Discovery of cationic amphiphilic drugs that activate chaperone mediated-autophagy
Introduction: Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradation pathway characterized by the translocation of proteins to the lysosome mediated by the LAMP2A protein and the hsc70 chaperone, wich recognize KFERQ-like peptide motifs in CMA protein substrates. Dysfunctional activity of CMA has been shown in several pathologies including neurodegenerative diseases and cancer. The knowledge of mechanisms of CMA is limited and there are no known molecules that modulate CMA directly. Cationic amphiphilic drugs (CADs) are a family of lysosomotropic drugs that have shown to modify several aspects of lysosomal activity; however their effects on CMA are unknown. Materials and Methods: We constructed a stable cell line reporter of CMA in NIH3T3 cells based in a previously described photoactivatable fluorescent protein reporter fused to a KFERQ sequence and performed a drug screening with a library of cationic amphiphilic drugs by high content imaging analysis. Results: We obtained a CMA reporter cell clone and validated his response to serum deprivation. Colocalization analysis of the reporter with lysosomes in single cells indicates increased localization of fluorescent reporter with lysosomes in response to serum deprivation that was not observed in control cells. Using this cell reporter a number of CADs were discovered that activates CMA at nanomolar concentrations. Analysis of the structures of positive and negative hits indicates structural similarities between active compounds. Treatment of α-synuclein expressing cells with these CADs identifies several compounds that induce degradation of this substrate by CMA. Discussion: According to our results some cationic amphiphilic drugs with a common structure are capable of modulating positively the activity of CMA and degrade CMA substrates. Preliminary results indicate a mechanism involving AMPK activation. Acknowledgements: Fondecyt 11161056, Proyecto FCPV Apoyo Basal AFB170004.
|Fecha de publicación:
|Año de Inicio/Término:
|18 de Octubre de 2018