Targeting LC3/GABARAP for degrader development and autophagy modulation

Abstract

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP byin silicodocking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

Exploring Arylidene-Indolinone Ligands of Autophagy Proteins LC3B and GABARAP

“…They found that 1 binds weakly to GABARAP-L2 and has weak, if any, binding to LC3B. 32 In that work, the authors suggested that these activities were too weak to account for the compound’s ability to mediate targeted degradation.…”

“…The similarity of compound 1-induced CSP patterns observed for GABARAPL2 (ref. 32) and GABARAP (this study) attests to the high conservation of the core LIR docking site and to the challenges of devising small-molecule ligands targeting individual family members. More broadly, this work adds to the growing evidence that these protein-protein interactions are highly likely to be druggable using orally bioavailable small molecules.…”

“…The binding affinity of 1 for recombinant LC3B was reported in different papers as 0.468 µM (measured by small molecule microarray with a scanning oblique-incidence reflectivity difference microscope), 19 8.9 µM (measured by surface plasmon resonance), 17 and greater than 200 µM (measured by 2D-NMR titration). 32 Overall, these contradictory findings called into question the actual binding affinity of 1 for LC3B and other family members. A recent report by Knapp, Rogov, and coworkers more directly addressed this question using competition fluorescence polarization, NMR titration, and NanoBRET assays.…”

Discovery of Novel PDEδ Autophagic Degraders: A Case Study of Autophagy-Tethering Compound (ATTEC)

“…The recent research suggests that compound 1 may not interact with LC3 . To further verify the direct interaction between compound 12c and LC3 protein, MicroScale Thermophoresis (MST) method was used to assess the binding affinity of compound 12c toward LC3.…”

“…The recent research suggests that compound 1 may not interact with LC3. 29 To further verify the direct interaction between compound 12c and LC3 protein, MicroScale Thermophoresis (MST) method was used to assess the binding affinity of compound 12c toward LC3. The determined K D value of 187 nM demonstrated the potent and effective binding capability of compound 12c with LC3 (Figure S3 in the Supporting Information).…”