(A) The isolation process of rat liver lysosomal fraction. autophagosome and lysosome but elevated lysosomal pH and impaired lysosomal enzymes activity. Using rat liver lysosome fraction and purified yeast V-ATPase, we found that toosendanin directly inhibited V-ATPase activity. By applying cellular thermal shift assay (CETSA), immunoprecipitation-coupled LC-MS/MS analysis, and biotin-toosendanin pull-down assay, we confirmed the direct binding between toosendanin and V-ATPase. Furthermore, toosendanin blocked chemotherapy-induced protective autophagy in cultured cancer cells and xenograft tumor tissues to significantly enhance anti-cancer activity. These results suggest that toosendanin has the potential to be developed into an anti-cancer drug by blocking chemotherapy-induced protective autophagy. Sieb. et Zucc which has been used to treat abdominal pain and used as the digestive tract parasiticide in ancient China for about 1500 years. Several studies have indicated that TSN shows potential anti-proliferative and pro-apoptotic effects on various human cancers 13-17, while the anti-cancer mechanism and drug target have not been fully comprehended. In this study, we identified a novel pharmacological effect of TSN: inhibition of V-ATPase activity to block late-stage autophagy. Furthermore, we studied the complementary effect of TSN around the anti-cancer efficacy of chemotherapy. Results showed that TSN inhibits camptothecin (CPT)-induced protective autophagy and significantly sensitizes cancer cells to CPT-induced toxicity in cellular and animal models, with good tolerance and safety. Results TSN inhibits autophagic flux To search for small molecules that block autophagic flux, we applied the high-content screening to observe the effect of compounds around the distribution of GFP-LC3 puncta in HeLa cell line with stable expression of GFP-LC3. GFP-LC3 is usually evenly distributed in the cytoplasm under normal conditions but dramatically accumulates to form puncta under either autophagy induction or autophagy maturation inhibition conditions. TSN was identified as the autophagy modulator by IN CELL Analyzer 2000 from our natural compound library ( 3000 compounds) and further confirmed by laser scanning Pindolol confocal microscopy (Physique ?(Figure1A).1A). TSN induced the dramatic accumulation of GFP-LC3 puncta in a pattern similar to that induced by autophagy inhibitor bafilomycin A1 (BAF), and was thus identified as an autophagy modulator. Open in a separate window Physique 1 The identification of the autophagy inhibitor TSN. (A) Results of high-content screening for chemical autophagy modulators. (B) WB analysis of LC3B-II and SQSTM1/p62 protein levels in HeLa and A549 cells treated with TSN (0.01~10 M) or TSN 1 M for (0~24 h) (tag inserted IGSF8 into its chromosomal DNA 3 of the gene encoding (Determine ?(Physique4C).4C). We confirmed that TSN inhibited the activity of purified yeast V-ATPase (Physique ?(Physique4E,4E, S1B). Open in a separate window Physique 4 TSN inhibits the V-ATPase activity. (A) The isolation process of rat liver lysosomal fraction. (B) WB analysis of LAMP1, CTSB and cytochrome C protein levels in the organelle pellet and lysosomal fraction. (C) The isolation process of yeast V-ATPase. (D)-(E) Rat liver lysosomal and purified yeast V-ATPase activity assay in TSN and BAF treatment groups (in vivoSieb. et Zucc., and showed anti-parasitic in the traditional application. In this study, we identified TSN as an autophagy modulator through a high-content screening model and further confirmed it blocks the autophagic flux and inhibits its substrates degradation. Interestingly, TSN showed highly potent autophagy inhibition activity in both Pindolol cancer cell lines and tumor xenograft nude mice models. We found that TSN can efficiently inhibit autophagic flux at a relative low concentration (10 nM in cell culture and 0.5 mg/kg (i.p.) in mice), indicating that TSN is usually a potent autophagy inhibitor. Drug target identification is usually a challenging task. V-ATPase contains a peripheral V1 domain name and an integral membrane V0 domain name. Each domain name comprises multiple subunits, which tethers together for ATP hydrolysis and proton translocation 37, 57. Therefore, it might not be easy to confirm the direct conversation between the TSN and the large V-ATPase complex through the technology of surface plasmon resonance (SPR) or isothermal titration microcalorimetry (ITC). Under this concern, we developed and applied biochemical analyses to confirm that V-ATPase complex can pull down TSN and biotin-TSN can also pull down V-ATPase complex, indicating that TSN actually binds and inhibits Pindolol V-ATPase activity. In this study, we used the NP40 lysis buffer in the identification of drug target, because SDS.