Copper pyrithione, a copper complex ATG4B and autophagy inhibitor, exhibits potent anticancer effects.

Inhibition of autophagy has been considered as a promising strategy for tumor therapy, discovery of small-molecule autophagy inhibitors suitable for clinical use would be of great significance. Since cysteine protease ATG4B plays a key role in the autophagy machinery by processing pro-LC3 and lipidated LC3 to drive the autophagy progress, inhibition of ATG4B may serve as a potential therapeutic strategy. We previously found that copper ions instead of other ions efficiently inhibited ATG4B activity, which was more potent than other ATG4B inhibitors reported. As copper ions are easily chelated, copper complexes may develop into novel ATG4B inhibitors. In this study we identified a copper complex antifouling agent, copper pyrithione (CuPT), which effectively inhibited ATG4B activity and blocked autophagy flux. By combining FRET-based assay in vitro and cell-based assays, we showed that CuPT effectively inhibited ATG4B activity with an IC(50) of 250.9 nM. CuPT (0.5, 1, 2 µM) dose-dependently promoted the formation of insoluble ATG4B and p62 aggregates in HeLa cells, which was similar to copper ions. Importantly, CuPT exhibited potent anticancer activities in vitro and in vivo: it potently suppressed the cell viability of 8 different cancer cell lines with IC(50) values less than 1 µM; administration of CuPT (1 mg/kg; i.p. every three days) significantly inhibited the tumor growth in colorectal xenograft mouse model without obvious organs damage. CuPT-induced cytotoxicity in HCT116 cells could be reversed by enhancing autophagy using rapamycin or Earle's Balanced Salt Solution (EBSS). Besides, we demonstrated that CuPT induced a novel copper-dependent cell death, cuproptosis, of cancer cells. Together, this study presents the first copper complex ATG4B inhibitor CuPT, a copper compound that can be further developed for the treatment of a wide range of cancers.