Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo

Abstract

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.

Targeting Autophagy in Cancer: Recent Advances and Future Directions

“…ATG4B inhibitors that have been reported, such as S130 (148) and FMK-9a (149), with biochemical and in vitro activity. In addition, the ATG4B inhibitor NSC185058 (150) has been shown to have both in vitro and in vivo antitumor activity.…”

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

“…Biochemical assays to measure the ATG4B protease activity are available (Figure 3). These range from simple fluorometric readouts [99], fluorescence resonance energy transfer (FRET)-based sensors [99,100] to gel electrophoresis assays [101]. Typically, assays to monitor ATG4B activity rely on the cleavage of its substrate LC3A or LC3B that can be monitored by a coupled enzymatic or fluorescent reaction.…”

“…More recently, computational docking studies have identified styrylquinolines as binders to ATG4B, and have subsequently been shown to reduce activity [106]. Another potent inhibitor called S130 was identified by a similar in silico approach and validated with the LC3B-GST assay [101]. Computational docking studies have also resulted in the identification of an agonist for ATG4B, Flubendazole [83].…”

“…Another study using computational docking for identification of ATG4B binding molecules presents results from an in silico screen using a library of 7249 diverse compounds, and identified S130 as a small compound that binds to a pocket in proximity to the active site of ATG4B [101]. S130 results in the accumulation of lipidated LC3 in cells, indicating disruption of autophagic flux, and has strong anti-proliferative activity in a variety of cancer cell lines, as well as in vivo growth of colorectal cancer xenografts [101]. Furthermore, a LC/MS-based assay to monitor the pharmacokinetic properties of S130 has been recently developed [120].…”

Autophagy and Cell Death: Antitumor Drugs Targeting Autophagy

“…This process induces Atg8/LC3-PE to be anchored to the membrane of autophagosomes to regulate autophagy. Fu et al [82] identified a novel ATG4B small-molecule inhibitor, S130, through in silico screening and in vitro high-throughput screening system for fluorescence resonance-energy transfer and showed that S130 did not affect the autophagosome function and autophagosome fusion with lysosomes. Instead, S130 inhibited the delipidation process of LC3-PE and ultimately blocked autophagy.…”