ATG4B inhibitor FMK-9a induces autophagy independent on its enzyme inhibition

Atg4 is essential for autophagosome formation and Atg8 recycle with the function of processing the precursor and the lipidated Atg8-family proteins. Abnormal autophagic activity is involved in a variety of pathophysiological diseases and ATG4B is of interest as a potential therapeutic target due to its key roles in autophagy process. So ATG4B inhibitors are highly needed. FMK-9a is the most potent inhibitor reported so far. In this study, we confirmed FMK-9a could suppress ATG4B activity in vitro and in cells, with an IC50 of 260 nM. Besides, FMK-9a could also attenuate the process of cleavage of pro-LC3 and the delipidation of LC3-PE. Importantly, FMK-9a could induce autophagy both in HeLa and MEF cells regardless of its inhibition on ATG4B activity. Moreover, FMK-9a induced autophagy required FIP200 and ATG5. In conclusion, we demonstrated that ATG4B inhibitor FMK-9a induces autophagy independent on its enzyme inhibition. Thus, FMK-9a may plays multiple roles in autophagy process and cannot simply take it as an ATG4B inhibitor.