Discovery of a Small Molecule Targeting Autophagy via ATG4B Inhibition and Cell Death of Colorectal Cancer Cells in Vitro and in Vivo

Autophagy. 2019 Feb;15(2):295-311. doi: 10.1080/15548627.2018.1517073. Epub 2018 Sep 20.


Human Atg4 homologs are cysteine proteases, which play key roles in the macroautophagy/autophagy process by cleaving Atg8 homologs for conjugation to lipid membranes and for deconjugation of Atg8 homologs from membranes. Expression of ATG4B is significantly increased in colorectal cancer cells compared to normal cells, suggesting that ATG4B may be important for cancer biology. Inhibition of ATG4B may reduce the autophagy activity, thereby sensitizing cancer cells to therapeutic agents. Thus, developing specific and potent ATG4B inhibitors for research as well as for potential therapeutic uses is highly needed. In this study, we integrated in silico screening and in vitro assays to discover a potent ATG4B inhibitor, named S130, from a noncommercial library. This chemical binds to ATG4B with strong affinity and specifically suppresses the activity of ATG4B but not other proteases. S130 did not cause the impairment of autophagosome fusion, nor did it result in the dysfunction of lysosomes. Instead, S130 might attenuate the delipidation of LC3-II on the autolysosomes to suppress the recycling of LC3-I, which normally occurs after LC3-II cleavage by ATG4B. Intriguingly, S130 induced cell death, which was accompanied with autophagy stress and could be further exacerbated by nutrient deprivation. Such cytotoxicity could be partially reversed by enhancing ATG4B activity. Finally, we found that S130 was distributed in tumor tissues in vivo and was also effective in arresting the growth of colorectal cancer cells. Thus, this study indicates that ATG4B is a potential anticancer target and S130 might be a novel small-molecule candidate for future cancer therapy.

Keywords: ATG4B; Anti-tumor; FRET assay; autophagy; cell death; colorectal cancer; delipidation; xenografts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Autophagy / drug effects*
  • Autophagy-Related Proteins / antagonists & inhibitors*
  • Autophagy-Related Proteins / metabolism
  • Cell Proliferation / drug effects
  • Colorectal Neoplasms / pathology*
  • Colorectal Neoplasms / ultrastructure
  • Cysteine Endopeptidases / metabolism
  • Female
  • Lysosomes / drug effects
  • Lysosomes / metabolism
  • Mice, Inbred BALB C
  • Mice, Nude
  • Microtubule-Associated Proteins / metabolism
  • Protein Binding / drug effects
  • Small Molecule Libraries / pharmacology*


  • Autophagy-Related Proteins
  • Microtubule-Associated Proteins
  • Small Molecule Libraries
  • light chain 3, human
  • ATG4B protein, human
  • Cysteine Endopeptidases

Grant support

This work was supported in part by the National Natural Science Foundation of China (31671437), the National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX09735010), the Natural Science Foundation of Guangdong Province, China (2016A030313335), the 111 project (B16047), and the Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030).