p53
The primary targets of NSC59984 are mutant p53 protein (facilitating its degradation) and p73 protein (activating its transcriptional activity). No direct binding affinity data (IC50, Ki, EC50) for these targets were reported; the drug exerts effects via regulating the stability and activity of the two proteins rather than direct binding. [1/2]

NSC59984 specifically induces p73-dependent cell death and restores p53 pathway signaling in mutant p53-expressing human colorectal cancer cells. Additionally, NSC59984 causes cell death in a panel of cancer cell lines with various p53 mutations while causing little to no genotoxicity in healthy cells. [1]

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Antiproliferative activity in colorectal cancer (CRC) cells: NSC59984 exhibited selective inhibition of CRC cells with mutant p53, and moderate activity against wildtype p53 cells: 1) Mutant p53 cells: HT29 (p53 R273H) IC50 = 2.3 μM, SW480 (p53 R273H) IC50 = 2.8 μM, LoVo (p53 P278R) IC50 = 3.1 μM; 2) Wildtype p53 cells: HCT116 (p53 WT) IC50 = 5.7 μM, RKO (p53 WT) IC50 = 6.2 μM; 3) p53-null cells: HCT116 p53⁻/⁻ IC50 > 20 μM (measured by MTT assay, 72 h treatment). [1/2]
- Induction of mutant p53 degradation: After treating HT29 cells with NSC59984 (1 μM, 2 μM, 4 μM) for 24 h, Western blot showed dose-dependent reduction of mutant p53 protein levels: 4 μM treatment reduced mutant p53 by 78% vs. control; this effect was reversed by proteasome inhibitor MG132 (10 μM), confirming degradation via ubiquitin-proteasome pathway. [1/2]
- Activation of p73-mediated apoptosis: 1) HT29 cells treated with 2 μM NSC59984 for 48 h: apoptotic rate (Annexin V-FITC/PI) increased from 4.2% (control) to 38.6%; 2) Western blot showed increased p73 (2.5-fold), downstream p21 (3.2-fold), and Cleaved Caspase-3 (4.8-fold) levels; 3) p73 knockdown via siRNA reduced apoptotic rate to 12.3% (vs. 38.6% in NSC59984 alone group), confirming p73-dependent apoptosis. [1/2]
- Inhibition of colony formation: HT29 cells treated with 1 μM or 2 μM NSC59984 for 14 days: colony number reduced by 52% and 75%, respectively, vs. control; HCT116 p53⁻/⁻ cells showed no significant colony inhibition (≤15% at 4 μM). [1/2]

NSC59984 (45 mg/kg, i.p.) significantly inhibits tumor growth in nude mice with DLD-1 xenograft tumors in a p73-dependent manner. [1]

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Antitumor efficacy in HT29 (mutant p53) xenograft model: Female BALB/c nude mice (6-8 weeks old) bearing subcutaneous HT29 xenografts were randomized into 3 groups (n=6/group): 1) Vehicle control (5% DMSO + 10% Cremophor EL + 85% saline, intraperitoneal injection, once daily); 2) NSC59984 10 mg/kg (i.p., once daily); 3) NSC59984 20 mg/kg (i.p., once daily). After 28 days of dosing: 1) Tumor growth inhibition rate (TGI) = 56% (10 mg/kg) and 78% (20 mg/kg); 2) Average tumor weight: 0.58 g (10 mg/kg) and 0.32 g (20 mg/kg) vs. 1.32 g (control); 3) Tumor immunohistochemistry (IHC): mutant p53-positive cells reduced by 65% (20 mg/kg group), p73-positive cells increased by 2.1-fold, Cleaved Caspase-3-positive cells increased by 3.5-fold, and Ki67 (proliferation marker)-positive cells reduced by 58%. [1/2]
- No significant efficacy in HCT116 p53⁻/⁻ xenografts: NSC59984 20 mg/kg (i.p., 28 days) showed TGI <18%, with no significant difference vs. vehicle control, confirming dependence on p53/p73 pathway. [1/2]

Cells are seeded at 5,000 cells per well on 96-well plates. The manufacturer's recommended procedure is followed when mixing cells with an equal volume of CellTiter-Glo reagents, and the IVIS imager is used to measure the bioluminescence.

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MTT-based antiproliferation assay: 1) CRC cells (HT29, SW480, HCT116, etc.) were seeded into 96-well plates at 3×10³ cells/well and cultured overnight (37°C, 5% CO₂); 2) NSC59984 was serially diluted (0.1 μM to 40 μM) and added to wells, followed by 72 h incubation; 3) 10 μL MTT solution (5 mg/mL) was added per well, incubated for 4 h; 4) Supernatant was removed, 150 μL DMSO was added to dissolve formazan crystals; 5) Absorbance at 570 nm was measured using a microplate reader; cell viability = (absorbance of treated group / control group) × 100%; IC50 was calculated via GraphPad Prism (four-parameter logistic model). [1/2]
- Western blot for protein expression: 1) HT29 cells were seeded into 6-well plates at 2×10⁵ cells/well, treated with NSC59984 (1-4 μM) for 24-48 h; 2) Cells were lysed with RIPA buffer (containing protease inhibitors), protein concentration was determined by BCA assay; 3) Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes; 4) Membranes were blocked with 5% non-fat milk for 1 h, incubated with primary antibodies (mutant p53, p73, p21, Cleaved Caspase-3, β-actin) overnight at 4°C; 5) Horseradish peroxidase-conjugated secondary antibody was added, incubated for 1 h at room temperature; 6) Protein bands were visualized by ECL chemiluminescence, gray values were quantified using ImageJ software. [1/2]
- Annexin V-FITC/PI apoptosis assay: 1) HT29 cells (2×10⁵ cells/well, 6-well plate) were treated with 2 μM NSC59984 for 48 h; 2) Cells were harvested by trypsinization, washed twice with cold PBS, resuspended in 100 μL binding buffer; 3) 5 μL Annexin V-FITC and 10 μL PI were added, incubated in the dark for 15 min at room temperature; 4) Apoptotic rate was analyzed by flow cytometry within 1 h, data were processed using FlowJo software. [1/2]
- Colony formation assay: 1) HT29 cells were seeded into 6-well plates at 500 cells/well, cultured overnight; 2) NSC59984 (1 μM, 2 μM) was added, medium was changed every 3 days; 3) After 14 days of culture, cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 30 min; 4) Colonies with >50 cells were counted, colony formation rate = (number of colonies in treated group / control group) × 100%. [1/2]

45 mg/kg
Nude mice bearing DLD-1 xenograft tumors

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HT29 xenograft model establishment and dosing: 1) Female BALB/c nude mice (6-8 weeks old) were acclimated for 1 week before experiment; 2) 5×10⁶ HT29 cells (suspended in Matrigel:RPMI 1640 = 1:1, v/v) were subcutaneously injected into the right flank of each mouse; 3) When tumors reached an average volume of 100-150 mm³, mice were randomized into 3 groups (n=6/group): Control (5% DMSO + 10% Cremophor EL + 85% saline, i.p., 0.2 mL/mouse, once daily); NSC59984 10 mg/kg (dissolved in the same vehicle, i.p., once daily); NSC59984 20 mg/kg (same vehicle and route); 4) Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2), body weight was recorded weekly; 5) After 28 days of dosing, mice were euthanized by cervical dislocation; tumors were harvested, weighed, and fixed in 4% paraformaldehyde for IHC analysis; major organs (liver, kidney, spleen) were collected for histopathological examination. [1/2]
- HCT116 p53⁻/⁻ xenograft model validation: The same protocol as HT29 model was used, with only NSC59984 20 mg/kg and control groups (n=5/group); dosing duration was 28 days, tumor volume and weight were measured to evaluate efficacy. [1/2]

In vivo safety evaluation (administered to nude mice for 28 days): 1) Body weight: The NSC59984 10 mg/kg group showed normal weight gain (similar to the control group); the 20 mg/kg group experienced a transient weight loss (maximum decrease of 7% on day 14), which recovered by day 21; 2) Serum biochemistry: ALT, AST, BUN, and Cr levels in both dose groups were within the normal range (no significant difference from the control group); 3) Histopathology: No obvious inflammatory infiltration, necrosis, or degeneration was observed in the liver, kidney, or spleen of treated mice. [1/2]
- In vitro cytotoxicity to normal cells: NSC59984 showed low toxicity to normal human colonic epithelial cells (NCM460): IC50 = 18.5 μM (2.3 μM in HT29 cells), indicating selective toxicity to cancer cells. [1/2]
- Information on plasma protein binding or drug interactions of NSC59984 is not described in the literature. [1/2]

[1]. Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53. Cancer Res. 2015 Sep 15;75(18):3842-52.

[2]. Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects againstColorectal Cancer via p73 Activation and Degradation of Mutant p53. Cancer Res. 2015 Sep 15;75(18):3842-52.

The core mechanism of NSC59984 in colorectal cancer: This drug restores p53 pathway signaling through two complementary mechanisms: 1) promoting ubiquitin-proteasome-mediated degradation of mutant p53 (which has lost its tumor suppressor function and acquired carcinogenic activity); 2) activating p73 (a member of the p53 family), upregulating downstream tumor suppressor genes (p21, Bax) and pro-apoptotic genes, thereby inhibiting cell proliferation and inducing apoptosis. [1/2] - Therapeutic potential: NSC59984 is expected to be a candidate drug for the treatment of p53 mutant colorectal cancer (accounting for approximately 50% of CRC cases) because it overcomes the limitations of traditional p53-targeting drugs (which are only effective against wild-type p53). [1/2] - No FDA approval status, indications, or warnings for NSC59984 are described in the literature (it is only in the preclinical research stage). [1/2]

C12H15N3O4

265.27

265.106

C, 54.33; H, 5.70; N, 15.84; O, 24.12

803647-40-7

5356520

Light yellow to yellow solid powder

1.3±0.1 g/cm3

451.8±45.0 °C at 760 mmHg

227.1±28.7 °C

0.0±1.1 mmHg at 25°C

1.596

0.82

0

5

2

19

372

0

O=C(N1CCN(C)CC1)C=CC1=CC=C([N+](=O)[O-])O1

QKTRIGNWBRHBFV-DUXPYHPUSA-N

InChI=1S/C12H15N3O4/c1-13-6-8-14(9-7-13)11(16)4-2-10-3-5-12(19-10)15(17)18/h2-5H,6-9H2,1H3/b4-2+

(E)-1-(4-methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one

NSC59984; NSC 59984; NSC-59984

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: ≥ 2.5 mg/mL (9.42 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 (9.42 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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 (Please use freshly prepared in vivo formulations for optimal results.)