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.

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.…”

Targeting LC3/GABARAP for degrader development and autophagy modulation

“…16 However, discovery of potent LC3/GABARAP ligands has remained challenging, possibly due to the conformational plasticity of LC3/GABARAP resulting in at least partial occlusion of the LDS. 17 First, LC3A/B targeting reversible ligands such as the antibiotic Novobiocin 18 , covalent lysine targeting ligands 19 as well as a number of low molecular weight fragments (overviewed in Figure 1C) have been described binding to the LDS, 20 but no highly potent ligands or ligands for the four remaining LC3/GABARAPs have been described. Interestingly, in 2019, first ATTECs have been reported suggesting that the autophagy degradation pathway can be exploited for the design of selective degrader small molecules.…”