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

BackgroundMammalian 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 FindingsUsing an automated cell-based assay, we screened a collection of &gt;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/SignificanceThe 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.<br/

The role of PKCε-dependent signaling for cardiac differentiation

Protein kinase Cepsilon (PKCε) exerts a well-known cardio-protective activity in ischemia–reperfusion injury and plays a pivotal role in stem cell proliferation and differentiation. Although many studies have been performed on physiological and morphological effects of PKCε mis-expression in cardiomyocytes, molecular information on the role of PKCε on early cardiac gene expression are still lacking. We addressed the molecular role of PKCε in cardiac cells using mouse cardiomyocytes and rat bone marrow mesenchymal stem cells. We show that PKCε is modulated in cardiac differentiation producing an opposite regulation of the cardiac genes NK2 transcription factor related, locus 5 (nkx2.5) and GATA binding protein 4 (gata4) both in vivo and in vitro. Phospho-extracellular regulated mitogen-activated protein kinase 1/2 (p-ERK1/2) levels increase in PKCε over-expressing cells, while pkcε siRNAs produce a decrease in p-ERK1/2. Indeed, pharmacological inhibition of ERK1/2 rescues the expression levels of both nkx2.5 and gata4, suggesting that a reinforced (mitogen-activated protein kinase) MAPK signaling is at the basis of the observed inhibition of cardiac gene expression in the PKCε over-expressing hearts. We demonstrate that PKCε is critical for cardiac cell early gene expression evidencing that this protein is a regulator that has to be fine tuned in precursor cardiac cells