CX-5461

Catalog No.S2684

CX-5461 Chemical Structure

Molecular Weight(MW): 513.61

CX-5461 is an inhibitor of rRNA synthesis, selectively inhibits Pol I-driven transcription of rRNA with IC50 of 142 nM in HCT-116, A375, and MIA PaCa-2 cells, has no effect on Pol II, and possesses 250- to 300-fold selectivity for inhibition of rRNA transcription versus DNA replication and protein translation.

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Cited by 18 Publications

5 Customer Reviews

  • Pharmacological and siRNA perturbations in HEK293 cells suggest that mTORC1 also modulates cytoplasmic rheology through ribosome crowding in mammals.

    Cell, 2018, 174(2):338-349. CX-5461 purchased from Selleck.

  • The specific Pol I inhibitor CX-5461 causes nucleolar disruption, blocks LTP maintenance and Fsk-induced synthesis of new rRNA. DAPI staining (blue; A, B,C) shows the area of the nucleus. Nomarski images (grey; G, H,I) show the area of the nuclei and nucleoli (arrowheads). Application of Pol I specific inhibitor CX-5461 (200 nM) causes nucleolar disruption as indicated by the distribution of fibrillarin (green); compare A, D to B, E and C, F.

    PLoS One 2014 9(8), e104364. CX-5461 purchased from Selleck.

  • Representative western blot evaluation and densitormetric analysis of p53 expression in control and CX-5461 (CX)-treated HCT116, HepG2, MCF7 and LoVo cells. Cells were exposed to 1 μM CX-5461 for 12 h.

    Oncogene, 2015, 10.1038/onc.2015.147. CX-5461 purchased from Selleck.

  • Cu(CX-5461) exhibits enhanced circulation lifetime of CX-5461 in vivo. When injected intravenously, over 95% of the injected free CX-5461 has left the plasma compartment within an hour (A). When prepared as Cu-CX5461 inside liposomes, about 10% of the drug was still detected via HPLC at 24 h post-injection. Cu(CX-5461) was cleared over a 48 h period as shown (B) and the copper concentration was reduced similarly (C), suggesting that the Cu(CX-5461) complex dissociated and CX-5461 was lost from the lipid vesicles over time. All data are plotted as mean ± SEM.

    J Control Release, 2018, 286:1-9. CX-5461 purchased from Selleck.

  • (B) After CX-5461 treatment at different concentrations for 72 hours, whole cell lysates from OS cells were subjected to Western blot analysis using LC3B-II and actin antibodies. (C) The cells were incubated with 2 mM CX-5461 with or without 2.5 mM 3-MA for 72 hours. The percentage of cell death was determined by CellTiter-Glo™ Luminescent Cell Viability Assay. The data represent the mean of three separate experiments performed in triplicate; bars represent SD. *P<0.05; **P<0.01. +, treatment; −, no treatment. Abbreviations: 3-MA, 3-methyladenine; OS, osteosarcoma; SD, standard deviation.

    Onco Targets Ther, 2016, 9:5985-5997.. CX-5461 purchased from Selleck.

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Biological Activity

Description CX-5461 is an inhibitor of rRNA synthesis, selectively inhibits Pol I-driven transcription of rRNA with IC50 of 142 nM in HCT-116, A375, and MIA PaCa-2 cells, has no effect on Pol II, and possesses 250- to 300-fold selectivity for inhibition of rRNA transcription versus DNA replication and protein translation.
Targets
Pol I-driven transcription of rRNA [1]
(HCT-116, A375, MIA PaCa-2 cells)
142 nM
In vitro

CX-5461 is found to selectively inhibit rRNA synthesis (Pol I IC50=142 nM; Pol II IC50 > 25 μM; selectivity ~200-fold) in the HCT-116 cells. Selective inhibition of rRNA synthesis by CX-5461 is confirmed in two other human solid tumor cell lines; melanoma A375 (Pol I IC50 = 113 nM; Pol II IC50 > 25 μM) and pancreatic carcinoma MIA PaCa-2 (Pol I IC50=54 nM; Pol II IC50 ~25 mM). CX-5461 possesses 250- to 300-fold selectivity for inhibition of rRNA transcription versus DNA replication and protein translation. CX-5461 exhibits broad antiproliferative potency in a panel of cancer cell lines in human cancer cell lines, but has minimal effect on viability of nontransformed human cells. The median EC50 across all tested cell lines is 147 nM, yet all normal cell lines have EC50 values of approximately 5, 000 nM. Evaluation of the antiproliferative dose response for HCT-116, A375, and MIA PaCa-2 cell lines yield EC50 values of 167, 58, and 74 nM. CX-5461 induces autophagy and senescence in solid tumor cancer cells, rather than apoptosis, through a p53-independent process. [1]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
MNNG NX31TYFLS2WubDD2bYFjcWyrdImgZZN{[Xl? NXHUWoRFPzJiaB?= MVHJR|UxRTBwNT2xMlUhyrWP NGjaS|MzPzd{OUiwOy=>
U2-OS M2PZN2NmdGxidnnhZoltcXS7IHHzd4F6 NF\iWIc4OiCq NHr3VYVKSzVyPUCuOU0yNjViwsXN NEPXU2YzPzd{OUiwOy=>
RS4;11 MWLQdo9tcW[ncnH0bY9vKGG|c3H5 M4S5bVI2OCCwTR?= NXrrR|FtOjRiaB?= NV:4fYJDfGmvZTDk[ZBmdmSnboSg[IVkemWjc3WgbY4heHKxbHnm[ZJifGmxbjDy[YxifGm4ZTD0c{B1cGWrcjDEUXNQKHS{ZXH0[YQh[2:wdILvcJM> MYeyOlQ4OjFyOB?=
SEM NWS1c5ZYWHKxbHnm[ZJifGmxbjDhd5NigQ>? MnrUNlUxKG6P MkjzNlQhcA>? MorIeIlu\SCmZYDlcoRmdnRiZHXjdoVie2ViaX6gdJJwdGmoZYLheIlwdiC{ZXzheIl3\SC2bzD0bIVqeiCGTWPPJJRz\WG2ZXSgZ49vfHKxbIO= NWTWNI5nOjZ2N{KxNFg>
KOPN-8 NXvhN4wyWHKxbHnm[ZJifGmxbjDhd5NigQ>? MXmyOVAhdk1? NYP6SW1yOjRiaB?= NVi1RWM6fGmvZTDk[ZBmdmSnboSg[IVkemWjc3WgbY4heHKxbHnm[ZJifGmxbjDy[YxifGm4ZTD0c{B1cGWrcjDEUXNQKHS{ZXH0[YQh[2:wdILvcJM> MUGyOlQ4OjFyOB?=
NALM-6 MWDQdo9tcW[ncnH0bY9vKGG|c3H5 NYf5cWZXOjVyIH7N M4HkV|I1KGh? Mn7UeIlu\SCmZYDlcoRmdnRiZHXjdoVie2ViaX6gdJJwdGmoZYLheIlwdiC{ZXzheIl3\SC2bzD0bIVqeiCGTWPPJJRz\WG2ZXSgZ49vfHKxbIO= MWCyOlQ4OjFyOB?=

... Click to View More Cell Line Experimental Data

Assay
Methods Test Index PMID
Western blot
Cyclin D1 / p53 / p16; 

PubMed: 29631594     


The cell cycle related proteins cyclin D1, p53, p16 were detected by immunoblotting after treatment with CX-5461 for 48 h, and β-actin was used as loading control.

53BP1 / γ-H2AX / p-ATM; 

PubMed: 29631594     


After treatment for 48 h, CX-5461 induced a strong increase of γ-H2AX, 53BP1, and p-ATM, which are hallmarks of DNA double-strand break response.

Bcl-2 / Bax / Caspase3 ; 

PubMed: 29631594     


The expression of Bcl-2 was significantly decreased, meanwhile the activities of cleaved caspase-3 and Bax were markedly elevated compared with the control group

cleaved PARP / cleaved Caspase-9 / cleaved caspase-3 ; 

PubMed: 28369725     


Immunoblotting of markers of apoptosis including cleaved PARP, caspase-9, and caspase-3 after treatment with 1000 nM CX-5461.

p-AMPK / AMPK / p-mTOR / mTOR; 

PubMed: 27729807     


After OS cells were treated with CX-5461 in a dose-dependent manner for 72 hours, the phosphorylation levels of Akt (Ser473), AMPK (Thr172), mTOR (Ser2448), and their total protein expression were determined by Western blot analysis. 

EG5 / Histone H3 / p-Histone H3 (S10); 

PubMed: 30049386     


Expression of miR-101 target molecules in HCT116 cells after exposure to CX-5461, analyzed by immunoblot analysis at the indicated time points. Concentrations of CX-5461 and incubation times are shown at the top of the image. Data are representative of two independent experiments.

29631594 28369725 27729807 30049386
Immunofluorescence
Vimentin / Phalloidin / Snail1; 

PubMed: 31068593     


Immunostaining of Vimentin (green), Phalloidin (green) and Snail1 (green) ± TGFβ ± CX-5461 in NMuMG cells.

Rictor / Calnexin; 

PubMed: 31068593     


Immunostaining of Rictor (green), Calnexin (red), Rictor/Calnexin (white arrows, yellow) and DAPI (blue) ± TGFβ ± CX-5461 in NMuMG cells.

Fibrillarin; 

PubMed: 25089620     


DAPI staining (blue; A, B,C) shows the area of the nucleus. Nomarski images (grey; G, H,I) show the area of the nuclei and nucleoli (arrowheads). Application of Pol I specific inhibitor CX-5461 (200 nM) causes nucleolar disruption as indicated by the distribution of fibrillarin (green); compare A, D to B, E and C, F.

LC3; 

PubMed: 27729611     


A. U251 cells were transfected with GFP-LC-3 plasmid for 24 h, and then treated with CX-5461 at the indicated concentrations for another 24 h. The cells were imaged under a confocal microscope (scale bar = 10 μm); C. U251 cells were transfected with GFP-LC-3 plasmid for 24 h, and then treated with 250 nM of CX-5461. The cells were imaged under a confocal microscope at the indicated time points post-treatment (scale bar = 10 μm).

γH2AX / 53BP1 / RPA / RAD51; 

PubMed: 28211448     


The formation of γ-H2AX, 53BP1, RPA and RAD51 foci was monitored upon CX-5461, CX-3543 and BMH-21 treatment at 10−7 M in U2OS cells. Drug treatment time is 24 h for all drugs. Scale bar, 10 μM.

chromosome / telomere; 

PubMed: 28211448     


Effect of CX-5461 on telomere fragility in BRCA2+/+ and BRCA2−/−HCT116 cells. Arrows point to telomere defects with either fragile telomeres or missing telomeres. N=3, >100 cells each replica. The data were modelled using a logistic regression model. Scale bar, 5 μM. 

F-actin / Aurora B; 

PubMed: 28369725     


In SKOV3ip1 cells, (C) DAPI (blue) labeling showed failed cytokinesis and an accumulation of multinucleated cells after CX-5461 treatment, with F-actin (red) indicating shared cytoplasm. (D) Aurora-B kinase (red) detection showing normal localization to the cleavage furrow during cytokinesis in control SKOV3ip1 cells. After treatment with CX-5461 Aurora-B mislocalization was apparent in an accumulation of multinucleated cells.

31068593 25089620 27729611 28211448 28369725
Growth inhibition assay
Cell viability; 

PubMed: 26061708     


All eight ALL cell lines showed marked decrease in proliferation after a 3 day treatment with CX-5461.

26061708
In vivo CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. CX-5461 demonstrates significant MIA PaCa-2 TGI with TGI equal to 69% on day 31, comparable to that of gemcitabine (63% TGI). Gemcitabine is a positive control which is administered intraperitoneally once every 3 days at 120 mg/kg. Likewise, CX- 5461 demonstrates significant A375 TGI with TGI equal to 79% on day 32. [1]

Protocol

Kinase Assay:[1]
- Collapse

Pol I and Pol II Transcription Assay:

Two short-lived RNA transcripts (half-lives ~20-30 minutes), one produced by Pol I and another by Pol II, are quantitated by qRT-PCR as a measure of CX-5461-related effects on transcription. The 45S pre-rRNA served as the Pol I transcript and the mRNA for the protooncogene c-myc served as the comparator Pol II transcript. Both Pol I and Pol II transcription are known to be affected by general cellular stress. To minimize the potential effects of such stress, cells are exposed to test agents for only a short period of time (2 hours). This is sufficient time for these transcripts to be reduced by greater than 90% if CX-5461 affects their synthesis.
Cell Research:[1]
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  • Cell lines: panel of cancer and normal cell lines
  • Concentrations: 0-2 μM
  • Incubation Time: 96 hours
  • Method: Cells are plated on 96-well plates and treated the next day with dose response of CX-5461 for 96 hours. Cell viability is determined using Alamar Blue and CyQUANT assays
    (Only for Reference)
Animal Research:[1]
- Collapse
  • Animal Models: 5 × 106 MIA Paca-2 and A375 cancer cells are subcutaneously inoculated in the right flank of 5- to 6- week-old female athymic mice
  • Formulation: CX-5461 is dissolved in 50 mM NaH2PO4 (pH 4.5).
  • Dosages: 50 mg/kg
  • Administration: CX-5461 is administered orally once daily or every 3 days.
    (Only for Reference)

Solubility (25°C)

In vitro DMF 3 mg/mL warmed (5.84 mM)
DMSO 0.02 mg/mL (0.03 mM)
Water Insoluble

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 513.61
Formula

C27H27N7O2S

CAS No. 1138549-36-6
Storage powder
in solvent
Synonyms N/A

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Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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Frequently Asked Questions

  • Question 1:

    I want to make it for further in vivo treatment. I

  • Answer:

    The solubility of this compound is poor in common vehicles. It can be dissolved in DMF at 3 mg/ml with warming.

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID