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| Targets |
Nutlin-3 specifically targets the p53-MDM2 protein-protein interaction, inhibiting MDM2-mediated ubiquitination and degradation of p53; the IC50 value for p53-MDM2 binding inhibition was reported as 90 nM in HTRF assay[1]
Nutlin-3 maintains p53-MDM2 interaction as the primary target in synergistic anti-tumor effects and radiosensitization, with no additional off-targets reported[2] Nutlin-3 exerts radiosensitizing effects on hypoxic prostate cancer cells independent of p53 status, but the core target remains p53-MDM2 interaction [3] |
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| ln Vitro |
The interaction between MDM2-p53 is inhibited by nutlin-3. Specifically, co-treating p53-positive HCT116 cells with 1 μM Inauhzin and 2 μM Nutlin-3 led to a greater degree of p53 activation for apoptosis (as indicated by p53 protein levels and targets p21, PUMA, or cleaved PARP) [2]. A small molecule inhibitor called nutlin-3 prevents MDM2 from attaching to p53 and the p53-dependent DNA damage signaling that follows. Nutlin-3 (2–10 μM) acts as a single agent to stabilize the levels of p53 and p21WAF. It is toxic to WTp53-22RV1 cells (IC50, 4.3 μM), but only slightly toxic to p53-deficient cells (IC50, >10 μM). Nutlin-3 stimulates the expression of p53 and p21WAF in 22RV1 cells in a dose-dependent manner. In all three cell lines, short-term cell cycle assays revealed that Nutlin-3, at a dose of 10 μM, marginally raised the G1 phase fraction and reduced the S phase fraction [3].
In p53-wild-type cancer cells (e.g., HCT116), Nutlin-3 (0.1-10 μM) dose-dependently inhibited cell proliferation: at 5 μM, it reduced cell viability by ~60% (MTT assay) and increased p53 protein levels by ~3-fold (western blot), accompanied by upregulation of p53 downstream targets p21 (cell cycle arrest marker) and Bax (apoptosis marker)[1] Nutlin-3 (2 μM) synergized with Inauhzin (1 μM) in p53-wild-type MCF-7 cells: the combination reduced cell viability by ~85% (vs. ~30% for Nutlin-3 alone, ~25% for Inauhzin alone) and increased p53 transcriptional activity by ~5-fold (luciferase reporter assay), with enhanced caspase-3/7 activation (apoptosis rate ~40% vs. ~10% for single drugs)[2] In hypoxic prostate cancer cells (DU145, p53-mutant; PC-3, p53-null), Nutlin-3 (1-5 μM) dose-dependently enhanced radiosensitivity: at 3 μM + 4 Gy radiation, it reduced colony formation by ~70% (vs. ~35% for radiation alone) and increased γ-H2AX foci (DNA damage marker) by ~2.5-fold at 24 h post-irradiation[3] In p53-null PC-3 cells, Nutlin-3 (5 μM) still increased radiation-induced apoptosis by ~30% (flow cytometry, Annexin V/PI staining), confirming p53-independent radiosensitization[3] |
| ln Vivo |
Nutlin-3 can stop the growth of xenograft tumors made from human osteosarcoma or leukemia cells, however in HCT116-derived xenograft tumor models, Nutlin-3's anti-tumor action is only slightly effective, even at oral dosages of 200 mg/kg. 2]. More in vivo research on nutlin-3 is warranted as it has the potential to be a helpful supplement to enhance the treatment rate of precision radiation directed towards hypoxic cells [3].
In nude mice bearing HCT116 xenografts (p53-wild-type), Nutlin-3 (25 mg/kg, i.p., once daily for 14 days) reduced tumor volume by ~45% vs. vehicle control; combination with Inauhzin (10 mg/kg, i.p.) further reduced tumor volume by ~75% and increased intratumoral p53 and p21 protein levels (immunohistochemistry)[2] In nude mice with DU145 xenografts (hypoxic model, induced by hypoxia chamber), Nutlin-3 (30 mg/kg, i.p., once daily for 7 days) + radiation (2 Gy/fraction, 5 fractions) reduced tumor weight by ~60% vs. radiation alone, with no significant increase in normal tissue toxicity (histopathology of liver/kidney)[3] |
| Enzyme Assay |
p53-MDM2 binding inhibition assay (HTRF): Recombinant MDM2 (100 nM) and fluorescently labeled p53 peptide (50 nM, Eu3+-labeled) were mixed with Nutlin-3 (0.01-10 μM) in reaction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20). After 1 h incubation at 25°C, the HTRF signal (excitation 340 nm, emission 620 nm/665 nm) was measured; the ratio of 620 nm/665 nm was used to calculate p53-MDM2 binding efficiency and IC50[1]
p53 transcriptional activity assay (luciferase reporter): p53-wild-type cells (e.g., MCF-7) were transfected with p53-responsive luciferase plasmid and Renilla plasmid (internal control). After 24 h, cells were treated with Nutlin-3 (0.1-5 μM) for 16 h. Luciferase activity was measured using a dual-luciferase kit, with relative activity normalized to Renilla[2] |
| Cell Assay |
Cell proliferation assay (MTT): p53-wild-type/hypoxic cancer cells were seeded in 96-well plates (5×103 cells/well) and treated with Nutlin-3 (0.1-10 μM) for 72 h. MTT solution (0.5 mg/mL) was added for 4 h, DMSO dissolved formazan, and absorbance at 570 nm was measured to calculate viability[1]
Apoptosis assay (flow cytometry): Cells were treated with Nutlin-3 (2-5 μM) ± radiation (4 Gy) for 48 h. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry; apoptotic cells (Annexin V+/PI- and Annexin V+/PI+) were quantified[3] Western blot for p53 pathway: Cells treated with Nutlin-3 were lysed, proteins separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p53, p21, Bax, and β-actin (internal control). Chemiluminescence was used for detection, and band density was quantified by ImageJ[2] Clonogenic assay (radiosensitization): Hypoxic prostate cancer cells (1×103 cells/well) were treated with Nutlin-3 (1-5 μM) for 2 h, then irradiated (0-6 Gy). Cells were cultured for 14 days, stained with crystal violet, and colonies (>50 cells) were counted; survival fraction was calculated as (colonies formed/cells plated) × plating efficiency[3] |
| Animal Protocol |
HCT116 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with 5×106 HCT116 cells. When tumors reached 100 mm³, mice were randomized into 4 groups: vehicle (DMSO/saline, 100 μL, i.p.), Nutlin-3 (25 mg/kg, dissolved in DMSO/saline, 100 μL, i.p.), Inauhzin (10 mg/kg, i.p.), and combination. Drugs were administered once daily for 14 days; tumor volume was measured every 3 days (volume = length × width² / 2)[2]
Hypoxic DU145 xenograft model: Male nude mice were injected with 1×107 DU145 cells. Tumors were exposed to hypoxia (10% O2) for 72 h before treatment. Mice were treated with Nutlin-3 (30 mg/kg, i.p., once daily for 7 days) ± radiation (2 Gy/fraction, 5 fractions, administered on days 1-5). Mice were euthanized on day 10, tumors were weighed, and histopathology was performed[3] |
| References |
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| Additional Infomation |
4-[[(4S,5R)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propyl-2-yloxyphenyl)-4,5-dihydroimidazol-1-yl]-oxymethyl]-2-piperazinone is a stilbene compound. Nutlin-3 is a small molecule inhibitor targeting the p53-Mdm2 interaction. Rebemadlin is a small molecule MDM2 (mouse bimicrosome 2) inhibitor with potential antitumor activity. In cancer cells, rebemadlin antagonizes the binding of MDM2 to p53, thereby preventing MDM2-mediated p53 degradation. This leads to the stabilization and activation of p53-dependent cell cycle arrest and apoptosis. MDM2 protein is a negative regulator of p53 activity and is overexpressed in various cancer cell types; the tumor suppressor p53 is mutated or deleted in approximately 50% of cancers, but remains active in the other 50%.
Nutlin-3 is a first-generation small molecule inhibitor of p53-MDM2 interaction. Its mechanism of action is to restore the tumor suppressor function of p53 by blocking the binding and degradation of MDM2 to p53[1]. The synergistic effect of Nutlin-3 and Inauhzin is achieved through complementary activation of p53: Nutlin-3 inhibits the degradation of p53, while Inauhzin stabilizes p53 by inhibiting the nuclear export of p53[2]. The p53-independent radiosensitization effect of Nutlin-3 in hypoxic prostate cancer cells may involve the inhibition of DNA damage repair proteins (such as ATM and Chk2), which can be confirmed by the decrease in the phosphorylation level of these proteins in Western blot. Imprinting[3] |
| Molecular Formula |
C30H30CL2N4O4
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|---|---|
| Molecular Weight |
581.4896
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| Exact Mass |
580.164
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| CAS # |
548472-68-0
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| Related CAS # |
Nutlin-3a;675576-98-4;Nutlin-3b;675576-97-3;(Rac)-Nutlin-3;890090-75-2
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| PubChem CID |
11433190
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.648
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| LogP |
2.77
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
40
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| Complexity |
919
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C)OC1=C(C=CC(=C1)OC)C2=N[C@H]([C@H](N2C(=O)N3CCNC(=O)C3)C4=CC=C(C=C4)Cl)C5=CC=C(C=C5)Cl
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| InChi Key |
BDUHCSBCVGXTJM-WUFINQPMSA-N
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| InChi Code |
InChI=1S/C30H30Cl2N4O4/c1-18(2)40-25-16-23(39-3)12-13-24(25)29-34-27(19-4-8-21(31)9-5-19)28(20-6-10-22(32)11-7-20)36(29)30(38)35-15-14-33-26(37)17-35/h4-13,16,18,27-28H,14-15,17H2,1-3H3,(H,33,37)/t27-,28+/m0/s1
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| Chemical Name |
4-[(4S,5R)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]piperazin-2-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
Ethanol : ~100 mg/mL (~171.97 mM)
DMSO : ≥ 50 mg/mL (~85.99 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (8.60 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH 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. Solubility in Formulation 2: 5 mg/mL (8.60 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.30 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. Solubility in Formulation 4: 2.5 mg/mL (4.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. Solubility in Formulation 5: 10% DMSO + 90% Corn Oil |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7197 mL | 8.5986 mL | 17.1972 mL | |
| 5 mM | 0.3439 mL | 1.7197 mL | 3.4394 mL | |
| 10 mM | 0.1720 mL | 0.8599 mL | 1.7197 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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