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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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Effect of active chemicals on membrane integrity and apoptosis.TSC2+/+ MEFs were incubated for 4 h with DMSO or with 3 µM niclosamide, 30 µM amiodarone, 3 µM rottlerin or 10 µM perhexiline in the cell culture media indicated. Chemicals and media were washed away and the cells were incubated for 24 h in complete medium without chemicals. The extent of cell death and apoptosis was determined by measuring propidium iodide uptake (PI, y axis; relative fluorescence intensity measured in FL2 channel) and FITC Annexin V staining (x axis; relative fluorescence intensity measured in FL1 channel) by flow cytometry. Live cells (PI- and Annexin V-negative) appear in the lower left part of the scatter plots while cells that have died of apoptosis (PI- and Annexin V-positive) appear in the upper right hand part. The numbers in each box indicate the percent of dead or dying (PI- and/or Annexin V-positive) cells.
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pone-0007124-g010: Effect of active chemicals on membrane integrity and apoptosis.TSC2+/+ MEFs were incubated for 4 h with DMSO or with 3 µM niclosamide, 30 µM amiodarone, 3 µM rottlerin or 10 µM perhexiline in the cell culture media indicated. Chemicals and media were washed away and the cells were incubated for 24 h in complete medium without chemicals. The extent of cell death and apoptosis was determined by measuring propidium iodide uptake (PI, y axis; relative fluorescence intensity measured in FL2 channel) and FITC Annexin V staining (x axis; relative fluorescence intensity measured in FL1 channel) by flow cytometry. Live cells (PI- and Annexin V-negative) appear in the lower left part of the scatter plots while cells that have died of apoptosis (PI- and Annexin V-positive) appear in the upper right hand part. The numbers in each box indicate the percent of dead or dying (PI- and/or Annexin V-positive) cells.

Mentions: The observation that some chemicals that inhibit mTORC1 signaling and stimulate autophagy also selectively killed cells in starvation conditions was not anticipated given the established role of autophagy in promoting survival during starvation. Therefore, we next investigated the death mechanism involved. TSC2+/+ MEFs were exposed to chemicals for 4 h in complete medium or in starvation conditions. The drugs were then washed away and after 24 h, the cells were stained with FITC-Annexin V and propidium iodide. Annexin V binds to phosphatidylserine on the outer leaflet of the plasma membrane and labels cells actively undergoing apoptosis while propidium iodide is excluded by viable cells with an intact plasma membrane and therefore labels dead cells. Flow cytometry analysis revealed that in starvation conditions, the four chemicals caused a considerable increase in cells labeled with FITC-Annexin and propidium iodide (Fig. 10). Autophagic cell death is not associated with increased Annexin V binding [56], indicating that the chemicals trigger apoptotic cell death in starvation conditions.


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)

Effect of active chemicals on membrane integrity and apoptosis.TSC2+/+ MEFs were incubated for 4 h with DMSO or with 3 µM niclosamide, 30 µM amiodarone, 3 µM rottlerin or 10 µM perhexiline in the cell culture media indicated. Chemicals and media were washed away and the cells were incubated for 24 h in complete medium without chemicals. The extent of cell death and apoptosis was determined by measuring propidium iodide uptake (PI, y axis; relative fluorescence intensity measured in FL2 channel) and FITC Annexin V staining (x axis; relative fluorescence intensity measured in FL1 channel) by flow cytometry. Live cells (PI- and Annexin V-negative) appear in the lower left part of the scatter plots while cells that have died of apoptosis (PI- and Annexin V-positive) appear in the upper right hand part. The numbers in each box indicate the percent of dead or dying (PI- and/or Annexin V-positive) cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007124-g010: Effect of active chemicals on membrane integrity and apoptosis.TSC2+/+ MEFs were incubated for 4 h with DMSO or with 3 µM niclosamide, 30 µM amiodarone, 3 µM rottlerin or 10 µM perhexiline in the cell culture media indicated. Chemicals and media were washed away and the cells were incubated for 24 h in complete medium without chemicals. The extent of cell death and apoptosis was determined by measuring propidium iodide uptake (PI, y axis; relative fluorescence intensity measured in FL2 channel) and FITC Annexin V staining (x axis; relative fluorescence intensity measured in FL1 channel) by flow cytometry. Live cells (PI- and Annexin V-negative) appear in the lower left part of the scatter plots while cells that have died of apoptosis (PI- and Annexin V-positive) appear in the upper right hand part. The numbers in each box indicate the percent of dead or dying (PI- and/or Annexin V-positive) cells.
Mentions: The observation that some chemicals that inhibit mTORC1 signaling and stimulate autophagy also selectively killed cells in starvation conditions was not anticipated given the established role of autophagy in promoting survival during starvation. Therefore, we next investigated the death mechanism involved. TSC2+/+ MEFs were exposed to chemicals for 4 h in complete medium or in starvation conditions. The drugs were then washed away and after 24 h, the cells were stained with FITC-Annexin V and propidium iodide. Annexin V binds to phosphatidylserine on the outer leaflet of the plasma membrane and labels cells actively undergoing apoptosis while propidium iodide is excluded by viable cells with an intact plasma membrane and therefore labels dead cells. Flow cytometry analysis revealed that in starvation conditions, the four chemicals caused a considerable increase in cells labeled with FITC-Annexin and propidium iodide (Fig. 10). Autophagic cell death is not associated with increased Annexin V binding [56], indicating that the chemicals trigger apoptotic cell death in starvation conditions.

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