Drug Repurposing Screening Identifies Tioconazole as an ATG4 Inhibitor that Suppresses Autophagy and Sensitizes Cancer Cells to Chemotherapy

Abstract

Background: Tumor cells require proficient autophagy to meet high metabolic demands and resist chemotherapy, which suggests that reducing autophagic flux might be an attractive route for cancer therapy. However, this theory in clinical cancer research remains controversial due to the limited number of drugs that specifically inhibit autophagy-related (ATG) proteins.Methods: We screened FDA-approved drugs using a novel platform that integrates computational docking and simulations as well as biochemical and cellular reporter assays to identify potential drugs that inhibit autophagy-required cysteine proteases of the ATG4 family. The effects of ATG4 inhibitors on autophagy and tumor suppression were examined using cell culture and a tumor xenograft mouse model.Results: Tioconazole was found to inhibit activities of ATG4A and ATG4B with an IC50 of 1.3 µM and 1.8 µM, respectively. Further studies based on docking and molecular dynamics (MD) simulations supported that tioconazole can stably occupy the active site of ATG4 in its open form and transiently interact with the allosteric regulation site in LC3, which explained the experimentally observed obstruction of substrate binding and reduced autophagic flux in cells in the presence of tioconazole. Moreover, tioconazole diminished tumor cell viability and sensitized cancer cells to autophagy-inducing conditions, including starvation and treatment with chemotherapeutic agents.Conclusion: Tioconazole inhibited ATG4 and autophagy to enhance chemotherapeutic drug-induced cytotoxicity in cancer cell culture and tumor xenografts. These results suggest that the antifungal drug tioconazole might be repositioned as an anticancer drug or chemosensitizer.

Knockdown ATG4C inhibits gliomas progression and promotes temozolomide chemosensitivity by suppressing autophagic flux

“…*p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant proliferation of primarily cultured glioblastoma cells from patients (Additional file 3: Figure S3). A large body of evidence suggests that inhibition of the activity of other ATG4 family members, including ATG4A, ATG4B and ATG4D, could suppress tumor progression and enhance chemosensitivity or the efficacy of radiotherapy [42][43][44][45][46][47][48][49]. These findings consistently implied that the ATG4 family played an important role in tumorigenesis and cancer therapy.…”

Combination of an Autophagy Inducer and an Autophagy Inhibitor: A Smarter Strategy Emerging in Cancer Therapy

“…Moreover, clinical trials showed that autophagy is unable to be completely inhibited by CQ in vivo. These warrant us that searching more potent autophagy inhibitors is critical for promoting an opportunity to apply the combination therapeutic strategy in clinical studies (Lalita Guntuku et al, 2019), especially agents that specifically inhibit autophagyrelated (ATG) proteins (Pei-Feng Liu1 et al, 2018).…”

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

“…ATG4B expression was found elevated in colorectal cancer, and knockdown of ATG4B resulted in reduced cell cycle progression and inhibition of colorectal cancer cell lines [92]. Some ATG4B inhibitors identified to date have been tested in xenograft models of colorectal cancer cells, typically HCT-116 cells, and demonstrated a significant benefit in reducing tumour growth, in particular when combined with chemotherapeutic agents [93].…”

“…Another luciferase-based sensor utilises the complementation of a split luciferase that is flanking LC3. In this arrangement, the uncleaved LC3 displays high levels of luminescence that is lost when cleaved by ATG4B [93]. This assay has been used to validate ATG4B inhibitors in cells, but the dynamic range of this assay is limited due to a high background activity.…”

“…Tioconazole was identified as an ATG4A and ATG4B inhibitor using a combination of in silico screening of a collection of FDA-approved drugs and validation with biochemical assays, notably a GABARAPL2-PLA assay and a split-luciferase reporter [93]. Tioconazole was found to dock to the active site of ATG4B, and also to a second site that is required for binding of LC3 at the N-terminus.…”