MDMX (via transcriptional inhibition) [1]
The primary target of XI-011 is MDMX (murine double minute X, also known as MDM4), a key negative regulator of p53. XI-011 inhibits the MDMX gene promoter activity, downregulating MDMX mRNA and protein levels, thereby blocking MDMX-mediated suppression of p53. This mechanism leads to increased p53 protein stability and half-life, activation of p53 transcriptional activity, and upregulation of downstream target genes such as p21 and PUMA, ultimately inducing apoptosis in tumor cells. Additionally, in cervical cancer, XI-011 has been shown to inhibit HPV E6-E6AP-mediated p53 degradation by interfering with the MDMX-E6AP interaction.

NSC146109 is small molecule strongly activates p53 while selectively inhibiting growth of transformed cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy. However, the mechanism(s) by which NSC146109 activates p53 and the effects of NSC146109 on growth of breast cancer cells are currently unknown. Here, we report that NSC146109 promoted breast cancer cells to undergo apoptosis through activating p53 and inducing expression of proapoptotic genes. Importantly, we found that activation of p53 by this small molecule was achieved through a novel mechanism, that is, inhibition of MDMX expression. NSC146109 repressed the MDMX promoter, resulting in down-regulation of MDMX messenger RNA level in MCF-7 cells. In line with these results, NSC146109 decreased the viability of breast cancer cells expressing low levels of MDMX in a less-efficient manner. Interestingly, NSC146109 acted additively with the MDM2 antagonist Nutlin-3a to inhibit growth of breast cancer cells. We conclude that NSC146109 belongs to a novel class of small-molecule p53 activators that target MDMX and could be of value in treating breast cancer.

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NSC146109 activates p53 and induces apoptosis in p53 wild-type MCF-7 breast cancer cells. It increases p53 protein levels, stabilizes p53 (extends half-life), and upregulates p53 target genes (p21, PUMA, BAX, PIG3) at mRNA and protein levels. It inhibits MDMX expression by repressing MDMX promoter activity and reducing RNA polymerase II occupancy on the MDMX promoter. In MCF-7 cells, 0.5 µM XI-011 induces apoptosis (over 40% apoptotic cells after 5 days), shown by TUNEL staining, PARP cleavage, and sub-G0/G1 accumulation. Its proapoptotic effect is p53-dependent and impaired by p53 knockdown. XI-011 is less effective in decreasing viability of breast cancer cell lines expressing low MDMX levels (ZR-75-1, ZR-75-30, MD-175VII). It acts additively with Nutlin-3a (an MDM2 inhibitor) to activate p53 and reduce cell viability. It does not induce DNA damage at concentrations up to 20 µM. [1]
In vitro studies have demonstrated that XI-011 exhibits significant anti-proliferative activity across multiple tumor cell lines. In breast cancer MCF-7 cells, XI-011 activates p53 by inhibiting MDMX expression, inducing apoptosis-related gene expression and reducing cell viability. In head and neck cancer cells, XI-011 suppresses cell growth in a dose-dependent manner, with the strongest effects observed in MDMX-overexpressing tumor cells. In cervical cancer cell lines, XI-011 shows robust anti-proliferation activity, inducing apoptosis through p53 stabilization and transcriptional activation. Furthermore, XI-011 acts additively with the MDM2 antagonist Nutlin-3a and synergistically with cisplatin, enhancing cisplatin-induced cytotoxicity. Experimental concentrations typically range from 0.5-10 μM with treatment durations of 24-72 hours.

In vivo studies have confirmed the antitumor activity of XI-011. In head and neck cancer xenograft mouse models, XI-011 monotherapy inhibited tumor growth, and combination with cisplatin showed synergistic antitumor effects. In cervical cancer HeLa cell xenograft-bearing mice, XI-011 similarly suppressed tumor growth and enhanced the in vivo antitumor activity of cisplatin. These in vivo efficacy data support XI-011 as a candidate compound targeting the MDMX-p53 pathway, with potential applications particularly in the treatment of p53 wild-type tumors.

Regarding cell-free binding assays for XI-011, existing literature primarily focuses on functional mechanistic studies rather than direct receptor binding affinity measurements. XI-011 is known to exert its effects mainly through inhibition of MDMX gene expression rather than direct binding to MDMX protein. Typical mechanistic approaches include: using MDMX promoter-reporter systems (luciferase reporter) to detect XI-011's inhibitory effect on MDMX promoter activity; Western blot analysis for MDMX protein level changes; and quantitative real-time PCR (qRT-PCR) for MDMX mRNA level measurement. Additionally, cycloheximide chase assays can be used to assess changes in p53 protein half-life to evaluate the impact of XI-011 on p53 stability.

NSC146109 Activates p53 in Breast Cancer Cells
\nThe pseudourea derivative NSC146109 was identified as one of the most potent active hits in a reporter-based screening for small-molecule p53 activators . We therefore sought to determine whether this small molecule could activate p53 in p53 wild-type MCF-7 breast cancer cells. Toward this end, we treated MCF-7 cells with NSC146109 and measured expression levels of p53 and its downstream target gene p21 using immunoblot assays. As controls, we also treated cells with Nutlin-3a and RITA—two well-characterized small-molecule p53 activators. Interestingly, NSC146109 increased the cellular p53 protein level as efficient as the two known p53 activators. Moreover, NSC146109 treatments resulted in a dose-dependent increase in the p21 protein level, suggesting that p53 was indeed activated by the small molecule . To corroborate these results, we carried out quantitative reverse transcription-PCR (qRT-PCR) assays and found that NSC146109 dramatically increase messenger RNA (mRNA) levels of p21 as well as p53-targeted proapoptotic genes including PUMA, BAX, and PIG3. These results indicated that NSC146109 could strongly activate p53 in breast cancer cells. Moreover, NSC146109 significantly extended the half-life of p53 , consistent with a notion that this small-molecule p53 activator could increase the stability of p53 in breast cancer cells.[1]

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\n\nNSC146109 Induces Apoptosis in Breast Cancer Cells
\np53 activation can lead to cell cycle arrest or apoptotic cell death. To determine the consequence of p53 activation induced by NSC146109, we treated MCF-7 cells with 0.5 µM of NSC146109 and carried out flow cytometry analysis to examine cell cycle progression. It has been shown that activation of p53 by the MDM2 inhibitor Nutlin-3a results in only cell cycle arrest but not apoptosis in cancer cells overexpressing MDMX (e.g., MCF-7) . Indeed, we found that Nutlin-3a induced a significant decrease in the number of S-phase cells. In contrast, NSC146109 did not appear to diminish the S-phase subpopulation . However, NSC146109 treatments resulted in apparent accumulation of cells in sub-G0/G1 phase , suggesting that this small molecule could rather induce MCF-7 cells to undergo apoptosis. To confirm this important finding, we used TUNEL staining to specifically label apoptotic cells. In line with the notion that NSC146109 could induce apoptosis, numbers of TUNEL-positive cells were largely increased after NSC146109 treatments. This finding was further confirmed by the observations that cleavage of PARP—a biochemical marker for apoptosis—was induced by NSC146109 in a dose-dependent manner. Indeed, more than 40% of MCF-7 cells were apoptotic after treatments with 0.5 µMNSC146109 for 5 days . These results thus indicate that one of the major effects of NSC146109 on breast cancer cells was to induce apoptosis. This effect was in sharp contrast to the effect conferred by Nutlin-3a, suggesting that NSC146109 might activate p53 through a mechanism distinct from the MDM2 inhibitor.[1]

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\nNSC146109 Induces Apoptosis through Activating p53
\nTo demonstrate that the apoptosis-inducing activity of NSC146109 was a consequence of p53 activation, we knocked down p53 expression in MCF-7 cells using a p53-specific shRNA and determined PARP cleavage induced by NSC146109. Indeed, NSC146109-induced PARP cleavage was largely impaired in p53-downregulated cells . Moreover, the NSC146109-induced expression of proapoptotic genes (i.e., PUMA, BAX, and PIG3) was significantly diminished by knockdown of p53 expression.[1]

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\nNSC146109 Activates p53 and Induces Apoptosis through Inhibiting MDMX Expression
\nIt has been suggested that the failure in induction of apoptosis by Nutlin-3a in MCF-7 cells was likely due to MDMX overexpression. Because we have shown that NSC146109 was distinct from Nutlin-3a and could induce MCF-7 cells to undergo apoptosis, we tested a hypothesis that NSC146109 activates p53 through targeting MDMX. Interestingly, accompanied by p53 activation, the MDMX expression level was dramatically decreased after MCF-7 cells were treated with NSC146109 . Inhibition of MDMX expression by NSC146109 occurred 4 to 8 hours after treatment when p53 activation started to become notable. These results suggest a strong possibility that p53 activation by NSC146109 could be a consequence of MDMX inhibition. To explore this possibility, we knocked down MDMX expression in MCF-7 cells with an MDMX-specific shRNA and determined p53 activation by immunoblot analysis and qRT-PCR assays. Indeed, activation of p53 and induction of expression of p53 target genes (i.e., PUMA, BAX, and PIG3) were largely impaired in cells expressing a low level of MDMX. Moreover, the cleavage of PARP induced by NSC146109 was also impaired by knockdown of MDMX expression . Furthermore, a small interfering RNA (siRNA) specific to MDMX but targeting a sequence different from the shRNA used above also impaired p53 activation induced by NSC146109 . These results thus indicate that NSC146109 activated p53 and induced apoptosis through inhibiting MDMX expression. Of note, knockdown of MDMX expression by shRNA alone seemed insufficient to activate p53 and induce apoptosis —an observation in agreement with several but in contrast to other reports . The reason for this apparent discrepancy might be related to the fact that the p53 activity is regulated by a complicated network and thus effects of MDMX shRNA might be cell context-dependent.[1]

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MCF-7 cells were treated with NSC146109 (XI-011) at indicated concentrations (e.g., 0.2–2 µM) for various durations. For immunoblot analysis, cells were lysed after treatment and proteins (p53, p21, MDMX, PARP, β-actin) were detected using specific antibodies. For qRT-PCR, total RNA was extracted, reverse-transcribed, and mRNA levels of p21, PUMA, BAX, PIG3, and MDMX were quantified using real-time PCR. For cell cycle and apoptosis analysis, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry; apoptotic cells were also detected by TUNEL staining. For viability assays, cells were treated with XI-011 for 4 days and assessed by MTT assay. For cycloheximide chase assays, cells were treated with XI-011, then cycloheximide was added, and cells were harvested at time points for immunoblot analysis to determine protein stability. For chromatin immunoprecipitation, cross-linked cells were sonicated, immunoprecipitated with RNA pol II antibody, and bound DNA was analyzed by real-time PCR to assess promoter occupancy. [1]

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A typical in vitro cell-based assay protocol for XI-011 is as follows. Cell culture: Tumor cell lines (e.g., MCF-7, HeLa, HepG2) are maintained in DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin, in a 37°C, 5% CO₂ incubator. Cell plating: Cells are seeded at appropriate densities, such as 3×10⁴ cells per well in 96-well plates or 2×10⁵ cells per well in 6-well plates. Drug treatment: After cell attachment, various concentrations of XI-011 (typically 0.5-10 μM) are added for 24-72 hours. Cell viability assessment: MTT or CCK-8 assays are performed, with absorbance read using a microplate reader. Apoptosis detection: Apoptosis is evaluated by flow cytometry (Annexin V/PI double staining) or by Western blot detection of PARP and Caspase-3 cleavage bands.

A typical in vivo animal experiment protocol for XI-011 is as follows. Animal model: Immunodeficient mice (e.g., nude mice) are used to establish xenograft tumor models by subcutaneously inoculating tumor cells (e.g., HeLa or head and neck cancer cells) into the flank region. Grouping and dosing: When tumor volumes reach a certain size (e.g., approximately 100 mm³), mice are randomized into control and treatment groups. XI-011 is typically administered via intraperitoneal injection or oral gavage, with dosage and frequency determined by the specific study design (e.g., once daily). In combination therapy studies, XI-011 is used together with cisplatin to evaluate synergistic effects. Efficacy assessment: Tumor volumes (length × width² × 0.5) and body weights are measured 2-3 times weekly. At study completion, mice are euthanized, tumor tissues are excised and weighed, and samples are collected for mechanistic studies including Western blot, immunohistochemistry, or TUNEL staining.

Based on its structural characteristics (a pseudourea derivative with molecular weight of approximately 296.4, and hydrochloride salt formulation to enhance water solubility), it is expected to have some oral bioavailability. In vivo studies typically administer XI-011 via intraperitoneal injection, indicating that this route achieves effective systemic exposure. Comprehensive PK parameters including plasma protein binding, half-life, clearance, and tissue distribution require further experimental investigation.

NSC146109 does not induce genotoxicity or DNA damage at concentrations up to 20 µM, which is higher than the concentration required for p53 activation (0.2–1 µM). It selectively inhibits the growth of transformed cells without producing general toxicity to non-transformed cells. [1]

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A key safety feature of XI-011 is that it does not induce genotoxicity, meaning it does not cause DNA damage. This represents a potential safety advantage over many conventional chemotherapeutic agents. The compound selectively inhibits the growth of transformed (tumor) cells while having less effect on normal cells. No significant body weight loss or other overt systemic toxicities have been reported in available studies. However, it should be noted that comprehensive toxicological studies of XI-011 (including maximum tolerated dose, target organ toxicity, long-term toxicity, etc.) are currently incomplete and represent areas requiring further evaluation as this compound progresses toward clinical application.

[1]. A small-molecule p53 activator induces apoptosis through inhibiting MDMX expression in breast cancer cells. Neoplasia. 2011 Jul;13(7):611-9.

See also: (10-methyl-9-anthrayl)methyliminothiocarbamate (note moved to).

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NSC146109 (XI-011) is a pseudourea derivative identified as a p53 activator through high-content screening. It inhibits MDMX expression at the transcriptional level, representing a novel p53 activation mechanism. It synergizes with MDM2 inhibitors (e.g., Nutlin-3a) to inhibit breast cancer cell growth. Its parent compound, pseudourea (NSC56054), showed cytotoxicity but was withdrawn from clinical trials due to severe toxicity; XI-011 may be better tolerable due to its non-induction of DNA damage. [1]

C17H16N2S

280.38734

316.08

C, 64.44; H, 5.41; Cl, 11.19; N, 8.84; S, 10.12

59474-01-0

740031-90-7;59474-01-0 (HCl);

16759161

White to yellow solid powder

1.23g/cm3

493.6ºC at 760 mmHg

252.3ºC

1.665

5.985

3

2

3

21

333

0

N=C(N)SCC1=C2C=CC=CC2=C(C)C3=CC=CC=C13.[H]Cl

VIBMUYOXJUCEMA-UHFFFAOYSA-N

InChI=1S/C17H16N2S.ClH/c1-11-12-6-2-4-8-14(12)16(10-20-17(18)19)15-9-5-3-7-13(11)15;/h2-9H,10H2,1H3,(H3,18,19);1H

(10-methylanthracen-9-yl)methyl carbamimidothioate;hydrochloride

NSC-146109; NSC 146109; XI-011 HCl; XI-011 HCl; XI011; XI-011 hydrochloride; XI011; NSC146109; NSC 146109 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~83.3 mg/mL (~263.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.89 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.56 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 3: ≥ 2.08 mg/mL (6.56 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 20.8 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.)