Exploring Arylidene-Indolinone Ligands of Autophagy Proteins LC3B and GABARAP

Abstract

Herein we report the first structure-activity studies of compoundGW5074which has demonstrated binding affinity to autophagy-related proteins LC3B and GABARAP. The literature has conflicting information on the binding affinities of this compound to LC3B and GABARAP, and there is some debate regarding its use as a component of autophagy-targeting chimeras (AUTACs) or autophagosome-tethering chimeras (ATTECs). We developed an AlphaScreen binding assay to compare the potencies of these compounds for inhibiting binding of known peptide ligands to LC3B and GABARAP. 38 analogs were synthesized and tested against both proteins. Inhibitory potencies were found to be mid to high micromolar, and 2D-NMR data revealed the binding site as hydrophobic pocket 1, where the native peptide ligands bind with an aromatic side chain. Our results suggest thatGW5074does bind LC3B and GABARAP in the micromolar range, but it may not be a good candidate for potent autophagy inhibition. These affinities could support further exploration in targeted protein degradation, but only if off-target effects can be appropriately controlled for.