Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of 4E-BP phosphorylation and function by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were incubated with 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone, 3 µM rottlerin or DMSO for 1 h or 4 h in complete medium. (a) EGFP-LC3 processing and mTORC1 and mTORC2 activation were monitored by immunoblotting as described in the legend to Fig. 3. Lysates were also probed with antisera to phosphorylated (Thr37/46 or Ser65) 4E-BPs and total 4E-BP1. (b) eIF4E was purified from cell extracts by m7GTP affinity chromatography as described in Materials and Methods and the purified material was probed with antisera against eIF4E, eIF4G and 4E-BP1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2742736&req=5

pone-0007124-g006: Inhibition of 4E-BP phosphorylation and function by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were incubated with 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone, 3 µM rottlerin or DMSO for 1 h or 4 h in complete medium. (a) EGFP-LC3 processing and mTORC1 and mTORC2 activation were monitored by immunoblotting as described in the legend to Fig. 3. Lysates were also probed with antisera to phosphorylated (Thr37/46 or Ser65) 4E-BPs and total 4E-BP1. (b) eIF4E was purified from cell extracts by m7GTP affinity chromatography as described in Materials and Methods and the purified material was probed with antisera against eIF4E, eIF4G and 4E-BP1.

Mentions: mTORC1 signaling also mediates the phosphorylation of multiple residues on 4E-BP1, including Thr37/46 and Ser65. Perhexiline, niclosamide, amiodarone and rottlerin, but not DMSO, strongly inhibited phosphorylation at Ser65 at 1 h and completely abolished it at 4 h as judged by the decreased binding of phospho-specific antibody and increased electrophoretic mobility of 4E-BP1 (Fig. 6a). These chemicals also decreased the phosphorylation of Thr37/46 in 4E-BP1 and 4E-BP2 (Fig. 6a). The phosphorylation of Ser65 requires both amino acids and growth factors, whereas phosphorylation of Thr37/46 is strongly stimulated by amino acids alone [34]. To examine whether perhexiline, niclosamide, amiodarone and rottlerin inhibited the amino acid-dependent phosphorylation of Thr37/46, MCF-7 cells were exposed to perhexiline, rottlerin, amiodarone or niclosamide in medium lacking serum for 1 h or 4 h. All four chemicals reduced the amino acid-mediated phosphorylation of Thr37/46 in 4E-BP1 and 4E-BP2, although not completely (Fig. S3b).


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)

Inhibition of 4E-BP phosphorylation and function by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were incubated with 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone, 3 µM rottlerin or DMSO for 1 h or 4 h in complete medium. (a) EGFP-LC3 processing and mTORC1 and mTORC2 activation were monitored by immunoblotting as described in the legend to Fig. 3. Lysates were also probed with antisera to phosphorylated (Thr37/46 or Ser65) 4E-BPs and total 4E-BP1. (b) eIF4E was purified from cell extracts by m7GTP affinity chromatography as described in Materials and Methods and the purified material was probed with antisera against eIF4E, eIF4G and 4E-BP1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007124-g006: Inhibition of 4E-BP phosphorylation and function by perhexiline, niclosamide, amiodarone and rottlerin.MCF-7 cells stably expressing EGFP-LC3 were incubated with 10 µM perhexiline, 10 µM niclosamide, 50 µM amiodarone, 3 µM rottlerin or DMSO for 1 h or 4 h in complete medium. (a) EGFP-LC3 processing and mTORC1 and mTORC2 activation were monitored by immunoblotting as described in the legend to Fig. 3. Lysates were also probed with antisera to phosphorylated (Thr37/46 or Ser65) 4E-BPs and total 4E-BP1. (b) eIF4E was purified from cell extracts by m7GTP affinity chromatography as described in Materials and Methods and the purified material was probed with antisera against eIF4E, eIF4G and 4E-BP1.
Mentions: mTORC1 signaling also mediates the phosphorylation of multiple residues on 4E-BP1, including Thr37/46 and Ser65. Perhexiline, niclosamide, amiodarone and rottlerin, but not DMSO, strongly inhibited phosphorylation at Ser65 at 1 h and completely abolished it at 4 h as judged by the decreased binding of phospho-specific antibody and increased electrophoretic mobility of 4E-BP1 (Fig. 6a). These chemicals also decreased the phosphorylation of Thr37/46 in 4E-BP1 and 4E-BP2 (Fig. 6a). The phosphorylation of Ser65 requires both amino acids and growth factors, whereas phosphorylation of Thr37/46 is strongly stimulated by amino acids alone [34]. To examine whether perhexiline, niclosamide, amiodarone and rottlerin inhibited the amino acid-dependent phosphorylation of Thr37/46, MCF-7 cells were exposed to perhexiline, rottlerin, amiodarone or niclosamide in medium lacking serum for 1 h or 4 h. All four chemicals reduced the amino acid-mediated phosphorylation of Thr37/46 in 4E-BP1 and 4E-BP2, although not completely (Fig. S3b).

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