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Fig. 1 · Structure
Cat. No.
A10657
CAS No.
548472-68-0
Formula
C30H30Cl2N4O4
Mol. Weight
581.5

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Nutlin-3

Mdm2 antagonist

MDM2 antagonist nutlin-3 is a potent inducer of apoptosis.

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Publications for Nutlin-3

4 publications citing this product

  1. Maxime Parisotto, .et al. , J Exp Med, 2018, Jun 4; 215(6): 1749-1763 PMID: 29743291
  2. Ohmuro-Matsuyama Y, .et al. , Anal Biochem, 2018, Dec 15;563:61-66 PMID: 30316750
  3. Alisa A. Garaeva, .et al. , Cell Cycle, 2016, 15(1): 64-71 PMID: 26771712
  4. A G Evstafieva, .et al. , Cell Death Dis., 2014, 5(11): e1511. PMID: 25375376
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Biological Activity

Bioactivity / Mechanism

Nutlin-3 activated p53-dependent transcription in HCT116 cells. In wild-type cells, it induced TP53INP1 and reduced ATF4 mRNA; pretreatment also attenuated the ATF4 response to myxothiazol. The combination response differed in p53-null cells. These experiments assess downstream p53 signaling rather than a biochemical MDM2-binding IC50. [1]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

In Vitro Studies

In the tested ALL cell-line panel, racemic Nutlin-3 reduced viability in wild-type-p53 lines, while mutant/null lines showed little comparable response. At 10 μM for 48 h, representative annexin-V plots showed 82% positive UOC-B3 cells and 6% positive EU-6 cells. These are representative flow-cytometry values, not population means. [2]

DNA-damage-response markers varied by model: Nutlin-3 increased γH2AX in mouse AJ02-NM0 colon cancer cells, whereas no increase was detected in human HCT116 cells under the reported 24 h conditions. Nutlin-3a, but not Nutlin-3b, reproduced the mouse-cell response. These results limit a blanket description of Nutlin-3 as having no DNA-damage-associated effects. [3]

p53-status-associated responses in ALL cell lines

Cell-based study · Pediatric ALL cell-line panel; representative UOC-B3 and Reh (wild-type p53), EU-4 (p53-null) and EU-6 (mutant p53). [2]

Nutlin-3 reduced viability in wild-type-p53 ALL lines; mutant/null lines lacked comparable responses. UOC-B3, unlike EU-6, showed prominent annexin-V positivity and caspase-3/PARP cleavage. This panel does not establish universal p53 dependence in all cell types.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Viability relative to control at 48 hdirection Dose-dependent reduction in UOC-B3/Reh; no comparable reduction in EU-4/EU-6[2]
Annexin-V-positive cells after 10 μM Nutlin-3 for 48 hpercentage, representative histograms UOC-B3: 82%; EU-6: 6%[2]

Literature-Reported Treatment Conditions

Treatment concentration
0–10 μM total racemate for viability; 10 μM for annexin-V and immunoblot examples
Exposure duration
48 h viability; 0, 8, 24 and 48 h annexin-V/immunoblot examples
Method
WST viability assay, annexin-V flow cytometry and immunoblotting
Vehicle
Ethanol; final concentration held constant within experiments
Reported limits
Final ethanol percentage not specified in the selected reagent paragraph. Representative annexin-V percentages have no replicate uncertainty stated in Figure 1c.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Nutlin-3, equimolar mixture of Nutlin-3a and Nutlin-3b (Cayman Chemical)

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 1a-d and caption; Results: Nutlin-3 induces cell death in ALL cell lines; Methods: reagents, cytotoxicity, apoptosis

Viability relative to control at 48 h: WST; mean percentage ± SD from three independent experiments — Figure 1b and caption

Annexin-V-positive cells after 10 μM Nutlin-3 for 48 h: Flow cytometry; printed representative percentages, not group means — Figure 1c and caption

p53 activation limits ATF4 transcription during mitochondrial stress

Cell-based study · HCT116 wild-type and isogenic p53−/− cells [1]

Nutlin-3 induced p53 and TP53INP1 in wild-type HCT116 cells and reduced ATF4 mRNA. Pretreatment attenuated ATF4 and associated transcriptional responses to short myxothiazol exposure; this attenuation was absent in p53−/− cells. Combination results are not attributed to Nutlin-3 alone.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
ATF4 mRNA and genotype-dependent response to combinationreported fold change and direction Nutlin-3 alone: twofold decrease in ATF4 mRNA in wild-type cells; myxothiazol-induced ATF4 response attenuated by Nutlin-3 in wild-type but not p53−/− cells[1]

Literature-Reported Treatment Conditions

Treatment concentration
10 μM Nutlin-3; 1 μM myxothiazol in combination conditions
Exposure duration
Nutlin-3: 16 h total; methods specify 12 h pretreatment followed by 4 h with myxothiazol
Method
Immunoblotting and RT-qPCR; untreated, single-agent, combination and p53−/− comparisons
Co-treatment
Myxothiazol in explicitly labeled combination conditions; separate Nutlin-3-only controls
Reported limits
Nutlin-3 vehicle, catalog, lot and stereochemical composition are not specified in the selected methods.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Nutlin-3 (AdooQ BioScience), stereochemistry unspecified

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 5a-e and caption; Results: Preliminary activation of p53 prevents induction of ATF4; Materials and methods: Cell cultures and treatments, RT-qPCR

ATF4 mRNA and genotype-dependent response to combination: RT-qPCR normalized to corresponding untreated control; mean and SD of three independent experiments per caption — Figure 5c-e; associated Results paragraph

DNA-damage-response markers in mouse colon cancer cells

Cell-based study · AJ02-NM0 mouse colon tumor-derived cells [3]

Racemic Nutlin-3 increased γH2AX-positive cells and p53 stabilization/phosphorylation compared with DMSO. The active enantiomer reproduced these responses; the inactive enantiomer did not. These are cellular marker results, not a direct DNA-binding or clinical toxicity assay.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
γH2AX-positive fraction versus DMSOdirection and reported significance Increased with Nutlin-3 and Nutlin-3a, not Nutlin-3b; P < 0.01 for marked comparisons[3]

Literature-Reported Treatment Conditions

Exposure duration
24 h
Method
γH2AX immunofluorescence with DAPI; p53/phospho-p53 immunoblotting
Vehicle
DMSO control
Reported limits
Figure 2 does not specify Nutlin-3 concentrations or replicate count/error-bar definition. A dose from another experiment has not been substituted.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Racemic Nutlin-3; Nutlin-3a and Nutlin-3b comparators (Cayman Chemical)

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 2a-c and caption; Results around Figure 2; Methods: Cell culture, immunofluorescence and immunoblotting

γH2AX-positive fraction versus DMSO: ANOVA with Bonferroni post-test as stated in caption; no exact bar heights extracted — Figure 2a-b and caption

Limited DNA-damage-marker response in human HCT116 cells

Cell-based study · Human HCT116 colon cancer cells [3]

Under the Figure 8 conditions, Nutlin-3 did not increase γH2AX staining relative to DMSO; 5-fluorouracil served as a positive comparator. This contrasts with the mouse-cell result and limits a general claim that Nutlin-3 necessarily produces a DNA-damage response.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
γH2AX response versus controlqualitative negative result No increase in γH2AX-positive cells with Nutlin-3 under the tested conditions[3]

Literature-Reported Treatment Conditions

Exposure duration
24 h
Method
γH2AX immunofluorescence and flow cytometry
Vehicle
DMSO control
Reported limits
Nutlin-3 concentration and replicate count are not specified in Figure 8 or its caption; figure labels do not assign replicate uncertainty.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Nutlin-3 (Cayman Chemical); no separate enantiomer label in Figure 8

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 8a-b and caption; Results: Nutlin-3 treatment and HCT116 DNA damage response; shared immunofluorescence methods

γH2AX response versus control: Immunofluorescence and gated flow cytometry; no exact population estimate extracted — Figure 8a-b and caption; adjacent Results paragraph

In Vivo Studies

Racemic Nutlin-3 reduced developmental retinal vascularization in neonatal mice. The authors reported reductions of 27.4% after nape delivery and 43.8% after periocular delivery. A separate adult-retina experiment detected no reduction in established vessels at 5 days. These findings distinguish developing from mature vessels and do not establish clinical efficacy or general ocular safety. [4]

Reduced developmental retinal vascularization

Animal study · Neonatal wild-type 129 S1-VIMJ mice [4]

Racemic Nutlin-3 reduced developing retinal vasculature relative to vehicle/sham injection. Delivery route influenced the reported reduction. Physiological vascular development is the endpoint, not tumor regression or clinical ocular efficacy.

Conditions shared by the study groups

Vehicle
100% DMSO
Study groups and reported outcomes
Group / doseComparatorObserved resultReference
Subcutaneous nape cohort
First injection 40 mg/kg; subsequent injections 80 mg/kg		Vehicle/sham-injected group27.4% reduction in retinal vessels; P < 0.05. Figure 7b: n = 6 Nutlin-3 and n = 4 sham; representative of two independent experiments.[4]
Periocular cohort
80 mg/kg per injection		Vehicle/sham-injected group43.8% fewer blood vessels; P < 0.01. Caption n = 8 each group; representative of two independent experiments.[4]
Subcutaneous nape cohort
Model: Neonatal wild-type 129 S1-VIMJ mice
Route: Subcutaneous, nape
Frequency: 5 injections total; spacing not specified
Treatment course: First administration within 12 h of birth; endpoint P3
Reported limits: Exact injection times not reported in the selected method; caption counting unit not independently resolved.
Periocular cohort
Model: Neonatal wild-type 129 S1-VIMJ mice
Route: Periocular area beneath fused eyelids
Frequency: 4 injections total; spacing not specified
Treatment course: First administration within 12 h of birth; endpoint P2
Reported limits: Exact injection times not reported in the selected method; caption counting unit not independently resolved.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Racemic Nutlin-3

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 7b-e and caption; retinal development Results; Methods: Subcutaneous and periocular injection for in vivo retinal development assay; Quantification of retinal vascular development model

Subcutaneous nape cohort · Figure 7b-c; retinal development Results and corresponding injection method

Periocular cohort · Figure 7d-e; retinal development Results and corresponding injection method

Mature retinal vessels show no detected reduction

Animal study · Adult 3-month-old 129 and C57BL6/129S mice [4]

A single intravitreal racemic Nutlin-3 treatment produced no detected reduction in established retinal vasculature at 5 days. Gross neuronal-layer thickness changes were not observed by H&E. This observation does not establish general retinal safety.

Measured endpoints — not treatment concentrations
EndpointReported resultReference
Established retinal vasculature compared with shamreported significance No detected difference; P > 0.05[4]

Conditions shared by the study groups

Route
Intravitreal; both eyes as stated in the adult assay method
Frequency
Single injection
Treatment course
Assessment 5 days after injection
Vehicle
DMSO (Figure 9 caption)
Study groups and reported outcomes
Group / doseComparatorObserved resultReference
Adult retinal racemic Nutlin-3 cohortVehicle (DMSO)/sham-injected adult miceNo detected reduction in established retinal vessels versus sham; P > 0.05. Figure 9c: n = 8 per group, mean ± SD, summation of two independent experiments. No general retinal-safety conclusion.[4]
Adult retinal racemic Nutlin-3 cohort
Model: Adult 3-month-old 129 and C57BL6/129S mice
Reported limits: Dose/concentration not reported in the selected adult method/caption. The method prints an implausible 1 ml injection volume; no corrected volume is assumed. Both eyes were injected; the analysis unit and within-animal handling are not resolved by these locations.
Methods and source locations

Compound reported in the paper: Nutlin-3; Compound label in the paper: Racemic Nutlin-3

Literature results for the reported material and model; no validation of A10657 lots or equivalence to isolated Nutlin-3a.

Figure 9a-c and caption; Results: established nonproliferating blood vessels; Methods: Intravitreal injection for in vivo adult retinal vessel assay

Established retinal vasculature compared with sham: Vascular pixel-ratio quantification; mean ± SD; caption n = 8 per group, summation of two independent experiments — Figure 9c and caption; adjacent Results

Adult retinal racemic Nutlin-3 cohort · Figure 9a-c and caption; Results paragraph beginning "Thus far, we examined"; Methods: Intravitreal injection for in vivo adult retinal vessel assay

Specifications

Manufacturer Adooq Bioscience
Actions Antagonist, Inducer
CAS Number 548472-68-0
Applications Mdm2 antagonist
Formula C30H30Cl2N4O4
Molecular Weight 581.5
SMILES
CC(C)OC1=C(C=CC(=C1)OC)C2=NC(C(N2C(=O)N3CCNC(=O)C3)C4=CC=C(C=C4)Cl)C5=CC=C(C=C5)Cl

Storage & Solubility

In Vitro Solubility

Reported Solubility

SolventReported Solubility
DMSO 87 mg/mL (149.61 mM)
Water Insoluble
Ethanol 26 mg/mL (44.71 mM)

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

  1. [1] Evstafieva AG, Garaeva AA, Khutornenko AA, et al. A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4 Cell death & disease (2014). PMID: 25375376 DOI: 10.1038/cddis.2014.469 Source material
  2. [2] Gu L, Zhu N, Findley HW, et al. MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2 Leukemia (2008). PMID: 18273046 DOI: 10.1038/leu.2008.11 Source material
  3. [3] Verma R, Rigatti MJ, Belinsky GS, et al. DNA damage response to the Mdm2 inhibitor nutlin-3 Biochemical pharmacology (2010). PMID: 19788889 DOI: 10.1016/j.bcp.2009.09.020 Source material
  4. [4] Chavala SH, Kim Y, Tudisco L, et al. Retinal angiogenesis suppression through small molecule activation of p53 The Journal of clinical investigation (2013). PMID: 24018558 DOI: 10.1172/jci67315 Source material

Publications

4 publications citing this product

  1. Maxime Parisotto, .et al. , J Exp Med, 2018, Jun 4; 215(6): 1749-1763 PMID: 29743291
  2. Ohmuro-Matsuyama Y, .et al. , Anal Biochem, 2018, Dec 15;563:61-66 PMID: 30316750
  3. Alisa A. Garaeva, .et al. , Cell Cycle, 2016, 15(1): 64-71 PMID: 26771712
  4. A G Evstafieva, .et al. , Cell Death Dis., 2014, 5(11): e1511. PMID: 25375376

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