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A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53.

Zhang Q, Zeng SX, Zhang Y, Zhang Y, Ding D, Ye Q, Meroueh SO, Lu H - EMBO Mol Med (2012)

Bottom Line: Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress.Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro.Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.

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Identification of INZ as a novel p53 activatorScreening for compounds that increase p53 levels in cells as detected by IB. H460 cells were harvested for IB after being treated with each of the top 50 compounds (10 µM) from computational-throughput screening for 18 h as shown in a representative blot here (number denotes each compound; INZ). Fifty micrograms of total proteins was used per lane (true for the following figures unless indicated).Chemical structures of INZ and its analogues 1–5.Cellular activity of INZ analogues was measured using IB that detects p53 levels in H460 and HCT116 cells. The induction levels of p53 were normalized with actin and plotted as percentage of the level of p53 in the cells treated with INZ (mean ± SD, n = 3).Dose-dependent activation of p53 pathway by INZ. Cells were treated with INZ or a control compound MI63 for 18 h and harvested for IB with the antibodies as indicated. * Indicates residual bands from p53 antibody.Cell growth inhibitory activity was evaluated by WST cell growth assays. IC50 values are represented as mean ± SD (n = 3).
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fig01: Identification of INZ as a novel p53 activatorScreening for compounds that increase p53 levels in cells as detected by IB. H460 cells were harvested for IB after being treated with each of the top 50 compounds (10 µM) from computational-throughput screening for 18 h as shown in a representative blot here (number denotes each compound; INZ). Fifty micrograms of total proteins was used per lane (true for the following figures unless indicated).Chemical structures of INZ and its analogues 1–5.Cellular activity of INZ analogues was measured using IB that detects p53 levels in H460 and HCT116 cells. The induction levels of p53 were normalized with actin and plotted as percentage of the level of p53 in the cells treated with INZ (mean ± SD, n = 3).Dose-dependent activation of p53 pathway by INZ. Cells were treated with INZ or a control compound MI63 for 18 h and harvested for IB with the antibodies as indicated. * Indicates residual bands from p53 antibody.Cell growth inhibitory activity was evaluated by WST cell growth assays. IC50 values are represented as mean ± SD (n = 3).

Mentions: Comparison of the structures of the MDM2–p53 and MDMX–p53 complexes (Kussie et al, 1996; Popowicz et al, 2007) revealed that the N-terminal hydrophobic pocket of MDMX for p53 binding is much shallower than that of MDM2. This information explained why MDM2 inhibitors failed to affect MDMX–p53 binding and also prompted us to initiate a computational structure-based screening using the AutoDock computer program (Morris et al, 2008) for the docking of virtual compounds that could distinguish the p53 binding sites on MDM2 and MDMX. From our initial computational screening of half a million of commercially available compounds from the ChemDiv chemical library, we selected and purchased 50 top candidates. These compounds were tested in cell-based assays at 10 µM for their ability to induce p53 levels in human lung carcinoma H460 cells using an immunoblotting (IB) analyses. To our delight, one small molecule, 10-[2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)butanoyl]-10H-phenothiazine (abbreviated as INZ; Fig 1B), induced p53 levels as effectively as actinomycin D (ActD; 10 nM) and in a much more pronounced manner than did the rest of the compounds tested (Fig 1A and data not shown).


A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53.

Zhang Q, Zeng SX, Zhang Y, Zhang Y, Ding D, Ye Q, Meroueh SO, Lu H - EMBO Mol Med (2012)

Identification of INZ as a novel p53 activatorScreening for compounds that increase p53 levels in cells as detected by IB. H460 cells were harvested for IB after being treated with each of the top 50 compounds (10 µM) from computational-throughput screening for 18 h as shown in a representative blot here (number denotes each compound; INZ). Fifty micrograms of total proteins was used per lane (true for the following figures unless indicated).Chemical structures of INZ and its analogues 1–5.Cellular activity of INZ analogues was measured using IB that detects p53 levels in H460 and HCT116 cells. The induction levels of p53 were normalized with actin and plotted as percentage of the level of p53 in the cells treated with INZ (mean ± SD, n = 3).Dose-dependent activation of p53 pathway by INZ. Cells were treated with INZ or a control compound MI63 for 18 h and harvested for IB with the antibodies as indicated. * Indicates residual bands from p53 antibody.Cell growth inhibitory activity was evaluated by WST cell growth assays. IC50 values are represented as mean ± SD (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376857&req=5

fig01: Identification of INZ as a novel p53 activatorScreening for compounds that increase p53 levels in cells as detected by IB. H460 cells were harvested for IB after being treated with each of the top 50 compounds (10 µM) from computational-throughput screening for 18 h as shown in a representative blot here (number denotes each compound; INZ). Fifty micrograms of total proteins was used per lane (true for the following figures unless indicated).Chemical structures of INZ and its analogues 1–5.Cellular activity of INZ analogues was measured using IB that detects p53 levels in H460 and HCT116 cells. The induction levels of p53 were normalized with actin and plotted as percentage of the level of p53 in the cells treated with INZ (mean ± SD, n = 3).Dose-dependent activation of p53 pathway by INZ. Cells were treated with INZ or a control compound MI63 for 18 h and harvested for IB with the antibodies as indicated. * Indicates residual bands from p53 antibody.Cell growth inhibitory activity was evaluated by WST cell growth assays. IC50 values are represented as mean ± SD (n = 3).
Mentions: Comparison of the structures of the MDM2–p53 and MDMX–p53 complexes (Kussie et al, 1996; Popowicz et al, 2007) revealed that the N-terminal hydrophobic pocket of MDMX for p53 binding is much shallower than that of MDM2. This information explained why MDM2 inhibitors failed to affect MDMX–p53 binding and also prompted us to initiate a computational structure-based screening using the AutoDock computer program (Morris et al, 2008) for the docking of virtual compounds that could distinguish the p53 binding sites on MDM2 and MDMX. From our initial computational screening of half a million of commercially available compounds from the ChemDiv chemical library, we selected and purchased 50 top candidates. These compounds were tested in cell-based assays at 10 µM for their ability to induce p53 levels in human lung carcinoma H460 cells using an immunoblotting (IB) analyses. To our delight, one small molecule, 10-[2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)butanoyl]-10H-phenothiazine (abbreviated as INZ; Fig 1B), induced p53 levels as effectively as actinomycin D (ActD; 10 nM) and in a much more pronounced manner than did the rest of the compounds tested (Fig 1A and data not shown).

Bottom Line: Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress.Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro.Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology and Cancer Center, Tulane University School of Medicine, Louisiana, LA, USA.

Show MeSH
Related in: MedlinePlus