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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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INZ inhibits SIRT1 activity and directly binds to SIRT1 in vitroA. INZ inhibits deacetylation of p53 at lysine 382 by SIRT1 in vitro in a dose-dependent fashion using acetylated p53 protein as a substrate as described in the Experimental Procedures Section of the Supporting Information.B-C. Purified SIRT1 was incubated at indicated concentrations with biotinylated INZ that was conjugated with avidin beads in the presence or the absence of 20 µM of non-biotinylated INZ. Purified SIRT7 was used as a negative control. The intensity of each band for bound SIRT1 as analysed using IB was quantified (B), and each sample was individually compared with the intensity of the samples without SIRT1. The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3).D. The inhibitory effects of INZ, Cambinol and Salermide on SIRT1 activity were measured by the increase of the levels of acetylated p53 at lysine 382 in vitro. The percentage of inhibition was calculated as described in the ‘Experimental Procedures’ Section of Supporting Information and shown in below. Values represent mean ± SD (n = 3).E-F. Effect of INZ, Cambinol, Salermide or Tenovin-6 on p53 acetylation and level in H460 (E) and HCT116+/+ (F) cells.
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fig06: INZ inhibits SIRT1 activity and directly binds to SIRT1 in vitroA. INZ inhibits deacetylation of p53 at lysine 382 by SIRT1 in vitro in a dose-dependent fashion using acetylated p53 protein as a substrate as described in the Experimental Procedures Section of the Supporting Information.B-C. Purified SIRT1 was incubated at indicated concentrations with biotinylated INZ that was conjugated with avidin beads in the presence or the absence of 20 µM of non-biotinylated INZ. Purified SIRT7 was used as a negative control. The intensity of each band for bound SIRT1 as analysed using IB was quantified (B), and each sample was individually compared with the intensity of the samples without SIRT1. The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3).D. The inhibitory effects of INZ, Cambinol and Salermide on SIRT1 activity were measured by the increase of the levels of acetylated p53 at lysine 382 in vitro. The percentage of inhibition was calculated as described in the ‘Experimental Procedures’ Section of Supporting Information and shown in below. Values represent mean ± SD (n = 3).E-F. Effect of INZ, Cambinol, Salermide or Tenovin-6 on p53 acetylation and level in H460 (E) and HCT116+/+ (F) cells.

Mentions: To validate this possibility, we measured the effect of INZ on SIRT1 activity by conducing in vitro assays using acetylated p53 protein as a substrate and purified His–SIRT1 (Fig S6A and B of Supporting Information) as described in the Experimental Procedures Section of the Supporting Information. As shown in Fig 6A, INZ inhibited SIRT1 deacetylase activity in a dose-dependent fashion and effectively inhibited this activity at 3 µM. This inhibition was specific to INZ and its chemical analogue INZ1 (methyl substituted R1), which activated p53 (Fig 1B and C) and decreased SIRT1 activity in a dose-dependent fashion (Fig S7A of Supporting Information). By contrast, the analogues INZ5 (bromide substituted on R3) and INZ 15 or INZ 18 (lack of G1, data not shown) that failed to induce p53 did not significantly inhibit SIRT1 activity even at the highest concentration we tested (20 µM).


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)

INZ inhibits SIRT1 activity and directly binds to SIRT1 in vitroA. INZ inhibits deacetylation of p53 at lysine 382 by SIRT1 in vitro in a dose-dependent fashion using acetylated p53 protein as a substrate as described in the Experimental Procedures Section of the Supporting Information.B-C. Purified SIRT1 was incubated at indicated concentrations with biotinylated INZ that was conjugated with avidin beads in the presence or the absence of 20 µM of non-biotinylated INZ. Purified SIRT7 was used as a negative control. The intensity of each band for bound SIRT1 as analysed using IB was quantified (B), and each sample was individually compared with the intensity of the samples without SIRT1. The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3).D. The inhibitory effects of INZ, Cambinol and Salermide on SIRT1 activity were measured by the increase of the levels of acetylated p53 at lysine 382 in vitro. The percentage of inhibition was calculated as described in the ‘Experimental Procedures’ Section of Supporting Information and shown in below. Values represent mean ± SD (n = 3).E-F. Effect of INZ, Cambinol, Salermide or Tenovin-6 on p53 acetylation and level in H460 (E) and HCT116+/+ (F) cells.
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fig06: INZ inhibits SIRT1 activity and directly binds to SIRT1 in vitroA. INZ inhibits deacetylation of p53 at lysine 382 by SIRT1 in vitro in a dose-dependent fashion using acetylated p53 protein as a substrate as described in the Experimental Procedures Section of the Supporting Information.B-C. Purified SIRT1 was incubated at indicated concentrations with biotinylated INZ that was conjugated with avidin beads in the presence or the absence of 20 µM of non-biotinylated INZ. Purified SIRT7 was used as a negative control. The intensity of each band for bound SIRT1 as analysed using IB was quantified (B), and each sample was individually compared with the intensity of the samples without SIRT1. The results shown are representative of three-independent experiments. Values represent mean ± SD (n = 3).D. The inhibitory effects of INZ, Cambinol and Salermide on SIRT1 activity were measured by the increase of the levels of acetylated p53 at lysine 382 in vitro. The percentage of inhibition was calculated as described in the ‘Experimental Procedures’ Section of Supporting Information and shown in below. Values represent mean ± SD (n = 3).E-F. Effect of INZ, Cambinol, Salermide or Tenovin-6 on p53 acetylation and level in H460 (E) and HCT116+/+ (F) cells.
Mentions: To validate this possibility, we measured the effect of INZ on SIRT1 activity by conducing in vitro assays using acetylated p53 protein as a substrate and purified His–SIRT1 (Fig S6A and B of Supporting Information) as described in the Experimental Procedures Section of the Supporting Information. As shown in Fig 6A, INZ inhibited SIRT1 deacetylase activity in a dose-dependent fashion and effectively inhibited this activity at 3 µM. This inhibition was specific to INZ and its chemical analogue INZ1 (methyl substituted R1), which activated p53 (Fig 1B and C) and decreased SIRT1 activity in a dose-dependent fashion (Fig S7A of Supporting Information). By contrast, the analogues INZ5 (bromide substituted on R3) and INZ 15 or INZ 18 (lack of G1, data not shown) that failed to induce p53 did not significantly inhibit SIRT1 activity even at the highest concentration we tested (20 µM).

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