressing mutant p53 compared to wild‑type controls, particularly for the p53R175 allele. In mouse fibroblast (10)3/175 cells (p53R175 mutant), the IC50 was 8 nM, whereas the IC50 for wild‑type Balb/c 3T3 fibroblasts was not reached. [1]

In WI38 human fibroblasts (p53 wild‑type), an IC50 for NSC319726 was not obtained. [1]

In isogenic mouse embryonic fibroblasts (MEFs) from p53+/+, p53−/−, and p53R172H/R172H mice, NSC319726 showed much higher sensitivity for the p53R172H/R172H cell line compared to p53+/+ and p53−/− controls. [1]

In human tumor cell lines with hotspot p53 mutations, cells carrying the p53R175 mutation exhibited IC50 values approximately 10‑fold, and in some instances 100‑fold, lower than cells with p53R248 or p53R273 mutations. [1]

NSC319726 induced apoptosis as measured by Annexin‑V staining, with a maximum increase in p53R175 mutant cells (10)3/175. In ovarian carcinoma cell lines, treatment with 1 μM NSC319726 for 24 hr resulted in >2‑fold higher apoptosis in TOV112D (p53R175H) compared to OVCAR3 (p53R248W) or SKOV3 (p53‑/‑). [1]

siRNA knockdown of p53R175 mutant protein in TOV112D cells markedly reduced sensitivity to NSC319726‑mediated growth inhibition, demonstrating partial dependence on the mutant protein. [1]

Immunofluorescence using conformation‑specific antibodies showed that NSC319726 induced a wild‑type‑like conformational change: PAB240 (mutant‑specific) fluorescence intensity decreased by 5‑fold, while PAB1620 (wild‑type‑specific) intensity increased by 2‑fold. Immunoprecipitation with PAB240 revealed >85% decrease in mutant p53 immunoreactivity after NSC319726 treatment. [1]

Western blot analysis showed that NSC319726 induced p21 protein in TOV112D (p53R175H) but not in SKOV3 (p53‑/‑). Etoposide failed to induce p21 in TOV112D. NSC319726 caused a reduction in mutant p53R175 protein levels, with lowest levels at 6 hr and return to pretreatment levels by 24 hr; this destabilization was not observed in p53R248 or p53R273 mutant cell lines. Nutlin‑3 (5 μM) abrogated the decrease in p53R175 stability induced by NSC319726. [1]

Chromatin immunoprecipitation (ChIP) demonstrated that NSC319726 restored site‑specific DNA binding of p53R175 mutant to the promoters of p21, PUMA, and MDM2 in TOV112D cells. [1]

qRT‑PCR showed that NSC319726 increased mRNA levels of p21, PUMA, and MDM2 in TOV112D cells, particularly the apoptotic gene PUMA. [1]

Luciferase reporter assay with the p21 promoter p53 response element showed a 2.5‑fold increase in luciferase activity upon NSC319726 treatment in TOV112D cells, but not in MEF cells expressing p53R248 or p53R273 alleles. [1]

Gene expression microarrays confirmed that NSC319726 produced a p53 target expression signature in TOV112D cells distinct from untreated controls. [1]

Addition of FeSO4 at concentrations above 15 μM completely abrogated the activity of NSC319726; below 15 μM, activity was inhibited in a dose‑dependent manner. [1]

Addition of ZnCl2 at 5–15 μM enhanced NSC319726 activity by 2‑fold in TOV112D cells. Concentrations above 100 μM ZnCl2 alone were toxic independent of p53 status. [1]

NSC319726 treatment (1 μM) significantly decreased the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) at 1 hr (p=0.0057), 3 hr (p=0.0027), and 24 hr (p=0.001). [1]

The reducing agent N‑acetyl‑cysteine (5 mM) inhibited the apoptotic activity of NSC319726, while the oxidizing agent diamide (100 μM) enhanced it. [1]

NSC319726 (5mg/kg, 7days) exhibits greater toxicity for p53(R172H/R172H) mice, with only 30% survival, as opposed to 100% survival for p53+/+ and p53-/- mice. NSC319726 inhibits the growth of TOV112D-p53(R175H) xenografts, but not H460 (p53+/+) and MDAMB468-p53(R273W) xenografts.[1]

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In toxicity assays, p53R172H/R172H mice treated daily with intraperitoneal (i.p.) NSC319726 at 10 mg/kg/day all died by day 3 (7/7), whereas only 1 of 9 p53+/+ mice died by day 3. By day 4, survival of p53+/+ mice was 70% and p53+/- mice 30%. At a lower dose of 5 mg/kg/day i.p., by day 7 p53+/+ and p53‑/‑ mice exhibited 100% survival compared to only 30% survival of p53R172H/R172H mice. [1]

After two days of serial compound dilution treatment, 5,000 TOV112D cells (5,000 cells/well, in a 100 ml culture) are cultured in a 96-well plate and reach a confluence of 50% to 60%. Following a 3-day incubation period, the growth is assessed using the MTS reagent and a Victor Plate reader instrument.

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Viability assays are done. The cells are cultured in a 12-well plate at a density of 5×104 cells per well in a 1 ml of culture, and are treated with serial dilutions of the compound on the second day to reach a confluence of 50% to 60%. Guava ViaCount reagent and Guava PCA instrument are used to measure the growth after three days of incubation.

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Cell growth inhibition was measured by MTS assay: 5,000 cells/well in 96‑well plates were cultured to 50‑60% confluence, then treated with serial dilutions of NSC319726 (0.00001 to 10 μM) for 3 days, and growth was measured using MTS reagent and a plate reader. [1]

Viability was also assessed using the Guava ViaCount assay: 5×10^4 cells/well in 12‑well plates were cultured to 50‑60% confluence, treated with serial dilutions of compound for 3 days, and measured with ViaCount reagent and a Guava PCA instrument. [1]

Apoptosis was measured by Annexin‑V staining using Guava Nexin reagent: cells in 12‑well plates were treated with NSC319726 for 24 hr, stained with Nexin reagent, and Annexin‑positive cells detected with Guava PCA instrument. [1]

siRNA transfection was performed using Lipofectamine 2000 with SMARTpool p53 siRNA. [1]

Immunofluorescence: cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‑100, stained overnight with PAB1620 (1:50, recognizes wild‑type conformation) or PAB240 (1:200, recognizes mutant conformation), followed by secondary goat anti‑mouse IgG for 40 min. Fluorescence intensity was quantified using Adobe Photoshop. [1]

Immunoprecipitation: cell lysates (500 μg) were immunoprecipitated with PAB240 (4 μg) using protein matrix, and pull‑down was detected by western blot with p53 antibody (FL393). Image density was determined using Adobe Photoshop. [1]

Western blot: lysates or immunoprecipitated products were run on SDS‑PAGE, transferred to PVDF membranes, and detected using ECL. Antibodies used: p21, GAPDH, p53 (DO‑1), actin. Density was expressed as ratio to loading control. [1]

Chromatin immunoprecipitation (ChIP): performed using a commercial kit. Recovered ChIP DNA was subjected to PCR using primers flanking p53 response elements in p21, PUMA, and MDM2 genes. Control primers for GAPDH were used. [1]

qRT‑PCR: RNA extracted using a kit, gene expression measured by TaqMan assays, normalized with β‑actin, average presented with standard deviation from duplicates or triplicates. [1]

Luciferase reporter assay: cells transfected with p21 promoter p53 response element in pGL3 vector, treated with NSC319726, lysed, and luciferase activity measured according to manufacturer’s instructions. [1]

Microarray assay: RNA extracted from cells with or without NSC319726 treatment, hybridized to GeneChip Human Genome U133 Plus 2.0 arrays. [1]

Glutathione measurement: reduced (GSH) and oxidized (GSSG) levels measured using a glutathione assay kit following manufacturer’s instructions. [1]

5-10 mg/kg/day, up to 7 days DMSO
p53+/+, p53-/- and p53R172H knockin mice

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Mice (6‑10 weeks old) of genotypes p53R172H/R172H, p53R172H/+, p53‑/‑, and p53+/+ were treated daily by intraperitoneal (i.p.) injection with NSC319726 at 10 mg/kg or 5 mg/kg, or vehicle control (dimethyl sulfoxide), for up to 7 days, after which tissues were harvested. [1]

In p53R172H/R172H mice, daily i.p. administration of 10 mg/kg NSC319726 resulted in death of all 7 mice by day 3; at 5 mg/kg, only 30% survival by day 7. In contrast, p53+/+ mice showed 70% survival at day 4 with 10 mg/kg and 100% survival at day 7 with 5 mg/kg. [1]

NSC319726 at doses that inhibited p53R175 mutant xenograft tumor growth (1 mg/kg i.v.) was completely nontoxic to mice. At 0.1 mg/kg, only a small difference in tumor growth inhibition was observed, indicating a therapeutic window. [1]

The compound was remarkably nontoxic to WI38 human fibroblasts (p53 wild‑type) as an IC50 was not obtained, and nontoxic to Balb/c 3T3 fibroblasts at concentrations effective in p53R175 mutant cells. [1]

[1]. Cancer Cell . 2012 May 15;21(5):614-625.

NSC319726 was identified using an in silico screening methodology applied to the NCI60 anticancer drug screen, which ranked compounds based on enrichment of good responders in mutant p53 cell lines (especially hotspot codons 175, 248, 273) and depletion in wild‑type p53 cell lines. Two other thiosemicarbazones (NSC319725, NSC328784) also scored highly. [1]

The mechanism of action involves zinc ion chelation and redox changes. The p53R175 mutant fails to coordinate zinc, and the compound may serve as a zinc metallochaperone to allow refolding. Redox changes (decreased GSH/GSSG ratio) and oxidative stress contribute to the apoptotic mechanism. [1]

The p53R175 mutant is the third most common p53 missense mutant, accounting for an estimated 5.5% of all missense mutations. Using the IARC TP53 database, the annual incidence of cancer patients carrying the TP53R175 allele in the United States is estimated to be >32,000. [1]

The compound did not induce wild‑type p53 protein levels or transcriptional activity like etoposide, and its activity was not explained by ribonucleotide reductase inhibition because nontumor cells with wild‑type p53 showed little to no growth inhibition at the same doses. [1]

C11H14N4S

234.32

234.093

C, 56.38; H, 6.02; N, 23.91; S, 13.68

71555-25-4

5351307

Off-white to light yellow solid powder

1.3±0.1 g/cm3

367.7±34.0 °C at 760 mmHg

176.2±25.7 °C

0.0±0.8 mmHg at 25°C

1.659

0.17

1

3

2

16

286

0

S=C(N([H])/N=C(\C([H])([H])[H])/C1=C([H])C([H])=C([H])C([H])=N1)N1C([H])([H])C([H])([H])C1([H])[H]

XDHBUMNIQRLHGO-UKTHLTGXSA-N

InChI=1S/C11H14N4S/c1-9(10-5-2-3-6-12-10)13-14-11(16)15-7-4-8-15/h2-3,5-6H,4,7-8H2,1H3,(H,14,16)/b13-9+

N-[(E)-1-pyridin-2-ylethylideneamino]azetidine-1-carbothioamide

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 (8.88 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 (8.88 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.)