Abstract
The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4–28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.
Highlights
Autophagy has been associated with numerous disorders including neurodegenerative diseases, metabolic diseases, cardiovascular diseases, infectious diseases and cancer[1,2,3], resulting in subsequent efforts to discover and develop target-selective and potent modulators of the autophagy process[3,4,5,6,7,8,9]
Another study identified an association between HER2 and ATG4B and showed that HER2 positive breast cancer cells are sensitive to ATG4B inhibition under stress conditions[35]
Several crystal structures of ATG4B are publicly available, including crystal structures of the free, closed, inactive conformation[43,44] and LC3B complexes with catalytically inert ATG4B co-crystallized as open, “active” conformations[19,43]
Summary
Autophagy has been associated with numerous disorders including neurodegenerative diseases, metabolic diseases, cardiovascular diseases, infectious diseases and cancer[1,2,3], resulting in subsequent efforts to discover and develop target-selective and potent modulators of the autophagy process[3,4,5,6,7,8,9]. One hundred of the predicted best binding ATG4B inhibitors were obtained to test for effects on GFP-LC3B puncta levels in SKBR3-hrGFP-LC3B breast cancer cells cultured in standard fed conditions. From this set of compounds, five were found to both significantly affect LC3B puncta levels at one or more concentrations and timepoints (Fig. 2c,d; compounds are depicted with their screening codes and relate to NCI codes shown in Table S1) and to inhibit ATG4B enzyme activity in a dose dependent manner when subsequently titrated (Table S1).
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