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Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator.

Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, McCarthy A, Appleyard V, Murray KE, Baker L, Thompson A, Mathers J, Holland SJ, Stark MJ, Pass G, Woods J, Lane DP, Westwood NJ - Cancer Cell (2008)

Bottom Line: Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6.Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents.This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK. s.lain@dundee.ac.uk

ABSTRACT
We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

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Effect of Tenovin-1 on Cultured Tumor Cell Lines(A) Tenovin-1 structure.(B) MCF-7 cells (expressing wild-type p53) were treated with 10 μM tenovin-1 for the indicated times. p53 levels and p21 levels were detected using DO1 (Bartkova et al., 1993) and 118 (Fredersdorf et al., 1996) mouse monoclonal antibodies, respectively. An antibody against α-tubulin (Cat. No. T9026, Sigma) was used to monitor loading efficiency.(C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times, and p53 and p21 mRNA levels were analyzed by Taqman-PCR as described (Saville et al., 2004). Error bars correspond to standard deviation values (n = 3).(D) Toxicity of tenovin-1 on cultured tumor cells. Tumor cell lines were treated with DMSO (control) or with 10 μM tenovin-1 for 48 hr. Cell death (necrosis and apoptosis) was measured by annexin-V/propidium iodide labeling and FACS. Values correspond to the average of two independent experiments (±SD). p53 status in each cell line is indicated (DN, coexpressing the dominant-negative form of p53).
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fig1: Effect of Tenovin-1 on Cultured Tumor Cell Lines(A) Tenovin-1 structure.(B) MCF-7 cells (expressing wild-type p53) were treated with 10 μM tenovin-1 for the indicated times. p53 levels and p21 levels were detected using DO1 (Bartkova et al., 1993) and 118 (Fredersdorf et al., 1996) mouse monoclonal antibodies, respectively. An antibody against α-tubulin (Cat. No. T9026, Sigma) was used to monitor loading efficiency.(C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times, and p53 and p21 mRNA levels were analyzed by Taqman-PCR as described (Saville et al., 2004). Error bars correspond to standard deviation values (n = 3).(D) Toxicity of tenovin-1 on cultured tumor cells. Tumor cell lines were treated with DMSO (control) or with 10 μM tenovin-1 for 48 hr. Cell death (necrosis and apoptosis) was measured by annexin-V/propidium iodide labeling and FACS. Values correspond to the average of two independent experiments (±SD). p53 status in each cell line is indicated (DN, coexpressing the dominant-negative form of p53).

Mentions: Following a pilot study with 4,000 compounds (Berkson et al., 2005), we screened 30,000 drug-like small molecules from the Chembridge DIVERSet for their ability to activate p53 in a robust, simple, and cheap primary cell-based screening assay. For details on the primary assay, secondary assays, and criteria used for prioritizing compounds, see the Supplemental Data available online. Here we describe the characterization of one hit compound from this screen, tenovin-1 (see Figure 1A for structure). As shown in Figure 1B, tenovin-1 elevates the amount of p53 protein within 2 hr of treatment. This compound also increases the levels of the p53-downstream target p21CIP/WAF1 protein (Figure 1B) and mRNA (Figure 1C), confirming that tenovin-1 can induce expression from an endogenous p53-dependent promoter. Tenovin-1 treatment does not alter p53 mRNA levels (Figure 1C), but increases p53 levels when p53 is coexpressed with mdm2 (see below and Figures 6B and 6G). This suggests that tenovin-1 protects p53 from mdm2-mediated degradation with little effect on p53 synthesis.


Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator.

Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, McCarthy A, Appleyard V, Murray KE, Baker L, Thompson A, Mathers J, Holland SJ, Stark MJ, Pass G, Woods J, Lane DP, Westwood NJ - Cancer Cell (2008)

Effect of Tenovin-1 on Cultured Tumor Cell Lines(A) Tenovin-1 structure.(B) MCF-7 cells (expressing wild-type p53) were treated with 10 μM tenovin-1 for the indicated times. p53 levels and p21 levels were detected using DO1 (Bartkova et al., 1993) and 118 (Fredersdorf et al., 1996) mouse monoclonal antibodies, respectively. An antibody against α-tubulin (Cat. No. T9026, Sigma) was used to monitor loading efficiency.(C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times, and p53 and p21 mRNA levels were analyzed by Taqman-PCR as described (Saville et al., 2004). Error bars correspond to standard deviation values (n = 3).(D) Toxicity of tenovin-1 on cultured tumor cells. Tumor cell lines were treated with DMSO (control) or with 10 μM tenovin-1 for 48 hr. Cell death (necrosis and apoptosis) was measured by annexin-V/propidium iodide labeling and FACS. Values correspond to the average of two independent experiments (±SD). p53 status in each cell line is indicated (DN, coexpressing the dominant-negative form of p53).
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Related In: Results  -  Collection

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Show All Figures
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fig1: Effect of Tenovin-1 on Cultured Tumor Cell Lines(A) Tenovin-1 structure.(B) MCF-7 cells (expressing wild-type p53) were treated with 10 μM tenovin-1 for the indicated times. p53 levels and p21 levels were detected using DO1 (Bartkova et al., 1993) and 118 (Fredersdorf et al., 1996) mouse monoclonal antibodies, respectively. An antibody against α-tubulin (Cat. No. T9026, Sigma) was used to monitor loading efficiency.(C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times, and p53 and p21 mRNA levels were analyzed by Taqman-PCR as described (Saville et al., 2004). Error bars correspond to standard deviation values (n = 3).(D) Toxicity of tenovin-1 on cultured tumor cells. Tumor cell lines were treated with DMSO (control) or with 10 μM tenovin-1 for 48 hr. Cell death (necrosis and apoptosis) was measured by annexin-V/propidium iodide labeling and FACS. Values correspond to the average of two independent experiments (±SD). p53 status in each cell line is indicated (DN, coexpressing the dominant-negative form of p53).
Mentions: Following a pilot study with 4,000 compounds (Berkson et al., 2005), we screened 30,000 drug-like small molecules from the Chembridge DIVERSet for their ability to activate p53 in a robust, simple, and cheap primary cell-based screening assay. For details on the primary assay, secondary assays, and criteria used for prioritizing compounds, see the Supplemental Data available online. Here we describe the characterization of one hit compound from this screen, tenovin-1 (see Figure 1A for structure). As shown in Figure 1B, tenovin-1 elevates the amount of p53 protein within 2 hr of treatment. This compound also increases the levels of the p53-downstream target p21CIP/WAF1 protein (Figure 1B) and mRNA (Figure 1C), confirming that tenovin-1 can induce expression from an endogenous p53-dependent promoter. Tenovin-1 treatment does not alter p53 mRNA levels (Figure 1C), but increases p53 levels when p53 is coexpressed with mdm2 (see below and Figures 6B and 6G). This suggests that tenovin-1 protects p53 from mdm2-mediated degradation with little effect on p53 synthesis.

Bottom Line: Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6.Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents.This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK. s.lain@dundee.ac.uk

ABSTRACT
We have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.

Show MeSH
Related in: MedlinePlus