Metabolic stress induces autophagy as an alternative source of energy and metabolites. Insufficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. Here, we performed a functional screen in search of novel autophagy regulators from natural products. We showed that oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, is a potent autophagic flux inhibitor. Exposure to OC results in an increased number of autophagosomes and impaired degradation of SQSTM1/p62. Costaining of GFP-LC3B with LysoTracker Red or LAMP1 antibody demonstrates that autophagosome-lysosome fusion is blocked by OC treatment. Furthermore, OC inhibits lysosomal proteolytic activity by altering lysosomal acidification and downregulating the expression of lysosomal cathepsins. Importantly, OC can eliminate the tolerance of cancer cells to nutrient starvation. Starvation dramatically increases the susceptibility of cancer cells to OC-induced CASP3-dependent apoptosis in vitro. Subsequent studies in xenograft mouse model showed that OC has anticancer potency as revealed by increased staining of cleaved CASP3, LC3 puncta, and SQSTM1, as well as reduced expression of lysosomal cathepsins. Combined treatment with OC and caloric restriction potentiates anticancer efficacy of OC in vivo. Collectively, these data demonstrated that OC is a novel autophagic flux inhibitor and might be useful in anticancer therapy.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure−activity relationship (SAR) and protease selectivity are also discussed.KEYWORDS: ATG4B, autophagy, covalent inhibitor, fluoromethylketone, peptidomimetics A utophagy is an evolutionarily conserved process essential for cell homeostasis and housekeeping by catabolizing aggregated proteins and damaged cellular components. 1 The hallmark of autophagy is the formation of autophagosomes and subsequent fusion with lysosomes to achieve degradation of their contents. Dysregulation of autophagy has been recently described in the pathogenesis of a variety of diseases such as cancer, neurodegenerative and metabolic disorders, and viral infections. 2,3 Modulation of autophagy has become a very active area of preclinical and clinical research, and particularly, there is high interest to identify potent and specific autophagy inhibitors. 4 ATG4 or autophagins are a class of cytosolic cysteine proteases that cleave ATG8 family proteins, such as light chain 3 (LC3). ATG4 plays essential roles in the formation and maturation of autophagosomes. 5,6 Among all four human ATG4 orthologues, ATG4B is functionally dominant, and it is the sole enzyme reported to efficiently cleave LC3 precursors and to deconjugate lipid from membrane bound LC3-phosphatidylethanolamine (LC3-PE). 7 ATG4B has been considered as a potential therapeutic target in the development of chemosensitizer for the treatment of certain cancer types. Recently, a number of small molecule ATG4B inhibitors such as Z-L-Phe-chloromethylketone and NSC185058 were reported to have modulatory effects on the autophagy process. 8−10 However, because of its high chemical reactivity, the chloromethylketone moiety is usually associated with significant cytotoxicity. NSC185058, however, is only a weak ATG4B inhibitor with an IC 50 of 51 μM in an assay detecting the cleavage of LC3-GST. 10 Previously, we reported the identification of Z-FA-FMK (1, Figure 1) as a covalent active-site directed ATG4B inhibitor from a TR-FRET based focused library screening. 11 The hit expansion of 1 led to the discovery of Z-FG-FMK (2), which was 10-times more potent than 1 with an IC 50 of 1.2 μM in the biochemical assay. 11 Herein we disclose the structure-guided optimization of 2 toward highly potent fluoromethylketone (FMK)-based ATG4B inhibitors.As an early step in the autophagosome formation, ATG4B cleaves proLC3 to expose the C-terminal Gly120 for subsequent PE-conjugation and autophagosome membrane insertion. 5 The cocrystal structures of catalytically inert C74S or

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Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti-cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti-proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP-LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.