Avobenzone, Guaiazulene and Tioxolone identified as potent autophagy inducers in a high-throughput image based screen for autophagy flux

Autophagy is a conserved intracellular degradation pathway that is essential for maintaining cellular homeostasis. Given its critical role in several disease conditions, recent studies are focussed on identifying drugs/small molecules with autophagy modulating capacity for potential clinical applications. Here, we describe the development and characterisation of a quantitative image-based high content screening platform for autophagy flux measurements using the human melanoma A375 cell line that stably expresses the GFP-LC3-RFP probe. The GFP-LC3 is incorporated into autophagosomes, while RFP serves an internal control. The GFP/RFP fluorescence intensity ratio gives an accurate indication of autophagy induction (low ratio) vs blockage of autophagy flux (high ratio), and was validated with the autophagy inducer Torin1 and inhibitor Bafilomycin A1. This assay was used to screen the Spectrum collection library comprising of 2560 compounds, to identify autophagy modulators. In addition to known autophagy effectors, several novel autophagy inducers and inhibitors were identified in our study. Further three FDA approved drugs that are widely used in skin-care products: Avobenzone, Guaiazulene and Tioxolone, were validated as potent autophagy inducers that function in an mTOR independent manner.Abbreviations Baf, Bafilomycin A1; LC3, Microtubule-associated protein light chain 3; mTOR, mechanistic target of rapamycin.