A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Biology

Background:

• Autophagy is a crucial cellular degradation pathway regulated by autophagy-related proteins (Atgs).
• The mammalian Atg8 (mAtg8) family, including LC3 and GABARAP proteins, is central to autophagosome formation and cargo recognition via the LC3-interacting region (LIR).
• Targeting mAtg8 interactions with small molecules is key to understanding and manipulating autophagy.

Purpose of the Study:

• To develop novel small-molecule ligands that bind to LC3 proteins.
• To explore fragment and DNA-encoded library (DEL) screening as methods for discovering these ligands.
• To investigate the potential of these ligands for probing autophagy and engineering targeted degradation.

Main Methods:

• Unbiased fragment screening to identify initial binders.
• DNA-encoded library (DEL) screening for higher-affinity compounds.
• LC3A-fragment complex crystallography to determine binding modes.
• Analysis of DEL hits for potential applications like fluorescent probes or bivalent degraders.

Main Results:

• Both fragment and DEL screening yielded compounds that bind to LC3 proteins.
• Fragment hits preferentially occupied a hydrophobic pocket in mATG8 proteins.
• DEL screening identified higher-affinity binders, including potential reversible covalent ligands.
• DEL hits suggested strategies for developing fluorescent probes and targeted protein degraders.

Conclusions:

• Small molecules can effectively target the LC3 LIR-binding surface, although rational design for high affinity is challenging.
• DEL screening is a powerful approach for discovering potent LC3 ligands with diverse functionalities.
• These findings provide valuable chemical tools for advancing autophagy research and therapeutic development.