A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands

Abstract

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function.Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3Afragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Targeting LC3/GABARAP for degrader development and autophagy modulation

“…However, due to the presence of two carboxylic acid groups the identified ligands might be characterized by poor cell penetration and might therefore require optimization for ATTEC development. 20 As a second hit finding approach, we conducted a large-scale fragment screening campaign using X-ray crystallography by soaking a total of 1006 LC3B crystals with a diverse fragment library. This led to the collection of over 800 high quality diffraction datasets, which identified a total of 21 diverse hits in the binding cavities on LC3B after refinement of the structures (Figure 5A).…”

Emerging degrader technologies engaging lysosomal pathways

“…Many of these technologies mentioned above, such as SPR, OI-RD, MST, and FP, have been utilized to measure the affinity/kinetics of compound-LC3 interactions in ATTEC studies. 9,76,144,158 In addition, pull-down experiments have been applied to validate the compound-SQSTM1/p62 interactions in the AUTOTAC study. 8…”

“…4). [75][76][77] Meanwhile, their potential influence on LC3 functions and global autophagy need to be tested, and proof-of-concept degradation studies using them as warheads are desired.…”

“…They identified the compounds through DNA-encoded library (DEL) screening. 76 The reversible covalent bonds are formed between the compounds’ aldehyde groups and the LC3 protein. Meanwhile, the detailed binding residue (possibly Lys) has not been determined yet.…”

“…6 : fragments 1-1, 2-3, and 2-10) binding with LC3's HP2 pocket and revealed the binary binding structures by LC3A co-crystallization. 76 To discover novel LC3 binders, they performed unbiased fragment-based NMR screening and DEL screening. They found two lead series through high-throughput screening of 4605 fragments from 5 pools using NMR and SPR measurements.…”