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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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Visualization of punctate EGFP-LC3 in cells treated with autophagy-modulating compounds.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control), 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone or 3 µM rottlerin. Photographs show stacks of confocal sections spanning the entire cell thickness.
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pone-0007124-g002: Visualization of punctate EGFP-LC3 in cells treated with autophagy-modulating compounds.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control), 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone or 3 µM rottlerin. Photographs show stacks of confocal sections spanning the entire cell thickness.

Mentions: To verify that the punctate EGFP-LC3 fluorescence induced by the four chemicals represented autophagosome formation rather than, for instance, fluorescent drug precipitates, EGFP-LC3 fluorescence was examined at higher resolution by laser confocal microscopy. As expected, non-treated cells showed diffuse EGFP-LC3 fluorescence with few punctate structures (Fig. 2). Incubation with perhexiline, niclosamide, amiodarone or rottlerin for 4 h induced the appearance of a large number of EGFP-LC3-labeled cytoplasmic vesicles consistent with autophagosome formation (Fig. 2).


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)

Visualization of punctate EGFP-LC3 in cells treated with autophagy-modulating compounds.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control), 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone or 3 µM rottlerin. Photographs show stacks of confocal sections spanning the entire cell thickness.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007124-g002: Visualization of punctate EGFP-LC3 in cells treated with autophagy-modulating compounds.MCF-7 cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control), 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone or 3 µM rottlerin. Photographs show stacks of confocal sections spanning the entire cell thickness.
Mentions: To verify that the punctate EGFP-LC3 fluorescence induced by the four chemicals represented autophagosome formation rather than, for instance, fluorescent drug precipitates, EGFP-LC3 fluorescence was examined at higher resolution by laser confocal microscopy. As expected, non-treated cells showed diffuse EGFP-LC3 fluorescence with few punctate structures (Fig. 2). Incubation with perhexiline, niclosamide, amiodarone or rottlerin for 4 h induced the appearance of a large number of EGFP-LC3-labeled cytoplasmic vesicles consistent with autophagosome formation (Fig. 2).

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