Affiliation:
1. Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA
Abstract
Macroautophagy is a catabolic process wherein cytosolic cargo is engulfed in an autophagosome that fuses with a lysosome to degrade the cargo for recycling. Autophagy maintains cellular homeostasis and is involved in a myriad of illnesses ranging from cancer to neurodegenerative diseases, but its therapeutic potential remains elusive due to a lack of potent and specific autophagy modulators. To identify specific inhibitors of early autophagy, a target-based, compound-multiplexed, fluorescence polarization, high-throughput screen that targets the ATG5–ATG16L1 protein–protein interaction was developed. This interaction is critical for the formation of LC3-II, which is involved in phagophore maturation, and its disruption should inhibit autophagy. This assay is based on the polarization of light emitted by a fluorescent rhodamine tag conjugated to a peptide corresponding to the N-terminal region of ATG16L1 (ATG16L1-N). It was confirmed that this peptide binds specifically to ATG5, and the assay was validated by rapidly screening 4800 molecules through compound multiplexing. Through these initial screening efforts, a molecule was identified that disrupts the ATG5–ATG16L1 protein–protein interaction with micromolar potency, and this molecule will serve as a starting point for chemical optimization as an autophagy inhibitor.
Funder
center for clinical and translational science, university of illinois at chicago
University of Illinois at Chicago
Subject
Molecular Medicine,Biochemistry,Analytical Chemistry,Biotechnology
Cited by
6 articles.
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