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


RESULTS
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.


CONCLUSIONS
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.
Balgi
Aruna D
AD
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Fonseca
Bruno D
BD
Donohue
Elizabeth
E
Tsang
Trevor C F
TC
Lajoie
Patrick
P
Proud
Christopher G
CG
Nabi
Ivan R
IR
Roberge
Michel
M
D000098:Acetophenones / Q000494:pharmacology; D000638:Amiodarone / Q000494:pharmacology; D000969:Antinematodal Agents / Q000494:pharmacology; D001331:Automation; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D001578:Benzopyrans / Q000494:pharmacology; D002317:Cardiovascular Agents / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D008956:Models, Chemical; D046912:Multiprotein Complexes; D009534:Niclosamide / Q000494:pharmacology; D052756:Nutritional Sciences; D010480:Perhexiline / Q000494:pharmacology; D011506:Proteins; D015398:Signal Transduction / Q000187:drug effects*; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000098:Acetophenones; D000969:Antinematodal Agents; D001578:Benzopyrans; D002317:Cardiovascular Agents; D004791:Enzyme Inhibitors; D046912:Multiprotein Complexes; D011506:Proteins; D014157:Transcription Factors; D009534:Niclosamide; C085746:rottlerin; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D010480:Perhexiline; D000638:Amiodarone