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Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.

Balgi AD, Fonseca BD, Donohue E, Tsang TC, Lajoie P, Proud CG, Nabi IR, Roberge M - PLoS ONE (2009)

Bottom Line: The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly.By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.

Methodology/principal findings: Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.

Conclusion/significance: The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.

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Related in: MedlinePlus

Concentration-dependent stimulation of EGFP-LC3 processing and inhibition of mTORC1 signaling by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with chemicals at the indicated concentrations or with 20 nM rapamycin. EGFP-LC3 processing was monitored by probing lysates with anti-GFP antibody, mTORC1 activity by probing lysates with antisera against phosphorylated (Thr389) S6K and total S6K, and mTORC2 activity with antisera against phosphorylated (Ser473) PKB/Akt and total PKB/Akt. β-tubulin immunostaining was used as a protein loading control.
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pone-0007124-g003: Concentration-dependent stimulation of EGFP-LC3 processing and inhibition of mTORC1 signaling by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with chemicals at the indicated concentrations or with 20 nM rapamycin. EGFP-LC3 processing was monitored by probing lysates with anti-GFP antibody, mTORC1 activity by probing lysates with antisera against phosphorylated (Thr389) S6K and total S6K, and mTORC2 activity with antisera against phosphorylated (Ser473) PKB/Akt and total PKB/Akt. β-tubulin immunostaining was used as a protein loading control.

Mentions: Incubation with perhexiline at varying concentrations (3 to 10 µM) for 4 h caused a concentration-dependent accumulation of free EGFP (Fig. 3, top panel) as well as a small but appreciable increase in EGFP-LC3 lipidation. Niclosamide led to the accumulation of EGFP-LC3II at concentrations as low as 1 µM (Fig. 3, top panel). Free EGFP did not accumulate, but faster migrating bands corresponding to EGFP proteolysis products were readily detectable (Fig. 3). Lipidated EGFP-LC3 (EGFP-LC3II) was detected within 15 min and it continued to accumulate over time (Fig. 4b). Amiodarone also led to the accumulation of EGFP-LC3II (Fig. 4c, 5a) and a substantial increase in free EGFP at 10 µM or higher concentrations (Fig. 3, 5a) within 2 to 4 h (Fig. 4c). Like niclosamide and amiodarone, rottlerin caused the accumulation of EGFP-LC3II and free EGFP, as well as proteolytic fragments of EGFP at 0.3 µM and higher concentrations (Fig. 3). Lipidated EGFP-LC3 was visible within 30 min incubation while free EGFP could be detected between 2 and 4 h (Fig. 4d).


Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.

Balgi AD, Fonseca BD, Donohue E, Tsang TC, Lajoie P, Proud CG, Nabi IR, Roberge M - PLoS ONE (2009)

Concentration-dependent stimulation of EGFP-LC3 processing and inhibition of mTORC1 signaling by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with chemicals at the indicated concentrations or with 20 nM rapamycin. EGFP-LC3 processing was monitored by probing lysates with anti-GFP antibody, mTORC1 activity by probing lysates with antisera against phosphorylated (Thr389) S6K and total S6K, and mTORC2 activity with antisera against phosphorylated (Ser473) PKB/Akt and total PKB/Akt. β-tubulin immunostaining was used as a protein loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742736&req=5

pone-0007124-g003: Concentration-dependent stimulation of EGFP-LC3 processing and inhibition of mTORC1 signaling by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with chemicals at the indicated concentrations or with 20 nM rapamycin. EGFP-LC3 processing was monitored by probing lysates with anti-GFP antibody, mTORC1 activity by probing lysates with antisera against phosphorylated (Thr389) S6K and total S6K, and mTORC2 activity with antisera against phosphorylated (Ser473) PKB/Akt and total PKB/Akt. β-tubulin immunostaining was used as a protein loading control.
Mentions: Incubation with perhexiline at varying concentrations (3 to 10 µM) for 4 h caused a concentration-dependent accumulation of free EGFP (Fig. 3, top panel) as well as a small but appreciable increase in EGFP-LC3 lipidation. Niclosamide led to the accumulation of EGFP-LC3II at concentrations as low as 1 µM (Fig. 3, top panel). Free EGFP did not accumulate, but faster migrating bands corresponding to EGFP proteolysis products were readily detectable (Fig. 3). Lipidated EGFP-LC3 (EGFP-LC3II) was detected within 15 min and it continued to accumulate over time (Fig. 4b). Amiodarone also led to the accumulation of EGFP-LC3II (Fig. 4c, 5a) and a substantial increase in free EGFP at 10 µM or higher concentrations (Fig. 3, 5a) within 2 to 4 h (Fig. 4c). Like niclosamide and amiodarone, rottlerin caused the accumulation of EGFP-LC3II and free EGFP, as well as proteolytic fragments of EGFP at 0.3 µM and higher concentrations (Fig. 3). Lipidated EGFP-LC3 was visible within 30 min incubation while free EGFP could be detected between 2 and 4 h (Fig. 4d).

Bottom Line: The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly.By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.

Methodology/principal findings: Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.

Conclusion/significance: The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.

Show MeSH
Related in: MedlinePlus